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Rapid Chemical Ligation of DNA and Acyclic Threoninol Nucleic Acid (aTNA) for Effective Nonenzymatic Primer Extension

Cite this: J. Am. Chem. Soc. 2023, 145, 32, 17872–17880
Publication Date (Web):July 19, 2023
https://doi.org/10.1021/jacs.3c04979

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Abstract

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Previously, nonenzymatic primer extension reaction of acyclic l-threoninol nucleic acid (L-aTNA) was achieved in the presence of N-cyanoimidazole (CNIm) and Mn2+; however, the reaction conditions were not optimized and a mechanistic insight was not sufficient. Herein, we report investigation of the kinetics and reaction mechanism of the chemical ligation of L-aTNA to L-aTNA and of DNA to DNA. We found that Cd2+, Ni2+, and Co2+ accelerated ligation of both L-aTNA and DNA and that the rate-determining step was activation of the phosphate group. The activation was enhanced by duplex formation between a phosphorylated L-aTNA fragment and template, resulting in unexpectedly more effective L-aTNA ligation than DNA ligation. Under optimized conditions, an 8-mer L-aTNA primer could be elongated by ligation to L-aTNA trimers to produce a 29-mer full-length oligomer with 60% yield within 2 h at 4 °C. This highly effective chemical ligation system will allow construction of artificial genomes, robust DNA nanostructures, and xeno nucleic acids for use in selection methods. Our findings also shed light on the possible pre-RNA world.

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Introduction

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The origin of life is an “eternal” question to which scientists have eagerly tried to get plausible answers. (1) A widely accepted scenario is the RNA world hypothesis, which posits that at the early stage of life, RNA self-replicated without protein-based enzymes under primordial conditions. (2,3) To prove that the RNA emerged from abiotic ancient earth as a preliminary life form, research studies have tried to replicate synthesis of ribonucleotides (4) and nonenzymatic RNA extension reactions (5−9) under conditions thought to mimic primordial earth. The pre-RNA world hypothesis, another scenario of the RNA-first hypothesis, suggests that xeno nucleic acids (XNAs), which have simpler scaffolds than ribose, might have been the initial self-replicating molecules. (10) This model assumes that the preliminary genetic system involved XNA, followed by genetic takeover by RNA. Various XNAs with nonribose scaffolds have been synthesized; (11−14) among them are threose nucleic acid (TNA) (11) and glycol nucleic acid (GNA). (13) Both TNA and GNA are candidates for pre-RNA world genetic material because they can be readily synthesized and they cross-hybridize with RNA. (15,16) However, elongation of an XNA strand on an XNA template, the key process for a nonenzymatic XNA replication system in the XNA world, has not been thoroughly investigated. In our knowledge, modified GNA and modified PNA residues have been ligated on an XNA template, but these XNAs are not phosphodiester-based. (17,18)
Our group designed and synthesized acyclic l-threoninol nucleic acid (L-aTNA) as an acyclic XNA and showed that L-aTNA oligomers form a stable duplex with complementary DNA and RNA as well as L-aTNA (Figure 1a). (19) Recently, we demonstrated nonenzymatic primer extension of L-aTNA via template-directed chemical ligation using a random pool of L-aTNA trimers in the presence of N-cyanoimidazole (CNIm) and Mn2+ (Figure 1b). (20) In this experiment, trimer fragments were ligated to a growing chain. The primer, an 8-mer L-aTNA, was hybridized to a 17-mer L-aTNA template, and selective incorporation of three trimer fragments resulted in 17-mer L-aTNA with about 75% yield at 4 °C after 24 h. This was the first example of nonenzymatic template-directed synthesis of an acyclic XNA. While this template-directed elongation reaction was attributed to rapid ligation of L-aTNA, the reaction mechanism was unclear.

Figure 1

Figure 1. (a) Chemical structures of DNA and L-aTNA. (b) Schematic illustration of the chemical ligation reaction by CNIm and a divalent metal cation. (c) Sequences of L-aTNA and DNA used for chemical ligation. (d) Denaturing PAGE analysis of chemical ligation of 8-mer L-aTNA fragments (T8A/T8B-3′p) on the 16-mer template (T16t) in the presence of CNIm and chloride salt of an indicated divalent metal cation (MCl2). Reaction conditions: 0.9 μM T8A, 1.1 μM T8B-3′p, 1.0 μM T16t, 100 mM NaCl, 5 mM MCl2, 20 mM CNIm, 25 °C. PAGE conditions: 20% acrylamide, 8 M urea, 1 × TBE, 4 W at room temperature for 1.5 or 2 h. Negative control (lane 1) included only T8A and NaCl. (e) Yield as a function of time for ligation of T8A to T8B-3′p on the T16t template (left) and of D16A-3′p to D16B on the D32t template (right) in the presence of indicated divalent metal cations. The graphs are based on data shown from panel d and Figure S1. (f) Calculated kobs values for chemical ligation of indicated oligomers in the presence of indicated metal ions. Reaction conditions: 0.9 μM fragment A, 1.1 μM fragment B, 1.0 μM template, 100 mM NaCl, 5 mM MCl2, 20 mM CNIm, 25 °C. kobs values were calculated from linearized plots of -ln([Fragment A]/[Fragment A]0) assuming a pseudo-first-order reaction.

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Template-directed “native” chemical ligation to form phosphodiester bonds has previously been investigated for ligation of DNAs for construction of DNA nanomaterials, (21) for circularization of DNA (and RNA), (22) for DNA-templated chemistry, (23) and for generation of long DNA strands like genome sequences. (24,25) Carbodiimide has been used as a reagent for chemical ligation of nucleic acids. (24,26−28) Braun and co-workers demonstrated a DNA replication system using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). (29) Recently, Hud and co-workers optimized conditions for EDC-driven ligation. (30) However, a high concentration of EDC (250 mM) and 24 h incubation are necessary for a quantitative reaction even under optimized conditions. A side reaction induced by EDC is also problematic. (29) Cyanogen bromide (BrCN) has also been used to facilitate DNA ligation, but the high concentration required (100–500 mM), HBr released, and side reactions prevent practical use. (21a,31,32) Kanaya and Yanagawa reported that N-cyanoimidazole (CNIm) generated by a BrCN/M2+/imidazole system enabled effective chemical ligation with a relatively low amount of reagent (∼20 mM). (33,34) The ligation reaction by CNIm proceeds in the presence of Mn2+ or Zn2+. (35−37) Mariani and Sutherland performed RNA ligation in the CNIm/Mn2+ system for the elucidation of only 3′,5′-linked RNA emergence from the heterogeneous mixture. (38) However, the reaction is slow, and the mechanism remains unknown.
Herein, we report analysis of the kinetics of chemical, template-directed ligation of L-aTNA oligomers and of DNA oligonucleotides driven by CNIm and various divalent cations. We found that the use of Cd2+, Ni2+, or Co2+ remarkably improved the reaction rates and efficacies of ligations of both L-aTNA and DNA compared to the reaction in the presence of Mn2+. Under optimized conditions based on the kinetic analyses, an 8-mer L-aTNA primer could be elongated to a 29-mer by incorporation of seven L-aTNA trimers from the random trimer pool.

Results and Discussion

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Effect of Metal Ion Identity on the Ligation Reaction

Before performing detailed kinetic analyses, we first measured the rates of ligation of two 8-mer L-aTNA fragments on a 16-mer template (T8A/T8B-3′p/T16t, Figure 1c) in the presence of CNIm and various divalent cations at 25 °C. Sequences of all oligomers used in chemical ligation experiments are shown in Table S1. Interestingly, the reaction was essentially complete within only 5 min in the presence of Cd2+, Ni2+, or Co2+, but over 20 min was required for Mn2+ (Figure 1d,e). The kobs values were over 33 h–1 for Cd2+, Ni2+, and Co2+ and 4.0 h–1 for Mn2+ (Figure 1f). The same tendency was observed for DNA ligation (D16A-3′p/D16B/D32t, Figure 1c): Cd2+, Ni2+, and Co2+ remarkably accelerated the ligation reaction (kobs > 3 h–1) relative to Mn2+ (kobs < 0.5 h–1) (Figure 1e,f). Previously, we reported that ligation proceeds much faster when a primary OH attacks an activated monophosphate of a secondary OH. (20) Even the reactions of L-aTNA fragments T8A-1′p and T8B, in which a 1′-phosphate reacts with a 3′-OH, and of DNA fragments D16A and D16B-5′p, in which a 5′-phosphate reacts with a 3′-OH, were accelerated in the presence of these metal cations (Figures 1f, S1, and S2). Although a prolonged reaction over 4 h occasionally resulted in precipitate formation (Figure S1), kobs values with Cd2+, Ni2+, or Co2+ were much larger than those with Mn2+ for all L-aTNAs and DNAs tested (Figure 1f). In the presence of Zn2+ and Cu2+, ligation did not proceed quantitatively, probably because these metal ions induced precipitation (Figure 1d). Of note, both 3′-phosphorylated DNA and 5′-phosphorylated DNA were efficiently ligated: ligation was quantitative after a 4 h incubation with 5 mM Cd2+ and after a 1 h incubation with 20 mM Cd2+ at 25 °C (Figure S3). Thus, ligation in the presence of CNIm and Cd2+ is much faster than EDC-mediated chemical ligation. (21,30) Although some groups have reported template-directed ligation of DNA with the CNIm/Ni2+ system, efficiencies were not as high as we observed using Cd2+. (33,35,37) Since the 5′-phosphorylated DNA can be easily prepared by incubation with a kinase and ATP, this rapid chemical ligation system should be widely applicable.

NMR Analysis of the Phosphate Activation Mechanism of the Phosphate Group

Since ligation was optimal in the presence of the diamagnetic Cd2+, we followed the reaction by NMR. The chemical ligation reaction proceeds in three main steps: (i) formation of a duplex between fragments and template, (ii) activation of phosphate by binding of CNIm, and (iii) generation of a phosphodiester bond by nucleophilic attack of the activated phosphate by an OH (Figure 1b). We can ignore step (i) because the melting temperatures (Tms) of the nicked duplexes were approximately 50 °C in the presence of each metal cation (Table S2), well above the 25 °C reaction temperature. We performed NMR measurements in D2O using a simple model compound, phenyl phosphate (PhP), which has a monophosphate group, rather than a phosphorylated oligomer. The pH was adjusted to 5.9 (pD 6.3) (39) using aqueous HCl because the pH of reaction solution for the chemical ligation reaction was between pH 5.5 and pH 6.5 (Figure S4). In the reaction, PhP is first activated by CNIm (Figure 2a). This is followed by D2O attack, not by attack of the OH of the fragment, and PhP is regenerated accompanied by release of a byproduct (Figure 2a). In 1H and 31P spectra measured as a function of time, peak intensities assigned to CNIm gradually decreased and those assigned to imidazole (Im) increased (Figures 2b and S5). This indicated that PhP mediated the decomposition of CNIm to Im (Figure 2c). No signals not assigned to PhP, CNIm, or Im were observed during the reaction, and PhP signals did not change over time in either 31P or 1H NMR spectra. Direct decomposition of CNIm to Im induced by Cd2+ was ruled out by the fact that the CNIm signal did not change in the absence of PhP (Figures S6 and S7).

Figure 2

Figure 2. (a) Scheme of reaction of PhP and CNIm in the presence of Cd2+. (b) 1H NMR time course analysis. 1H NMR peaks colored brown, red, and green were assigned to PhP, CNIm, and Im, respectively. Reaction conditions: 5 mM PhP, CdCl2, and CNIm, 25 °C. (c) Possible reaction scheme and intermediates (i) suggested by NMR and (ii) not observed. (d) Normalized peak intensity of CNIm (red) and Im (green) calculated from NMR spectra.

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It should be noted that no signals suggestive of a PhP-CNIm intermediate were observed (Figure 2c (ii)). These results suggest that the interaction between CNIm and the phosphate is transient and that activation of phosphate is the rate-determining step. Kanaya and Yanagawa previously reported two possible intermediates: (i) direct binding of CNIm to the oxygen of the phosphate and (ii) imidazolide form (Figure 2c). (33) The latter intermediate was reported to be isolable, (40) but we did not observe signals characteristic of the imidazolide. We suggest that CNIm and Cd2+ activate the phosphate via direct binding (Figure 2c (i)), generating a highly reactive intermediate that is not detectable by NMR.
We calculated the rate constant for the activation step by assuming a bimolecular reaction between CNIm and phosphate. The reaction can be described as the pseudo-first-order as shown below because the concentration of PhP is constant:
− d [ CNIm ] d t = k 1 [ PhP ] 0 [ CNIm ] = k app [ CNIm ] ( w h e r e ⁢   k 1 = k app [ PhP ] 0 )
If the nucleophilic attack by D2O is considerably faster than the activation process, the rates of consumption of CNIm and generation of Im should be the same. Natural logarithmic plots of the concentrations of CNIm and Im as a function of time were linear and yielded the same rate constants within the experimental error: the kapp for consumption of CNIm was 1.3 h–1 and that for production of Im was 1.4 h–1 (Figure 2d). The bimolecular rate constant k1 was 2.6 × 102–2.8 × 102 M–1·h–1. We also examined the reaction of 2′-deoxycytidine-5′-monophosphate (dCMP) and CNIm in the presence of Cd2+. The kapp for consumption of CNIm was 1.6 h–1 and that for production of Im was 1.4 h–1. This demonstrated that the monophosphate group is activated and nucleophilic substitution occurs through the same pathway, irrespective of the substrate (Figures S8–S10).

Kinetic Analysis of the Chemical Ligation Reaction

To unravel the mechanism that results in rapid chemical ligation of L-aTNA, we examined concentration dependence of the reaction kinetics. Reaction rates were determined from PAGE analysis by changing the concentration of CNIm (Figures 3a–c and S11–S15 and Table S3). In most cases, the reaction rates were pseudo-first-order, and the apparent k1s (k1,apps) linearly increased with the concentration of CNIm (Figure 3a–c). This is consistent with the NMR results that indicated that the activation process was the rate-determining step. When CNIm is in large excess compared to the oligomer fragments, the reaction rate can be described as follows:
d [ Product ] d t = − d [ Fragment ] d t = k 1 [ CNIm ] 0 [ Fragment ] = k 1 , app [ Fragment ] ( w h e r e k 1 = k 1 , app [ CNIm ] 0 ) ( [ CNIm ] 0 ≫ [ Fragment ] )

Figure 3

Figure 3. (a) Plot of -ln([Fragment A]/[Fragment A]0) as a function of time over a range of CNIm concentrations. Reaction conditions: 0.9 μM T8A, 1.1 μM T8B-3′p, 1.0 μM T16t, 5 mM CdCl2, 1, 5, 10, or 20 mM CNIm, 25 °C. (b) Apparent k1 obtained assuming a pseudo-first-order reaction. k1,app of T8A/T8B-3′p/T16t and the other k1,apps were calculated from plots shown in Figures 3a and S11–S15, respectively. Reaction conditions: 0.9 μM fragment A, 1.1 μM fragment B, 1.0 μM template, 5 mM CdCl2, 1, 5, 10, or 20 mM CNIm, 25 °C. (c) Plots of apparent k1 values for each component versus CNIm concentration based on the Ostwald isolation method. (d) Energy-minimized structures of T8B-3′p and template (top) and T8A-1′p and template (bottom) illustrating interactions of phosphate with the neighboring amide group. (e) Correlation between the concentration of Cd2+ ions and the rate constant k1. (f) Schematic illustration of the predicted mechanism involving divalent metal cations in activation of the phosphate group and rates for steps 1 and 2 and the overall reaction.

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We calculated the bimolecular rate constant k1 (M–1·h–1) in 5 mM Cd2+ from pseudo-first-order rate constants (k1,apps) for each component using the Ostwald isolation method (Figures 3c and S16). For ligation of T8A-1′p to T8B on the T16t template and of D16A-3′p to D16B on the D32t template, k1 values were 2.1 × 102 and 1.7 × 102 M–1·h–1, respectively (Figure 3c). These values are in accordance with that obtained from NMR analyses (2.6 × 102–2.8 × 102 M–1·h–1). Although nucleophilic attack by H2O to activated phosphate possibly competes with the ligation, it was ignorable as we expect that bond formation between the activated phosphate and the hydroxyl group will be rapid. In contrast, k1 for ligation of T8A to T8B-3′p on template T16t (1.7 × 103 M–1·h–1) was considerably larger than that determined by NMR analysis.
We initially hypothesized that the rate of activation of the phosphate would depend on the terminal chemical structure of each strand. To test this, we measured the activation rate of the phosphate groups on four trimers: 3′p-L-aTNA-T3, 1′p-L-aTNA-T3, 5′p-DNA-T3, and 3′p-DNA-T3 (Figures S17–S22). In contrast to our predictions, observed k1 values for all oligomers were almost the same (2.6 × 102–3.3 × 102 M–1·h–1) and were comparable to k1 for activation of PhP and dCMP (Figures 2 and S10). These results suggest that the activation of phosphate does not depend on the chemical structure. Presumably, the duplex between the 3′-phosphorylated L-aTNA fragment and the template has a conformation suitable for activation of the phosphate group by CNIm. The energy-minimized structure of the 3′-phosphorylated L-aTNA fragment on an L-aTNA template revealed a hydrogen bond between the phosphate and an amide group that was not observed in the duplex with the 1′-phosphorylated L-aTNA (Figure 3d). The conformation stabilized by this hydrogen bond likely led to rapid activation of the phosphate. The slow ligation of D16A to D16B-5′p on the D32t template might be due to the low nucleophilicity of secondary 3′-OH of D16A (k1 = 54 M–1 h–1). (20,24)
We also investigated the effect of the concentration of Cd2+. The k1 increased almost linearly with the concentration of Cd2+ (Figures 3e and S11–S14), indicating that the metal ion is also necessary for the activation step. This reaction rate can be approximated by the following formula because k1 is proportional to the concentration of the divalent ion at time zero ([M2+]0):
k 1 ′ = k 1 [ M 2 + ] 0 = k 1 , app [ M 2 + ] 0 [ CNIm ] 0
d [ Product ] d t = k 1 ′ [ CNIm ] 0 [ M 2 + ] 0 [ Fragment ] ( w h e r e [ CNIm ] 0 , [ M 2 + ] 0 ≫ [ Fragment ] )
This equation suggests that the complex of metal ion, phosphate, and CNIm is required for the activation process (Figure 3e). The stability constants between the imidazole derivative and metal ions are in the order of Cu2+ > Ni2+ > Cd2+, Co2+, and Zn2+ > Mn2+. (41) Furthermore, the stabilities of the complexes between phosphate and metal ions are in the order of Cu2+ > Ni2+, Cd2+, Co2+, and Zn2+ > Mn2+ based on stabilities of the complexes of these divalent ions with ethylenedinitrilotetra(methylphosphonic acid). (42) These orders are in accordance with the efficiencies of ligation (Ni2+, Cd2+, and Co2+ > Mn2+), supporting our hypothesis. Note that although Cu2+ was intrinsically more reactive than the other metal ions, it caused precipitation during the reaction, resulting in low reproducibility (Figures 1e and S3).

Chemical Primer Extension To Yield Longer L-aTNA Oligomers under Optimized Conditions

Next, we attempted primer extension of an L-aTNA template under the improved conditions in the presence of a pool of all possible trimers. Use of Cd2+ instead of Mn2+ would be expected to improve the efficacy of the chemical primer extension. Moreover, we hypothesized that the rate of elongation would depend on the direction of elongation. Previously, elongation was carried out in the 3′ → 1′ direction (Figure 4a). (20) In this case, either the terminal phosphate group of the trimer bound on the template is activated or an activated trimer binds to the template. However, unlike the ligation of two long strands (Figure 1), binding of trimers is so weak, resulting in only a small amount of the 3′-phosphate group with the proper conformation for the activation. In contrast, 1′ → 3′ elongation should accelerate the activation of the 3′-phosphate group on the primer and the elongating strand. To study the elongation reaction, we used systems with fluorescently labeled L-aTNA primer strands (T8A or Rev-T8A-3′p), 17-mer L-aTNA templates (T17t or Rev-T17t), and random trimer fragments (T3Bmix) (Figure 4a,b).

Figure 4

Figure 4. (a and b) Schematic illustrations of elongation via chemical ligation of an L-aTNA primer in (a) 3′ → 1′ direction and (b) 1′ → 3′ direction. (c) Denaturing PAGE of L-aTNA elongation reaction products in the presence of Cd2+ with ligation in the 1′ → 3′ direction. (d) Comparison of yields of full-length elongation products in different conditions. Reaction conditions: 0.9 μM primer, 100 μM T3Bmix, 1.0 μM template, 100 mM NaCl, 5 mM CdCl2 or 20 mM MnCl2, 20 mM CNIm, 4 °C for 6 h in the presence of Cd2+ or for 24 h in the presence of Mn2+. PAGE conditions: 20% acrylamide, 8 M urea, 1 × TBE, 3 h, 4 °C, 4 W. Negative control (lane 1) included only primer, NaCl, and divalent metal cation salt (Lane 1). Markers were prepared by the reaction using complementary fragments (Figure S23).

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First, we evaluated 3′ → 1′ elongation in the presence of Cd2+ (Figures 4d and S24) to compare to the previously reported primer extension reaction with Mn2+. (20) As expected, primer extension was considerably faster in the presence of Cd2+ than Mn2+; the yield of the 17-mer full-length product was 60% after 2 h in the presence of Cd2+ (Figures 4d and S24), whereas the yield was only 17% after 3 h in the presence of Mn2+. (20) Next, we studied 1′ → 3′ elongation. Efficiency was remarkably higher for 1′ → 3′ elongation (81% yield after 1 h) than for 3′ → 1′ elongation (28% after 1 h) with Cd2+ at 4 °C (Figures 4c,d and S24). The same acceleration for 1′ → 3′ elongation was also observed in the presence of Mn2+ (Figure S25). (20) We also examined elongation reaction with another symmetrical 17-mer template (UT17t) to eliminate the effect of different sequences between two directions (Figures S26 and S27). Again, 1′ → 3′ elongation was much faster than 3′ → 1′ in the presence of both Mn2+ and Cd2+ (Figure 4d).
Finally, we attempted 1′ → 3′ elongation of L-aTNA using longer templates, 23-mer (T23t) and 29-mer (T29t), under the optimized conditions in the presence of Cd2+ (Figure 5a). The full-length product was generated in 84% yield after 2 h with T23t as the template and in 60% yield after 2 h with T29t as the template (Figure 5b). Sequence-specific elongation was confirmed by analyses of reaction products using MALDI-TOF mass spectroscopy. The spectra indicated generation of full-length products (Figures 5c and S28). Thus, the optimized conditions enabled rapid and effective chemical primer extension to yield a 29-mer product due to ligation of an 8-mer primer and seven trimer fragments in a sequence-specific manner.

Figure 5

Figure 5. (a) Schematic illustration of chemical primer extension on 23-mer and 29-mer L-aTNA templates with optimized conditions. (b) Denaturing PAGE of the reaction with T23t as the template (left) and T29t as the template (right). Reaction conditions: 0.45 μM Rev-T8A-3′p, 200 μM T3Bmix for T23t or 400 μM T3Bmix for T29t, 1.0 μM T23t or T29t, 100 mM NaCl, 5 mM CdCl2, 20 mM CNIm, 4 °C for 4 h. PAGE conditions: 20% acrylamide, 8 M urea, 1 × TBE, 3 h with T23t as the template or 3.5 h with T29t as the template, 15 °C, 4 W. Negative controls (lanes 1 and 6) included only Rev-T8A-3′p, NaCl, and CdCl2. (c) MALDI-TOF MS analyses of products of the elongation reaction for 2 h with T23t as the template (left) and for 4 h with T29t as the template (right).

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Conclusions

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Our experiments demonstrated that the chemical ligation reactions of L-aTNA and DNA were efficiently driven by CNIm in the presence of Cd2+, Ni2+, and Co2+. Reactions in the presence of these divalent ions were more efficient than those in the presence of Mn2+, and side reactions were rarely observed. Importantly, even DNA fragments with a 5′-phosphate and a 3′-OH were efficiently ligated. NMR and kinetic analyses showed that the activation of phosphate was the rate-determining step. Although the mechanism was different from that of the pre-activated imidazolide-mediated system, (43) the present reaction system was remarkably efficient. This reaction system is potentially the prebiotic strand-elongation pathway.
The local structure of the nicked duplex formed by the 3′-phosphorylated L-aTNA primer, fragment, and the template influenced efficiency of the chemical ligation. Hence, the 1′ → 3′ ligation rate was faster than the 3′ → 1′ ligation rate. Under optimized conditions in the presence of Cd2+ with ligation in the 1′ → 3′ direction, a complementary L-aTNA was efficiently replicated from a 29-mer L-aTNA template.
Highly efficient chemical primer extension of L-aTNA was achieved in the presence of a simple condensation agent and a divalent metal ion, conditions that likely existed on the primordial earth. Nonenzymatic copying of an L-aTNA using chemical ligation will serve as the model of a primitive genetic system and artificial life and will also enable XNA-based SELEX (in vitro selection). (44) Furthermore, highly effective chemical ligation of DNA can be employed for artificial genome synthesis and construction of robust DNA nanostructures.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.3c04979.

  • Experimental details for preparation of experimental materials, sequences, pH measurements, melting temperatures, and additional figures and tables (PDF)

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Author Information

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  • Corresponding Authors
  • Authors
    • Hikari Okita - Graduate School of Engineering, Nagoya University, Nagoya 464-8603, Japan
    • Shuto Kondo - Graduate School of Engineering, Nagoya University, Nagoya 464-8603, Japan
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This work was supported by a JST (JPMJFR2226), JSPS KAKENHI grants Grant-in-Aid for Transformative Research Areas “Molecular Cybernetics” JP20H05970 (K.M.), 20H05968 (K.M.), and JP21H05025 (H.A.). AMED under Grant Number 23am0401007 (H.A.) is also acknowledged.

References

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    A review. The general notion of an "RNA World" is that, in the early development of life on the Earth, genetic continuity was assured by the replication of RNA and genetically encoded proteins were not involved as catalysts. There is now strong evidence indicating that an RNA World did indeed exist before DNA- and protein-based life. However, arguments regarding whether life on Earth began with RNA are more tenuous. It might be imagined that all of the components of RNA were available in some prebiotic pool and that these components assembled into replicating, evolving polynucleotides without the prior existence of any evolved macromols. A thorough consideration of this "RNA-first" view of the origin of life must reconcile concerns regarding the intractable mixts. that are obtained in expts. designed to simulate the chem. of the primitive Earth. Perhaps these concerns will eventually be resolved and recent exptl. findings provide some reason for optimism. However, the problem of the origin of the RNA World is far from being solved and it is fruitful to consider the alternative possibility that RNA was preceded by some other replicating, evolving mol., just as DNA and proteins were preceded by RNA.
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  3. 3
    McCall, M. J.; Hendry, P.; Jennings, P. A. Minimal Sequence Requirements for Ribozyme Activity. Proc. Natl. Acad. Sci. U. S. A. 1992, 89, 5710– 5714,  DOI: 10.1073/pnas.89.13.5710
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    3
    Minimal sequence requirements for ribozyme activity
    McCall, Maxine J.; Hendry, Philip; Jennings, Philip A.
    Proceedings of the National Academy of Sciences of the United States of America (1992), 89 (13), 5710-14CODEN: PNASA6; ISSN:0027-8424.
    The hammerhead ribozyme, as engineered by J. Haseloff and W. L. Gerlach (1988), is an RNA mol. contg. 2 regions of conserved nucleotides, a double helix (helix II), which connects the 2 conserved regions, and flanking arms of variable sequence, which hydridize the ribozyme to its specific target. Here, it is shown that this ribozyme may be reduced in size and still retain cleavage activity by replacing helix II with just a few nucleotides that cannot form Watson-Crick base pairs between themselves. Furthermore, the nucleotides replacing helix II and the nucleotides in the flanking arms may be substituted with DNA, and this small, DNA-contg. ribozyme was fully as active as the original, full-size ribozyme. The cleavage activity of the minimized ribozyme (minizyme) depended on the no. and sequence of the few nucleotides that replaced helix II; optimal activity, thus far, was achieved by 4 or 5 deoxyribopyrimidines. The minizyme was active as a monomer, as shown by its nearly const. activity over a concn. range varying 25,000-fold, by the mobility of the minizyme-substrate complex in nondenaturing polyacrylamide gels as compared with other nucleic acid mols. of known size, and by other observations. These minizymes provide an excellent model system for studying the structure and mechanism of catalytic RNA, and they may also be useful in a variety of biol. applications.
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  4. 4
    Becker, S.; Feldmann, J.; Wiedemann, S.; Okamura, H.; Schneider, C.; Iwan, K.; Crisp, A.; Rossa, M.; Amatov, T.; Carell, T. Unified Prebiotically Plausible Synthesis of Pyrimidine and Purine RNA Ribonucleotides. Science 2019, 366, 76– 82,  DOI: 10.1126/science.aax2747
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    4
    Unified prebiotically plausible synthesis of pyrimidine and purine RNA ribonucleotides
    Becker, Sidney; Feldmann, Jonas; Wiedemann, Stefan; Okamura, Hidenori; Schneider, Christina; Iwan, Katharina; Crisp, Antony; Rossa, Martin; Amatov, Tynchtyk; Carell, Thomas
    Science (Washington, DC, United States) (2019), 366 (6461), 76-82CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
    Theories about the origin of life require chem. pathways that allow formation of life's key building blocks under prebiotically plausible conditions. Complex mols. like RNA must have originated from small mols. whose reactivity was guided by physico-chem. processes. RNA is constructed from purine and pyrimidine nucleosides, both of which are required for accurate information transfer, and thus Darwinian evolution. Sep. pathways to purines and pyrimidines have been reported, but their concurrent syntheses remain a challenge. We report the synthesis of the pyrimidine nucleosides from small mols. and ribose, driven solely by wet-dry cycles. In the presence of phosphate-contg. minerals, 5'-mono- and diphosphates also form selectively in one-pot reactions. The pathway is compatible with purine synthesis, allowing the concurrent formation of all Watson-Crick bases.
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  5. 5
    (a) Inoue, T.; Orgel, L. E. Substituent Control of the Poly(C)-Directed Oligomerization of Guanosine 5’-Phosphoroimidazolide. J. Am. Chem. Soc. 1981, 103, 7666– 7667,  DOI: 10.1021/ja00415a051
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    5a
    Substituent control of the poly(C)-directed oligomerization of guanosine 5'-phosphoroimidazolide
    Inoue, Tan; Orgel, Leslie E.
    Journal of the American Chemical Society (1981), 103 (25), 7666-7CODEN: JACSAT; ISSN:0002-7863.
    The poly(C)-directed oligomerization of closely-related guanosine 5'-phosphorimidazolides demonstrates the extreme sensitivity of the efficiency and regioselectivity of the reactions to minor changes in the nature of the imidazole moiety. The reaction of the 2-methylimidazole deriv. generates 89% of oligomers 4 or more units long and the product is predominantly 3'-5'-linked, while related alkyl derivs. give much lower yields of mixed 2'-5'- and 3'-5'-linked isomers.
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    (b) Tam, C. P.; Zhou, L.; Fahrenbach, A. C.; Zhang, W.; Walton, T.; Szostak, J. W. Synthesis of a Nonhydrolyzable Nucleotide Phosphoroimidazolide Analogue that Catalyzes Nonenzymatic RNA Primer Extension. J. Am. Chem. Soc. 2018, 140, 783– 792,  DOI: 10.1021/jacs.7b11623
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    5b
    Synthesis of a Non-hydrolyzable Nucleotide Phosphoroimidazolide Analogue That Catalyzes Nonenzymatic RNA Primer Extension
    Tam, Chun Pong; Zhou, Lijun; Fahrenbach, Albert C.; Zhang, Wen; Walton, Travis; Szostak, Jack W.
    Journal of the American Chemical Society (2018), 140 (2), 783-792CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    We report the synthesis of guanosine 5'-(4-methylimidazolyl)phosphonate (ICG), the third member of a series of nonhydrolyzable nucleoside 5'-phosphoro-2-methylimidazolide (2-MeImpN) analogs designed for mechanistic studies of nonenzymic RNA primer extension. The addn. of a 2-MeImpN monomer to a primer is catalyzed by the presence of a downstream activated monomer, yet the three nonhydrolyzable analogs do not show catalytic effects under std. mildly basic primer extension conditions. Surprisingly, ICG, which has a pKa similar to that of 2-MeImpG, is a modest catalyst of nonenzymic primer extension at acidic pH. Here we show that ICG reacts with 2-MeImpC to form a stable 5'-5'-imidazole-bridged guanosine-cytosine dinucleotide, with both a labile nitrogen-phosphorus and a stable carbon-phosphorus linkage flanking the central imidazole bridge. Cognate RNA primer-template complexes react with this GC-dinucleotide by attack of the primer 3'-hydroxyl on the activated N-P side of the 5'-5'-imidazole bridge. These observations support the hypothesis that 5'-5'-imidazole-bridged dinucleotides can bind to cognate RNA primer-template duplexes and adopt appropriate conformations for subsequent phosphodiester bond formation, consistent with our recent mechanistic proposal that the formation of activated 5'-5'-imidazolium-bridged dinucleotides is responsible for 2-MeImpN-driven primer extension.
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  6. 6
    Li, L.; Prywes, N.; Tam, C. P.; O’Flaherty, D. K.; Lelyveld, V. S.; Izgu, E. C.; Pal, A.; Szostak, J. W. Enhanced Nonenzymatic RNA Copying with 2-Aminoimidazole Activated Nucleotides. J. Am. Chem. Soc. 2017, 139, 1810– 1813,  DOI: 10.1021/jacs.6b13148
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    6
    Enhanced nonenzymatic RNA copying with 2-aminoimidazole activated nucleotides
    Li, Li; Prywes, Noam; Tam, Chun Pong; O'Flaherty, Derek K.; Lelyveld, Victor S.; Izgu, Enver Cagri; Pal, Ayan; Szostak, Jack W.
    Journal of the American Chemical Society (2017), 139 (5), 1810-1813CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Achieving efficient nonenzymic replication of RNA is an important step toward the synthesis of self-replicating protocells that may mimic early forms of life. Despite recent progress, the nonenzymic copying of templates contg. mixed sequences remains slow and inefficient. Here we demonstrate that activating nucleotides with 2-aminoimidazole results in superior reaction kinetics and improved yields of primer extension reaction products. This new leaving group significantly accelerates monomer addn. as well as trimer-assisted RNA primer extension, allowing efficient copying of a variety of short RNA templates with mixed sequences.
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  7. 7
    Zhang, S. J.; Duzdevich, D.; Szostak, J. W. Potentially Prebiotic Activation Chemistry Compatible with Nonenzymatic RNA Copying. J. Am. Chem. Soc. 2020, 142, 14810– 14813,  DOI: 10.1021/jacs.0c05300
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    7
    Potentially Prebiotic Activation Chemistry Compatible with Nonenzymatic RNA Copying
    Zhang, Stephanie J.; Duzdevich, Daniel; Szostak, Jack W.
    Journal of the American Chemical Society (2020), 142 (35), 14810-14813CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The nonenzymic replication of RNA may have enabled the propagation of genetic information during the origin of life. RNA copying can be initiated in the lab. with chem. activated nucleotides, but continued copying requires a source of chem. energy for in situ nucleotide activation. Recent work has illuminated a potentially prebiotic cyanosulfidic chem. that activates nucleotides, but its application to nonenzymic RNA copying had not been demonstrated. Here, we report a novel pathway that activates RNA nucleotides in a manner compatible with template-directed nonenzymic copying. We show that this pathway, which we refer to as bridge-forming activation, selectively yields the reactive imidazolium-bridged dinucleotide intermediate required for copying. Our results will enable more realistic simulations of RNA propagation based on continuous in situ nucleotide activation.
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  8. 8
    Izgu, E. C.; Oh, S. S.; Szostak, J. W. Synthesis of Activated 3’-Amino-3’-Deoxy-2-Thio-Thymidine, a Superior Substrate for the Nonenzymatic Copying of Nucleic Acid Templates. Chem. Commun. 2016, 52, 3684– 3686,  DOI: 10.1039/C5CC10317G
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    8
    Synthesis of activated 3'-amino-3'-deoxy-2-thio-thymidine, a superior substrate for the non-enzymatic copying of nucleic acid templates
    Izgu, Enver Cagri; Oh, Seung Soo; Szostak, Jack W.
    Chemical Communications (Cambridge, United Kingdom) (2016), 52 (18), 3684-3686CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    We present a scalable synthesis of 3'-amino-3'-deoxy-2-thio-thymidine-5'-phosphoro-2-methylimidazolide, an activated monomer that can copy adenosine residues in nucleic acid templates rapidly without a polymerase. The sulfur atom substitution enhances the rate of template copying by 5-fold compared with the 3'-amino-3'-deoxy-T monomer, while the 3'-amino monomers exhibit a 2- to 30-fold enhancement compared with their ribonucleotide counterparts.
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  9. 9
    Ding, D.; Zhou, L.; Giurgiu, C.; Szostak, J. W. Kinetic Explanations for the Sequence Biases Observed in the Nonenzymatic Copying of RNA Templates. Nucleic Acids Res. 2022, 50, 35– 45,  DOI: 10.1093/nar/gkab1202
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    9
    Kinetic explanations for the sequence biases observed in the nonenzymatic copying of RNA templates
    Ding, Dian; Zhou, Lijun; Giurgiu, Constantin; Szostak, Jack W.
    Nucleic Acids Research (2022), 50 (1), 35-45CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    The identification of nonenzymic pathways for nucleic acid replication is a key challenge in understanding the origin of life. We have previously shown that nonenzymic RNA primer extension using 2-aminoimidazole (2AI) activated nucleotides occurs primarily through an imidazolium-bridged dinucleotide intermediate. The reactive nature and preorganized structure of the intermediate increase the efficiency of primer extension but remain insufficient to drive extensive copying of RNA templates contg. all four canonical nucleotides. To understand the factors that limit RNA copying, we synthesized all ten 2AI-bridged dinucleotide intermediates and measured the kinetics of primer extension in a model system. The affinities of the ten dinucleotides for the primer/template/helper complexes vary by over 7,000-fold, consistent with nearest neighbor energetic predictions. Surprisingly, the reaction rates at satg. intermediate concns. still vary by over 15-fold, with the most weakly binding dinucleotides exhibiting a lower maximal reaction rate. Certain noncanonical nucleotides can decrease sequence dependent differences in affinity and primer extension rate, while monomers bridged to short oligonucleotides exhibit enhanced binding and reaction rates. We suggest that more uniform binding and reactivity of imidazolium-bridged intermediates may lead to the ability to copy arbitrary template sequences under prebiotically plausible conditions.
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  10. 10
    Orgel, L. E. Prebiotic Chemistry and the Origin of the RNA World. Crit. Rev. Biochem. Mol. Biol. 2004, 39, 99– 123,  DOI: 10.1080/10409230490460765
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    10
    Prebiotic chemistry and the origin of the RNA world
    Orgel, Leslie E.
    Critical Reviews in Biochemistry and Molecular Biology (2004), 39 (2), 99-123CODEN: CRBBEJ; ISSN:1040-9238. (Taylor & Francis, Inc.)
    A review and discussion. The demonstration that ribosomal peptide synthesis is a ribozyme-catalyzed reaction makes it almost certain that there was once an RNA World. The central problem for origin-of-life studies, therefore, is to understand how a protein-free RNA World became established on the primitive Earth. The author 1st reviews the literature on the prebiotic synthesis of nucleotides, nonenzymic synthesis, the copying of polynucleotides, and the selection of ribozyme catalysts of a kind that might have facilitated polynucleotide replication. This leads to a brief outline of the "mol. biologists' dream" (MBD), an optimistic scenario for the origin of the RNA World. In the 2nd part of the review, the author points out the many unresolved problems presented by the MBD. This in turn leads to a discussion of genetic systems simpler than RNA that might have "invented" RNA. Finally, studies of prebiotic membrane formation are reviewed.
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  11. 11
    Schöning, K. -U.; Scholz, P.; Guntha, S.; Wu, X.; Krishnamurthy, R.; Eschenmoser, A. Chemical Etiology of Nucleic Acid Structure: The α-Threofuranosyl-(3′→2′) Oligonucleotide System. Science 2000, 290, 1347– 1351,  DOI: 10.1126/science.290.5495.1347
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    11
    Chemical etiology of nucleic acid structure: The α-threofuranosyl-(3'→2') oligonucleotide system
    Schoning, K.-U.; Scholz, P.; Guntha, S.; Wu, X.; Krishnamurthy, R.; Eschenmoser, A.
    Science (Washington, D. C.) (2000), 290 (5495), 1347-1351CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    TNAs [(L)-α-threofuranosyl oligonucleotides] contg. vicinally connected (3'→2') phosphodiester bridges undergo informational base pairing in antiparallel strand orientation and are capable of cross-pairing with RNA and DNA. Being derived from a sugar contg. only four carbons, TNA is structurally the simplest of all potentially natural oligonucleotide-type nucleic acid alternatives studied thus far. This, along with the base-pairing properties of TNA, warrants close scrutiny of the system in the context of the problem of RNA's origin.
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  12. 12
    Hendrix, C.; Rosemeyer, H.; Verheggen, I.; Seela, F.; Aerschot, A. V.; Herdewijn, P. 1′,5′-Anhydrohexitol Oligonucleotides: Synthesis, Base Pairing and Recognition by Regular Oligodeoxyribonucleotides and Oligoribonucleotides. Chem. – Eur. J. 1997, 3, 110– 120,  DOI: 10.1002/chem.19970030118
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    12
    1',5'-Anhydrohexitol oligonucleotides: synthesis, base pairing and recognition by regular oligodeoxyribonucleotides and oligoribonucleotides
    Hendrix, Chris; Rosemeyer, Helmut; Verheggen, Ilse; Seela, Frank; Van Aerschot, Arthur; Herdewijn, Piet
    Chemistry - A European Journal (1997), 3 (1), 110-120CODEN: CEUJED; ISSN:0947-6539. (VCH)
    Oligonucleotides constructed of 1',5'-anhydrohexitol nucleoside building blocks (hexitol nucleic acids, HNA) are completely stable towards 3'-exonuclease and form very stable self-complementary duplexes as well as sequence-selective stable duplexes with the natural DNA and RNA. Triple-helix formation has also been obsd. These hybridization characteristics are highly dependent on the base sequence and the exptl. conditions. When using a phosphate buffer contg. 0.1 M NaCl, a homopurine HNA dodecamer gives a δTm of +1.3°C/base pair with DNA as complement and a ΔTm of +3.0°C/base pair with RNA as complement. These oligomers may therefore be of considerable interest as antisense constructs.
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  13. 13
    (a) Zhang, L.; Peritz, A.; Meggers, E. A Simple Glycol Nucleic Acid. J. Am. Chem. Soc. 2005, 127, 4174– 4175,  DOI: 10.1021/ja042564z
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    13a
    A Simple Glycol Nucleic Acid
    Zhang, Lilu; Peritz, Adam; Meggers, Eric
    Journal of the American Chemical Society (2005), 127 (12), 4174-4175CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    A glycol nucleic acid (GNA) with an acyclic propylene glycol phosphodiester backbone forms stable antiparallel duplexes following the Watson-Crick base pairing rules.
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    (b) Meggers, E.; Zhang, L. Synthesis and Properties of the Simplified Nucleic Acid Glycol Nucleic Acid. Acc. Chem. Res. 2010, 43, 1092– 1102,  DOI: 10.1021/ar900292q
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    13b
    Synthesis and Properties of the Simplified Nucleic Acid Glycol Nucleic Acid
    Meggers, Eric; Zhang, Lilu
    Accounts of Chemical Research (2010), 43 (8), 1092-1102CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
    In the present review, the nucleosides of glycol nucleic acid (GNA), with the backbone comprising just the three carbons and one stereocenter of propylene glycol (1,2-propanediol), probably constitute the simplest possible building blocks for a chem. stable nucleic acid that contains phosphodiester bonds is analyzed. However, it was not until 2005 that the astonishing duplex formation properties of GNA homoduplexes were discovered in our lab. The R- and S-enantiomers of GNA, (R)-GNA and (S)-GNA, pair in like-sym. combinations to form highly stable antiparallel duplexes in a Watson-Crick fashion, with thermal and thermodn. stabilities exceeding those of analogous duplexes of DNA and RNA. Interestingly, (R)-GNA and (S)-GNA do not significantly cross-pair with each other, either in a parallel or antiparallel fashion. GNA discriminates strongly in favor of the Watson-Crick base-pairing scheme, with only slightly lower fidelity than DNA. Two (S)-GNA homoduplex structures recently detd. by X-ray crystallog., one a brominated 6-mer duplex and the other an 8-mer duplex contg. two copper(II) ions, reveal that the overall GNA double helix is distinct from canonical A- and B-form nucleic acids. The structure is perhaps best described as a helical ribbon loosely wrapped around the helix axis. Within the backbone, the propylene glycol nucleotides adopt two different conformations, gauche and anti, with respect to the torsional angles between the vicinal C3'-O and C2'-O bonds. A strikingly large backbone-base inclination results in extensive zipper-like interstrand and reduced intrastrand base-base interactions. This strong backbone-base inclination might explain the observation that neither the R- nor S-enantiomer of GNA cross-pairs with DNA, whereas (S)-GNA can interact with RNA strands that are devoid of G:C base pairs. Given the combination of structural simplicity, straightforward synthetic accessibility, and high duplex stability of GNA duplexes, GNA affords a promising nucleic acid scaffold for biotechnol. and nanotechnol. Along these lines, we describe the functionalization of GNA duplexes through the incorporation of metal-ion-mediated base pairs. Finally, the properties of GNA discussed here reinforce its candidacy as one of the initial genetic mols. formed during the origins of life on Earth.
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  14. 14
    (a) Leumann, C. J. DNA Analogues: From Supramolecular Principles to Biological Properties. Bioorg. Med. Chem. 2002, 10, 841– 854,  DOI: 10.1016/S0968-0896(01)00348-0
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    14a
    DNA analogues: From supramolecular principles to biological properties
    Leumann, Christian J.
    Bioorganic & Medicinal Chemistry (2002), 10 (4), 841-854CODEN: BMECEP; ISSN:0968-0896. (Elsevier Science Ltd.)
    A review, with refs. Mainly driven by the needs of antisense research, a large no. of oligonucleotide analogs have been prepd. and evaluated over the last 15 yr. Besides minor structural modifications of the building blocks of DNA and RNA itself, a considerable effort has been devoted to the de novo design of nucleoside analogs with improved binding properties. A particularly successful concept turned out to be that of conformational restriction. This review focuses on recent advances in this area and tries to summarize scope and limitations of this design principle.
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    (b) Petersen, M.; Wengel, J. LNA: a Versatile Tool for Therapeutics and Genomics. Trends Biotechnol. 2003, 21, 74– 81,  DOI: 10.1016/S0167-7799(02)00038-0
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    14b
    LNA: a versatile tool for therapeutics and genomics
    Petersen, Michael; Wengel, Jesper
    Trends in Biotechnology (2003), 21 (2), 74-81CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Science Ltd.)
    A review. Locked nucleic acid (LNA) is a nucleic acid analog that displays unprecedented hybridization affinity towards complementary DNA and RNA. Structural studies have shown LNA to be an RNA mimic, fitting seamlessly into an A-type duplex geometry. Several reports have revealed LNA as a most promising mol. for the development of oligonucleotide-based therapeutics. For example, Tat-dependent transcription and telomerase activity have been efficiently suppressed by LNA oligomers, and efficient cleavage of highly structured RNA has been achieved using LNA-modified DNAzymes ('LNAzyme'). Furthermore, convincing examples of the application of LNA to nucleic acid diagnostics have been reported, including high capturing efficiencies and unambiguous scoring of single-nucleotide polymorphisms.
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    (c) Zhang, S.; Switzer, C.; Chaput, J. C. The Resurgence of Acyclic Nucleic Acids. Chem. Biodiversity 2010, 7, 245– 258,  DOI: 10.1002/cbdv.200900281
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    14c
    The Resurgence of Acyclic Nucleic Acids
    Zhang, Su; Switzer, Christopher; Chaput, John C.
    Chemistry & Biodiversity (2010), 7 (2), 245-258CODEN: CBHIAM; ISSN:1612-1872. (Verlag Helvetica Chimica Acta)
    A review, examg. acyclic nucleoside analogs as therapeutic agents, potential progenitor candidates to RNA, and novel building blocks for nucleic-acid nanotechnol. Together, these areas of research provide new insights into the structural and functional properties of nucleic acids and suggest new paradigms for nucleic acid self-assembly.
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    (d) Pinheiro, V. B.; Holliger, P. The XNA World: Progress Towards Replication and Evolution of Synthetic Genetic Polymers. Curr. Opin. Chem. Biol. 2012, 16, 245– 252,  DOI: 10.1016/j.cbpa.2012.05.198
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    14d
    The XNA world: progress towards replication and evolution of synthetic genetic polymers
    Pinheiro, Vitor B.; Holliger, Philipp
    Current Opinion in Chemical Biology (2012), 16 (3-4), 245-252CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
    A review. Life's diversity is built on the wide range of properties and functions that can be encoded in natural biopolymers such as polypeptides and nucleic acids. However, despite their versatility, the range of chem. functionalities is limited, particularly in the case of nucleic acids. Chem. modification of nucleic acids can greatly increase their functional diversity but access to the full phenotypic potential of such polymers requires a system of replication. Here we review progress in the chem. and enzymic synthesis, replication and evolution of unnatural nucleic acid polymers, which promises to enable the exploration of a vast sequence space not accessible to nature and deliver ligands, catalysts and materials based on this new class of biopolymers.
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    (e) Chaput, J. C.; Herdewijn, P. What Is XNA?. Angew. Chem., Int. Ed. 2019, 58, 11570– 11572,  DOI: 10.1002/anie.201905999
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    14e
    What Is XNA?
    Chaput, John C.; Herdewijn, Piet
    Angewandte Chemie, International Edition (2019), 58 (34), 11570-11572CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. The term "xeno-nucleic acids", abbreviated XNA, has grown in popularity to the point that it has become a catch-all phrase for almost any unnatural nucleic acid, raising the question: what is XNA and how does it differ from chem. modified DNA.
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    (f) Murayama, K.; Asanuma, H. Design and Hybridization Properties of Acyclic Xeno Nucleic Acid Oligomers. ChemBioChem 2021, 22, 2507– 2515,  DOI: 10.1002/cbic.202100184
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    14f
    Design and Hybridization Properties of Acyclic Xeno Nucleic Acid Oligomers
    Murayama, Keiji; Asanuma, Hiroyuki
    ChemBioChem (2021), 22 (15), 2507-2515CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Xeno nucleic acids (XNAs) are analogs of DNA and RNA that have a non-ribose artificial scaffold. XNAs are possible prebiotic genetic carriers as well as alternative genetic systems in artificial life. In addn., XNA oligomers can be used as biol. tools. Acyclic XNAs, which do not have cyclic scaffolds, are attractive due to facile their synthesis and remarkably high nuclease resistance. To maximize the performance of XNAs, a neg. charged backbone is preferable to provide sufficient water soly.; however, acyclic XNAs contg. polyanionic backbones suffer from high entropy cost upon duplex formation, because of the high flexibility of the acyclic nature. Herein, we review the relationships between the structure and duplex hybridization properties of various acyclic XNA oligomers with polyanion backbones.
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  15. 15
    Taylor, A. I.; Houlihan, G.; Holliger, P. Beyond DNA and RNA: The Expanding Toolbox of Synthetic Genetics. Cold Spring Harbor Perspect. Biol. 2019, 11, a032490  DOI: 10.1101/cshperspect.a032490
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    15
    Beyond DNA and RNA: the expanding toolbox of synthetic genetics
    Taylor, Alexander I.; Houlihan, Gillian; Holliger, Philipp
    Cold Spring Harbor Perspectives in Biology (2019), 11 (6), a032490CODEN: CSHPEU; ISSN:1943-0264. (Cold Spring Harbor Laboratory Press)
    The remarkable physicochem. properties of the natural nucleic acids, DNA and RNA, define modern biol. at the mol. level and are widely believed to have been central to life's origins. However, their ability to form repositories of information as well as functional structures such as ligands (aptamers) and catalysts (ribozymes/DNAzymes) is not unique. A range of nonnatural alternatives, collectively termed xeno nucleic acids (XNAs), are also capable of supporting genetic information storage and propagation as well as evolution. This gives rise to a new field of "synthetic genetics," which seeks to expand the nucleic acid chem. toolbox for applications in both biotechnol. and mol. medicine. In this review, we outline XNA polymerase and reverse transcriptase engineering as a key enabling technol. and summarize the application of "synthetic genetics" to the development of aptamers, enzymes, and nanostructures.
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  16. 16
    (a) Bhowmik, S.; Krishnamurthy, R. The Role of Sugar-Backbone Heterogeneity and Chimeras in the Simultaneous Emergence of RNA and DNA. Nat. Chem. 2019, 11, 1009– 1018,  DOI: 10.1038/s41557-019-0322-x
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    16a
    The role of sugar-backbone heterogeneity and chimeras in the simultaneous emergence of RNA and DNA
    Bhowmik, Subhendu; Krishnamurthy, Ramanarayanan
    Nature Chemistry (2019), 11 (11), 1009-1018CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)
    Hypotheses of the origins of RNA and DNA are generally centered on the prebiotic synthesis of a pristine system (pre-RNA or RNA), which gives rise to its descendent. However, a lack of specificity in the synthesis of genetic polymers would probably result in chimeric sequences; the roles and fate of such sequences are unknown. Here, we show that chimeras, exemplified by mixed threose nucleic acid (TNA)-RNA and RNA-DNA oligonucleotides, preferentially bind to, and act as templates for, homogeneous TNA, RNA and DNA ligands. The chimeric templates can act as a catalyst that mediates the ligation of oligomers to give homogeneous backbone sequences, and the regeneration of the chimeric templates potentiates a scenario for a possible cross-catalytic cycle with amplification. This process provides a proof-of-principle demonstration of a heterogeneity-to-homogeneity scenario and also gives credence to the idea that DNA could appear concurrently with RNA, instead of being its later descendent.
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    (b) Kim, S. C.; O’Flaherty, D. K.; Giurgiu, C.; Zhou, L.; Szostak, J. W. The Emergence of RNA from the Heterogeneous Products of Prebiotic Nucleotide Synthesis. J. Am. Chem. Soc. 2021, 143, 3267– 3279,  DOI: 10.1021/jacs.0c12955
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    16b
    The Emergence of RNA from the Heterogeneous Products of Prebiotic Nucleotide Synthesis
    Kim, Seohyun Chris; O'Flaherty, Derek K.; Giurgiu, Constantin; Zhou, Lijun; Szostak, Jack W.
    Journal of the American Chemical Society (2021), 143 (9), 3267-3279CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    A review. Recent advances in prebiotic chem. are beginning to outline plausible pathways for the synthesis of the canonical ribonucleotides and their assembly into oligoribonucleotides. However, these reaction pathways suggest that many noncanonical nucleotides are likely to have been generated alongside the std. ribonucleotides. Thus, the oligomerization of prebiotically synthesized nucleotides is likely to have led to a highly heterogeneous collection of oligonucleotides comprised of a wide range of types of nucleotides connected by a variety of backbone linkages. How then did relatively homogeneous RNA emerge from this primordial heterogeneity. Here we focus on nonenzymic template-directed primer extension as a process that would have strongly enriched for homogeneous RNA over the course of multiple cycles of replication. We review the effects on copying the kinetics of nucleotides with altered nucleobase and sugar moieties, when they are present as activated monomers and when they are incorporated into primer and template oligonucleotides. We also discuss three variations in backbone connectivity, all of which are nonheritable and regenerate native RNA upon being copied. The kinetic superiority of RNA synthesis suggests that nonenzymic copying served as a chem. selection mechanism that allowed relatively homogeneous RNA to emerge from a complex mixt. of prebiotically synthesized nucleotides and oligonucleotides.
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  17. 17
    Chen, J. J.; Cai, X.; Szostak, J. W. N2’→P3’ Phosphoramidate Glycerol Nucleic Acid as a Potential Alternative Genetic System. J. Am. Chem. Soc. 2009, 131, 2119– 2121,  DOI: 10.1021/ja809069b
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    17
    N2' → P3' phosphoramidate glycerol nucleic acid as a potential alternative genetic system
    Chen, Jesse J.; Cai, Xin; Szostak, Jack W.
    Journal of the American Chemical Society (2009), 131 (6), 2119-2121CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Glycerol nucleic acid (GNA) is an interesting base-pairing system with an acyclic, three-carbon backbone. In the present study, GNA analogs with N2' → P3' phosphoramidate linkages (npGNA) have been synthesized and their base-pairing properties examd. Thermal denaturation and CD studies show that npGNA can form stable duplexes with itself and with GNA. Furthermore, we show that npGNA can be assembled by template-directed ligation of 3'-imidazole-activated-2'-amino GNA dinucleotides. These results suggest that npGNA is a potential candidate for a self-replicating system based upon phosphoramidate linkages.
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    Brudno, Y.; Birnbaum, M. E.; Kleiner, R. E.; Liu, D. R. An In Vitro Translation, Selection and Amplification System for Peptide Nucleic Acids. Nat. Chem. Biol. 2010, 6, 148– 155,  DOI: 10.1038/nchembio.280
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    18
    An in vitro translation, selection and amplification system for peptide nucleic acids
    Brudno, Yevgeny; Birnbaum, Michael E.; Kleiner, Ralph E.; Liu, David R.
    Nature Chemical Biology (2010), 6 (2), 148-155CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
    Methods to evolve synthetic, rather than biol., polymers could significantly expand the functional potential of polymers that emerge from in vitro evolution. Requirements for synthetic polymer evolution include (i) sequence-specific polymn. of synthetic building blocks on an amplifiable template, (ii) display of the newly translated polymer strand in a manner that allows it to adopt folded structures, (iii) selection of synthetic polymer libraries for desired binding or catalytic properties and (iv) amplification of template sequences that survive selection in a manner that allows subsequent translation. Here we report the development of such a system for peptide nucleic acids (PNAs) using a set of 12 PNA pentamer building blocks. We validated the system by performing six iterated cycles of translation, selection and amplification on a library of 4.3 × 108 PNA-encoding DNA templates and obsd. >1,000,000-fold overall enrichment of a template encoding a biotinylated (streptavidin-binding) PNA. These results collectively provide an exptl. foundation for PNA evolution in the lab.
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    Murayama, K.; Kashida, H.; Asanuma, H. Acyclic L-Threoninol Nucleic Acid (L-aTNA) with Suitable Structural Rigidity Cross-Pairs with DNA and RNA. Chem. Commun. 2015, 51, 6500– 6503,  DOI: 10.1039/C4CC09244A
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    19
    Acyclic L-threoninol nucleic acid (L-aTNA) with suitable structural rigidity cross-pairs with DNA and RNA
    Murayama, Keiji; Kashida, Hiromu; Asanuma, Hiroyuki
    Chemical Communications (Cambridge, United Kingdom) (2015), 51 (30), 6500-6503CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    We report the hybridization properties of a novel artificial nucleic acid: acyclic L-threoninol nucleic acid (L-aTNA). L-ATNA formed a more stable duplex with DNA and RNA than either D-aTNA or serinol nucleic acid (SNA) as the rigidity of the L-form was more optimal for interaction with natural nucleic acids.
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  20. 20
    Murayama, K.; Okita, H.; Kuriki, T.; Asanuma, H. Nonenzymatic Polymerase-Like Template-Directed Synthesis of Acyclic L-threoninol Nucleic Acid. Nat. Commun. 2021, 12, 804,  DOI: 10.1038/s41467-021-21128-0
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    20
    Nonenzymatic polymerase-like template-directed synthesis of acyclic L-threoninol nucleic acid
    Murayama, Keiji; Okita, Hikari; Kuriki, Takumi; Asanuma, Hiroyuki
    Nature Communications (2021), 12 (1), 804CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Evolution of xeno nucleic acid (XNA) world essentially requires template-directed synthesis of XNA polymers. In this study, we demonstrate template-directed synthesis of an acyclic XNA, acyclic L-threoninol nucleic acid (L-aTNA), via chem. ligation mediated by N-cyanoimidazole. The ligation of an L-aTNA fragment on an L-aTNA template is significantly faster and occurs in considerably higher yield than DNA ligation. Both L-aTNA ligation on a DNA template and DNA ligation on an L-aTNA template are also obsd. High efficiency ligation of trimer L-aTNA fragments to a template-bound primer is achieved. Furthermore, a pseudo primer extension reaction is demonstrated using a pool of random L-aTNA trimers as substrates. To the best of our knowledge, this is the first example of polymerase-like primer extension of XNA with all four nucleobases, generating phosphodiester bonding without any special modification. This technique paves the way for a genetic system of the L-aTNA world.
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  21. 21
    (a) Kramer, M.; Richert, C. Enzyme-Free Ligation of 5’-Phosphorylated Oligodeoxynucleotides in a DNA Nanostructure. Chem. Biodiversity 2017, 14, e1700315  DOI: 10.1002/cbdv.201700315
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    (b) Weizenmann, N.; Scheidgen-Kleyboldt, G.; Ye, J.; Krause, C. B.; Kauert, D.; Helmi, S.; Rouillon, C.; Seidel, R. Chemical Ligation of an Entire DNA Origami Nanostructure. Nanoscale 2021, 13, 17556– 17565,  DOI: 10.1039/D1NR04225D
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    21b
    Chemical ligation of an entire DNA origami nanostructure
    Weizenmann, Nicole; Scheidgen-Kleyboldt, Gerda; Ye, Jingjing; Krause, Cordula B.; Kauert, Dominik; Helmi, Seham; Rouillon, Christophe; Seidel, Ralf
    Nanoscale (2021), 13 (41), 17556-17565CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
    Within the field of DNA nanotechnol., numerous methods were developed to produce complex two- and three-dimensional DNA nanostructures for many different emerging applications. These structures typically suffer from a low tolerance against non-optimal environmental conditions including elevated temps. Here, we apply a chem. ligation method to covalently seal the nicks between adjacent 5 phosphorylated and 3 amine-modified strands within the DNA nanostructures. Using a cost-effective enzymic strand modification procedure, we are able to batch-modify all DNA strands even of large DNA objects, such as origami nanostructures. The covalent strand linkage increases the temp. stability of the structures by ~ 10 K. Generally, our method also allows a 'surgical' introduction of covalent strand linkages at preselected positions. It can also be used to map the strand ligation into chains throughout the whole nanostructure and identify assembly defects. We expect that our method can be applied to a large variety of DNA nanostructures, in particular when full control over the introduced covalent linkages and the absence of side adducts and DNA damages are required.
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  22. 22
    (a) Prakash, G.; Kool, E. T. Structural Effects in the Recognition of DNA by Circular Oligonucleotides. J. Am. Chem. Soc. 1992, 114, 3523– 3527,  DOI: 10.1021/ja00035a056
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    22a
    Structural effects in the recognition of DNA by circular oligonucleotides
    Prakash, Gautam; Kool, Eric T.
    Journal of the American Chemical Society (1992), 114 (9), 3523-7CODEN: JACSAT; ISSN:0002-7863.
    It was recently reported that certain pyrimidine-rich circular DNA oligomers can bind strongly and specifically to purine-rich DNA or RNA strands by forming bimol. triple helical complexes. In this study, the effects of structural variations on the strength of binding for this new class of nucleotide-binding ligand are investigated. The no. of loop nucleotides (nt) optimal for bridging the two binding domains of a circle is examd. Comparing loop sizes of 3, 4, 5, 6, and 10 nt, the optimum no. of nucleotides in a loop is 5 for the sequences studied. To test the method of construction and the ability of these compds. to bind sites of varied length, circles of varied size were synthesized. Circles over the size range 24-46 nt were successfully constructed. Varying the target site length shows that oligomers of 4, 8, 12, and 18 nucleotides can be complexed strongly by circles, with melting temps. (Tm) 17° to >33° higher at pH 7.0 than the corresponding Watson-Crick duplexes of the same length. Also studied is the effect of the covalently closed circular structure in comparison to linear oligomers having the same sequence; a covalently closed circle has considerably higher binding affinity than do three different nicked circles (linear oligomers) which contain the same bases. The high binding affinities of these circles are thus attributed to the entropic benefit of preorganization. Finally, the ability of such circles to bind to complementary sites within longer oligomers, the ends of which must pass beyond the loops of a circle, is confirmed by melting studies with synthetic target strands 36 bases in length.
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    (b) Petkovic, S.; Müller, S. RNA Circularization Strategies In Vivo and In Vitro. Nucleic Acids Res. 2015, 43, 2454– 2465,  DOI: 10.1093/nar/gkv045
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    22b
    RNA circularization strategies in vivo and in vitro
    Petkovic, Sonja; Mueller, Sabine
    Nucleic Acids Research (2015), 43 (4), 2454-2465CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    A review. In the plenitude of naturally occurring RNAs, circular RNAs (circRNAs) and their biol. role were underestimated for years. However, circRNAs are ubiquitous in all domains of life, including eukaryotes, archaea, bacteria and viruses, where they can fulfill diverse biol. functions. Some of those functions, as for example playing a role in the life cycle of viral and viroid genomes or in the maturation of tRNA genes, have been elucidated; other putative functions still remain elusive. Due to the resistance to exonucleases, circRNAs are promising tools for in vivo application as aptamers, trans-cleaving ribozymes or siRNAs. How are circRNAs generated in vivo and what approaches do exist to produce ring-shaped RNAs in vitro. In this review we illustrate the occurrence and mechanisms of RNA circularization in vivo, survey methods for the generation of circRNA in vitro and provide appropriate protocols.
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    (c) Liang, X.; Chen, H.; Li, L.; An, R.; Komiyama, M. Ring-Structured DNA and RNA as Key Players In Vivo and In Vitro. Bull. Chem. Soc. Jpn. 2021, 94, 141– 157,  DOI: 10.1246/bcsj.20200235
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    22c
    Ring-Structured DNA and RNA as Key Players In Vivo and In Vitro
    Liang, Xingguo; Chen, Hui; Li, Lin; An, Ran; Komiyama, Makoto
    Bulletin of the Chemical Society of Japan (2021), 94 (1), 141-157CODEN: BCSJA8; ISSN:0009-2673. (Chemical Society of Japan)
    A review. Ring-structured DNA and RNA exhibit a variety of unique features in chem., biol., medicine, material science, and so on, which cannot be accomplished by their non-cyclic counterparts. In this review, both naturally occurring DNA/RNA rings and artificially synthesized ones have been comprehensively covered, mainly to bridge these two growing fields. In the first part, the structures and functions of naturally occurring DNA/RNA rings (extrachromosomal circular DNA, circulating cell-free DNAs, cyclic RNAs, and others) are described. Their roles as biomarkers for disease diagnosis are esp. noteworthy. The second part mainly presents recent methods to synthesize DNA/RNA rings selectively and efficiently from oligonucleotide fragments. DNA/RNA rings of desired sequences and sizes are successfully prepd. in large amts. for versatile applications. Prodn. of RNA rings in cells using autocatalytic transcripts is also described. Lastly, practical applications of DNA/RNA rings are briefly reviewed. Crit. significance of the cooperation of these two areas for further developments, as well as strong potential for interdisciplinary studies, have been emphasized.
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    (d) Silverman, A. P.; Kool, E. T. Detecting RNA and DNA with Templated Chemical Reactions. Chem. Rev. 2006, 106, 3775– 3789,  DOI: 10.1021/cr050057+
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    22d
    Detecting RNA and DNA with templated chemical reactions
    Silverman, Adam P.; Kool, Eric T.
    Chemical Reviews (Washington, DC, United States) (2006), 106 (9), 3775-3789CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. Recent advances in the development of chem. reactions that are triggered by hybridization of an oligonucleotide (a synthetic short strand of DNA) to a target RNA or DNA strand are summarized. A description is given of how these reactions are being used, or could be used, in detecting and identifying such targets in a biol. sample or cell. The basic principles of DNA and RNA mol. recognition underlying this research are discussed. The historical development of such templated reactions is outlined and then the current state of the art is examd. Some exciting new technologies that are under development in this field and future areas of special promise are mentioned.
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    O’Reilly, R. K.; Turberfield, A. J.; Wilks, T. R. The Evolution of DNA-Templated Synthesis as a Tool for Materials Discovery. Acc. Chem. Res. 2017, 50, 2496– 2509,  DOI: 10.1021/acs.accounts.7b00280
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    The Evolution of DNA-Templated Synthesis as a Tool for Materials Discovery
    O'Reilly, Rachel K.; Turberfield, Andrew J.; Wilks, Thomas R.
    Accounts of Chemical Research (2017), 50 (10), 2496-2509CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
    A review on the processes involved in DTS (DNA templated synthesis) and highlights the challenges that remain in creating a general system for mol. discovery by evolution.
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    Shabarova, Z. A.; Dolinnaya, N. G.; Turkin, S. I.; Gromova, E. S. DNA-like Duplexes with Repetitions. I. Properties of Concatemer Duplexes Formed by d(T-G-C-A-C-A-T-G). Nucleic Acids Res. 1980, 8, 2413– 2430,  DOI: 10.1093/nar/8.11.2413
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    24
    DNA-like duplexes with repetitions. I. Properties of concatemer duplexes formed by d(T-G-C-A-C-A-T-G)
    Shabarova, Z. A.; Dolinnaya, N. G.; Turkin, S. I.; Gromova, E. S.
    Nucleic Acids Research (1980), 8 (11), 2413-29CODEN: NARHAD; ISSN:0305-1048.
    A new class of synthetic DNA duplexes contg. repeating oligonucleotide sequences, double-helical concatemers, is characterized. The UV absorption and CD of a concatemer formed in self-assocn. of d(T-G-C-A-C-T-G) were studied. The thermodn. parameters of complex formation are the following: ΔH10 = -9.2 kcal/mol, ΔS10 = -27 entropy units. The data show that pseudopolymeric duplexes having structures that are similar to DNA-B-type helixes are formed in solns. of d(T-G-C-A-C-A-T-G). Polymn. of 32P-labeled d(pT-G-C-A-C-A-T-G) induced by water-sol. carbodiimide was carried out under the conditions of concatemer stability. The yield of the dimer, a 16-member oligonucleotide, was 13%.
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    Kukwikila, M.; Gale, N.; El-Sagheer, A. H.; Brown, T.; Tavassoli, A. Assembly of a Biocompatible Triazole-Linked Gene by One-pot Click-DNA Ligation. Nat. Chem. 2017, 9, 1089– 1098,  DOI: 10.1038/nchem.2850
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    25
    Assembly of a biocompatible triazole-linked gene by one-pot click-DNA ligation
    Kukwikila, Mikiembo; Gale, Nittaya; El-Sagheer, Afaf H.; Brown, Tom; Tavassoli, Ali
    Nature Chemistry (2017), 9 (11), 1089-1098CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)
    The chem. synthesis of oligonucleotides and their enzyme-mediated assembly into genes and genomes has significantly advanced multiple scientific disciplines. However, these approaches are not without their shortcomings; enzymic amplification and ligation of oligonucleotides into genes and genomes makes automation challenging, and site-specific incorporation of epigenetic information and/or modified bases into large constructs is not feasible. Here we present a fully chem. one-pot method for the assembly of oligonucleotides into a gene by click-DNA ligation. We synthesize the 335 base-pair gene that encodes the green fluorescent protein iLOV from ten functionalized oligonucleotides that contain 5'-azide and 3'-alkyne units. The resulting click-linked iLOV gene contains eight triazoles at the sites of chem. ligation, and yet is fully biocompatible; it is replicated by DNA polymerases in vitro and encodes a functional iLOV protein in Escherichia coli. We demonstrate the power and potential of our one-pot gene-assembly method by prepg. an epigenetically modified variant of the iLOV gene.
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    Naylor, R.; Gilham, P. T. Studies on Some Interactions and Reactions of Oligonucleotides in Aqueous Solution. Biochemistry 1966, 5, 2722– 2728,  DOI: 10.1021/bi00872a032
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    Studies on some interactions and reactions of oligonucleotides in aqueous solution
    Naylor, R.; Gilham, P. T.
    Biochemistry (1966), 5 (8), 2722-8CODEN: BICHAW; ISSN:0006-2960.
    The interactions between thymidine and deoxyadenosine oligonucleotides of various chain lengths as measured by percentage hypochromicity have been studied in salt solns. at 0°. The amt. of complexing was found to be dependent on the chain lengths of the 2 interacting species, esp. with those with chain lengths of <8 nucleotides. However, no interaction could be detected with thymidine and deoxyadenosine oligonucleotides of chain lengths <5 and 4 nucleotides, resp. The amt. and types of interaction between thymidine penta- and hexanucleotides with poly(adenylic acid) have been investigated with a view to using the complexes formed to direct the synthesis of internucleotide linkages in aq. soln. Water-sol. carbodiimides have been studied as reagents for the activation of terminal phosphate groups of oligonucleotides in aq. soln. In model expts., these reagents were shown to be capable of effecting the rapid cyclization of nucleoside 2',3'-phosphates at pH 6, and to be capable of converting adenosine 5'-phosphate to its Et ester and to adenylyl-(5' → 5')-adenosine on reaction with the appropriate hydroxylic component. By using the complexes formed with poly(adenylic acid) to achieve favorable orientations of the oligonucleotides and by employing a water-sol. carbodiimide as an activating agent, thymidine penta- and hexanucleotides have been converted in aq. soln. to thymidine deca- and dodecanucleotides in yields of 3 and 5%, resp.
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  27. 27
    Uesugi, S.; Ts’o, P. O. P. Chemical Polymerization of Oligonucleotides Directed by a Complementary Polynucleotide. Preparation and Polymerization of Oligo(2’-O-methylinosine 3′-phosphate). Biochemistry 1974, 13, 3142– 3152,  DOI: 10.1021/bi00712a022
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    27
    Chemical polymerization of oligonucleotides directed by a complementary polynucleotide. Preparation and polymerization of oligo(2'-O-methylinosine 3'-phosphate)
    Uesugi, S.; Tso, P. O. P.
    Biochemistry (1974), 13 (15), 3142-52CODEN: BICHAW; ISSN:0006-2960.
    Substantial quantities of oligo(2'-O-methylinosinates) with 3'-terminal phosphates and defined chain lengths (n = 2-12) were prepd. by controlled hydrolysis of poly(2'-O-methylinosinate) with micrococcal nuclease, followed by DEAE-cellulose column chromatog. Two oligonucleotide fractions, hexa(2'-O-methylinosine 3'-phosphate) and penta(2'-O-methylinosine 3'-phosphate), were used as starting materials in a polymn. reaction directed by a poly(C) template. These reactions were carried out in aq. soln. at low temp. (0 or -15°) with a H2O-sol. carbodiimide as the activating agent. The abs. overall yield was 38-61%, and the relative overall yield based on the recovered material was 43-71%; the yield of the 30-mer fraction (product with 5-6 linkages) can be ≤15%. The stability of the 1:1 oligo(2'-O-methylinosine)-poly(C) complex was an important factor in detg. the extent of the polymn. and the chain length of the product.
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  28. 28
    Shabarova, Z. A.; Dolinnaya, N. G.; Drutsa, V. L.; Melnikova, N. P.; Purmal, A. A. DNA like Duplexes with Repetitions. III. Efficient Template-Guided Chemical Polymerization of d(TGGCCAAGCTp). Nucleic Acids Res. 1981, 9, 5747– 5761,  DOI: 10.1093/nar/9.21.5747
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    28
    DNA-like duplexes with repetitions. III. Efficient template-guided chemical polymerization of d(TGGCCAAGCTp)
    Shabarova, Z. A.; Dolinnaya, N. G.; Druca, V.; Mel'nikova, N. P.; Purmalis, A.
    Nucleic Acids Research (1981), 9 (21), 5747-61CODEN: NARHAD; ISSN:0305-1048.
    Self-assocn. of a decanucleotide d(TGGCCAAGCTp) in an aq. soln. is shown by UV and CD spectroscopy and sedimentation anal. to yield a pseudopolymeric (concatemeric) duplex having a geometry similar to that of DNA B-type. Under conditions where the concatemeric duplex is stable, a water-sol. carbodiimide induces efficient polymn. of the 3'- or 5'-phosphorylated decanucleotide, and the resulting polymers d(TGGCCAAGCTp)2-10 contain only natural phosphodiester bonds. In conditions optimal for template-guided polymn. of d(TGGCCAAGCTp), the overall yield of 20-100-member polynucleotides is >90%. The polymeric duplexes are cleaved by restriction endonuclease AluI, BsuRI, and HindIII to corresponding decamers which were isolated and sequenced.
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  29. 29
    Edeleva, E.; Salditt, A.; Stamp, J.; Schwintek, P.; Boekhoven, J.; Braun, D. Continuous Nonenzymatic Cross-Replication of DNA Strands with In Situ Activated DNA Oligonucleotides. Chem. Sci. 2019, 10, 5807– 5814,  DOI: 10.1039/C9SC00770A
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    29
    Continuous nonenzymatic cross-replication of DNA strands with in situ activated DNA oligonucleotides
    Edeleva, Evgeniia; Salditt, Annalena; Stamp, Julian; Schwintek, Philipp; Boekhoven, Job; Braun, Dieter
    Chemical Science (2019), 10 (22), 5807-5814CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    Continuous enzyme-free replication of oligonucleotides is central for open-ended evolution expts. that mimic the origin of life. Here, we studied a reaction system, whereby two 24mer DNA templates cross-catalyzed each other's synthesis from four 12mer DNA fragments, two of which were in situ activated with the condensing agent 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC). We circumvented the problem of product inhibition by melting the stable product duplexes for their reuse as templates in the following ligation step. The system reproduced itself through ligation/melting cycles and survived exponential diln. We quantified EDC-induced side reactions in a detailed kinetic model. The model allowed us to analyze the effects of various reaction rates on the system's kinetics and confirmed maximal replication under the chosen conditions. The presented system enables us to study nonenzymic open-ended evolution expts. starting from diverse sequence pools.
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  30. 30
    Obianyor, C.; Newnam, G.; Clifton, B. E.; Grover, M. A.; Hud, N. V. Towards Efficient Nonenzymatic DNA Ligation: Comparing Key Parameters for Maximizing Ligation Rates and Yields with Carbodiimide Activation. ChemBioChem 2020, 21, 3359– 3370,  DOI: 10.1002/cbic.202000335
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    30
    Towards Efficient Nonenzymatic DNA Ligation: Comparing Key Parameters for Maximizing Ligation Rates and Yields with Carbodiimide Activation
    Obianyor, Chiamaka; Newnam, Gary; Clifton, Bryce E.; Grover, Martha A.; Hud, Nicholas V.
    ChemBioChem (2020), 21 (23), 3359-3370CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Chem. ligation is an important tool for the generation of synthetic DNA structures, which are used for a wide range of applications. Surprisingly, reported chem. ligation yields can range from 30 to 95% for the same chem. activating agent and comparable DNA structures. We report a systematic study of DNA ligation by using a well-defined bimol. test system and a water-sol. carbodiimide (EDC) as a phosphate-activating agent. Our results emphasize the interplay between template-substrate complex stability and the rates of the chem. steps of ligation, with 3' phosphate substrates providing yields near 100% after 24 h for particularly favorable reaction conditions. Ligation rates are also shown to be sensitive to the identity of the base pairs flanking a nick site, with as much as threefold variation. Finally, the observation that DNA substrates are modified by EDC at rates that can be comparable with ligation rates emphasizes the importance of considering side reactions when designing protocols to maximize ligation yields.
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  31. 31
    (a) Sokolova, N. I.; Ashirbekova, D. T.; Dolinnaya, N. G.; Shabarova, Z. A. Chemical Reactions within DNA Duplexes Cyanogen Bromide as an Effective Oligodeoxyribonucleotide Coupling Agent. FEBS Lett. 1988, 232, 153– 155,  DOI: 10.1016/0014-5793(88)80406-X
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    Chemical reactions within DNA duplexes. Cyanogen bromide as an effective oligodeoxyribonucleotide coupling agent
    Sokolova, N. I.; Ashirbekova, D. T.; Dolinnaya, N. G.; Shabarova, Z. A.
    FEBS Letters (1988), 232 (1), 153-5CODEN: FEBLAL; ISSN:0014-5793.
    CNBr condensed oligodeoxyribonucleotides on a complementary template in aq. soln. Optimum conditions for this vigorous and effective reaction were developed. CNBr proved to be useful for incorporation of phosphoramidate or pyrophosphate internucleotide bonds in DNA duplexes.
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    (b) Fedorova, O. A.; Gottikh, M. B.; Oretskaya, T. S.; Shabarova, Z. A. Cyanogen Bromide-Induced Chemical Ligation: Mechanism and Op-timization of the Reaction Conditions. Nucleosides, Nucleotides Nucleic Acids 1996, 15, 1137– 1147,  DOI: 10.1080/07328319608007382
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    Vogel, H.; Gerlach, C.; Richert, C. Reactions of Buffers in Cyanogen Bromine-Induced Ligations. Nucleosides, Nucleotides Nucleic Acids 2013, 32, 17– 27,  DOI: 10.1080/15257770.2012.744036
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    Reactions of Buffers in Cyanogen Bromide-Induced Ligations
    Vogel, Heike; Gerlach, Claudia; Richert, Clemens
    Nucleosides, Nucleotides & Nucleic Acids (2013), 32 (1), 17-27CODEN: NNNAFY; ISSN:1525-7770. (Taylor & Francis Ltd.)
    Rapid, template-directed ligation reactions between a phosphate-terminated oligonucleotide and an un-phosphorylated reaction partner may be induced by cyanogen bromide (BrCN). Frequently, however, the reaction is low yielding, and even a large excess of the condensing agent can fail to induce quant. conversions. In this study, we used BrCN to induce chem. primer extension reactions. Here, we report that buffers contg. hydroxyl groups react with short oligodeoxynucleotides in the presence of BrCN. One stable adduct between HEPBS buffer and cytosine was characterized by mass spectrometry and NMR after HPLC purifn., indicating that a side reaction occurred at this nucleobase. Further, a first example of a primer extension reaction between an unmodified oligodeoxynucleotide as primer and dGMP is reported. Together, our results shed light on the potency, as well as the drawbacks of BrCN as a highly reactive condensing reagent for the ligation of unmodified nucleic acids.
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    Kanaya, E.; Yanagawa, H. Template-Directed Polymerization of Oligoadenylates Using Cyanogen Bomide. Biochemistry 1986, 25, 7423– 7430,  DOI: 10.1021/bi00371a026
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    Template-directed polymerization of oligoadenylates using cyanogen bromide
    Kanaya, Eiko; Yanagawa, Hiroshi
    Biochemistry (1986), 25 (23), 7423-30CODEN: BICHAW; ISSN:0006-2960.
    BrCN condensed oligoadenylates [oligo(A)] on a poly(uridylic acid) [poly(U)] template in an aq. soln. Imidazole and divalent metal ions such as Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Mg2+, and Fe2+ were required for the condensation. Chain length of oligo(A) and reaction temp. affected the coupling yield. Hexaadenylate [(pA)6] was converted to (pA)12, (pA)18, (pA)24, (pA)30, (pA)36, (pA)42, and (pA)48 in a 68% overall yield for 20 h at 25°. The coupling yield increased with increases in the poly(U) concn. Five to seven-fold molar excess of uridylyl residues of poly(U) to adenylyl residues of oligo(A) gave the best yield (68%). Metal ions affected the formation of linkage isomers of the phosphate bonds: The 2',5'- and 3',5'-phosphodiester bonds were predominant in the presence of Co2+, Zn2+, and Ni2+, and the 5',5'-pyrophosphate bond was predominant in the presence of Mn2+. In particular, Ni2+ gave the highest ratio of the 3',5'-phosphodiester bond (30%). n-Cyanoimidazole (I), N,N'-iminodiimidazole (II), and N-carboxamidoimidazole (III) were formed in a reaction of imidazole with BrCN in an aq. soln. I and II had much the same condensing activity for the polymn. of adenylates as BrCN. A reaction pathway was proposed in which I and II are not only intermediates for the prodn. of III but also the true condensing agent in the coupling reaction of oligo(A). Phosphorimidazolide deriv. was detected in a reaction of 5'-AMP with either I or II. The condensation would proceed by way of N-cyanoimidazole-phosphate adduct, or the phosphorimidazolide deriv., or both.
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    Luebke, K. J.; Dervan, P. B. Nonenzymatic Ligation of Oligodeoxyribonucleotides on a Duplex DNA Template by Triple-Helix Formation. J. Am. Chem. Soc. 1989, 111, 8733– 8735,  DOI: 10.1021/ja00205a033
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    Nonenzymatic ligation of oligodeoxyribonucleotides on a duplex DNA template by triple-helix formation
    Luebke, Kevin J.; Dervan, Peter B.
    Journal of the American Chemical Society (1989), 111 (23), 8733-5CODEN: JACSAT; ISSN:0002-7863.
    A double-stranded DNA template can direct the sequence-specific formation of a phosphodiester linkage between pyrimidine oligodeoxynucleotides in aq. soln. by juxtaposing the oligonucleotide termini head-to-tail in a triple helical complex. Within the context of the development of chem. systems for macromol. information transfer, triple helix-directed ligation can create sequences that are neither identical nor complementary in a Watson-Crick sense to the template, but rather new sequences of nucleic acids.
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  35. 35
    Ferris, J. P.; Huang, C. -H.; Hagan, W. J. N-Cyanoimidazole and Diimidazole Imine: Water-Soluble Condensing Agents for the Formation of the Phosphodiester Bond. Nucleosides Nucleotides 1989, 8, 407– 414,  DOI: 10.1080/07328318908054184
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    N-cyanoimidazole and diimidazole imine: water-soluble condensing agents for the formation of the phospho diester bond
    Ferris, James P.; Huang, Chun Hsien; Hagan, William J., Jr.
    Nucleosides & Nucleotides (1989), 8 (3), 407-14CODEN: NUNUD5; ISSN:0732-8311.
    The reaction of BrCN with imidazole results in the formation of N-cyanoimidazole and diimidazole imine. These compds. were shown to be useful condensing agents for the formation of the phosphodiester bond in aq. soln.
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    (a) Li, T.; Weinstein, D. S.; Nicolaou, K. C. The Chemical End-Ligation of Homopyrimidine Oligodeoxyribonucleotides within a DNA Triple Helix. Chem. Biol. 1997, 4, 209– 214,  DOI: 10.1016/S1074-5521(97)90290-8
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    36a
    The chemical end-ligation of homopyrimidine oligodeoxyribonucleotides within a DNA triple helix
    Li, Tianhu; Weinstein, David A.; Nicolaou, K. C.
    Chemistry & Biology (1997), 4 (3), 209-214CODEN: CBOLE2; ISSN:1074-5521. (Current Biology)
    Triple-helical nucleic acids, first reported in the late 1950s, are receiving attention for their possible involvement in controlling gene expression. Certain sequences of DNA are believed to form local triple-helical structures (H-form DNA), although this has not been directly obsd. in vivo. Studies carried out in our labs. have suggested that self-replicating oligonucleotides could have been involved in chem. evolution via triple-helical intermediates. In addn. to self-replication mechanisms, elucidating processes for the nonenzymic elongation of biol. relevant polymers remains an important challenge in understanding the origin of life. To this end, we have studied a novel ligation of oligodeoxyribonucleotides that lie within a triple helix. The chem. end-ligation of homopyrimidine oligodeoxyribonucleotides on a triple helix is reported. This selective process, induced by cyanoimidazole, is facilitated by a template effect of the DNA aggregate and occurs between the 3' end (hydroxyl) of the third minor-groove-bound strand and the 5' end (phosphate) of the antiparallel oligopyrimidine strand. Double-helical homopurine/homopyrimidine DNA can serve as a template for the elongation of oligonucleotides in a manner that has not been described previously. The end-ligation of homopyrimidine oligomers, a nonenzymic process, proceeds via a requisite triple-helical intermediate and constitutes an efficient and selective method for the template-directed elongation of nucleic acids. Such a process could conceivably have been involved in the elongation of primordial information-bearing biopolymers.
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    (b) Li, T.; Liu, D.; Chen, J.; Lee, A. H. F.; Qi, J.; Chan, A. S. C. Construction of Circular Oligodeoxyribonucleotides on the New Structural Basis of i-Motif. J. Am. Chem. Soc. 2001, 123, 12901– 12902,  DOI: 10.1021/ja011401x
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    Construction of Circular Oligodeoxyribonucleotides on the New Structural Basis of i-Motif
    Li, Tianhu; Liu, Dongsheng; Chen, Jian; Lee, Alex H. F.; Qi, Jianying; Chan, Albert S. C.
    Journal of the American Chemical Society (2001), 123 (51), 12901-12902CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    We now report for the first time that beyond the scope of the previous duplex and triplex strategies, the i-motif, a four stranded assembly, can direct the sequence-specific formation of a phosphodiester linkage and thus represents a new type of structural template for constructing circular oligonucleotides.
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    Chen, H.; Du, F.; Chen, G.; Streckenbach, F.; Yasmeen, A.; Zhao, Y.; Tang, Z. Template-Directed Chemical Ligation to Obtain 3′-3′ and 5′-5’ Phosphodiester DNA Linkages. Sci. Rep. 2014, 4, 4595,  DOI: 10.1038/srep04595
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    Template-directed Chemical Ligation to Obtain 3'-3' and 5'-5' Phosphodiester DNA Linkages
    Chen, Haodong; Du, Feng; Chen, Gangyi; Streckenbach, Frank; Yasmeen, Afshan; Zhao, Yun; Tang, Zhuo
    Scientific Reports (2014), 4 (), 4595/1-4595/6CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
    Up to now, the direct ligation of two DNA fragments with opposite directions to obtain 3'-3' or 5'-5' phosphate ester bonds is still challenging. The only way to obtain DNA oligonucleotides contg. a 3'-3' or 5'-5' inversion of polarity sites is based on professional DNA chem. synthesis. Herein, we demonstrate a convenient template-directed chem. ligation that enables 3'-3' and 5'-5' linkages of two DNA oligonucleotides. This method is based on the assembly of two oligonucleotides on a template in opposite directions through forming antiparallel and parallel duplexes simultaneously, followed by coupling with [ital: null]-Cyanoimidazole under mild condition. Moreover, on the basis of DNA oligonucleotides with 5'-5' linkage obtained through our template-directed chem. ligation, we developed a new cDNA display technique for [ital: null] selection of functional polypeptides.
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    Mariani, A.; Sutherland, J. D. Non-Enzymatic RNA Backbone Proofreading through Energy-Dissipative Recycling. Angew. Chem., Int. Ed. 2017, 56, 6563– 6566,  DOI: 10.1002/anie.201703169
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    Non-enzymatic RNA backbone proofreading through energy-dissipative recycling
    Mariani, Angelica; Sutherland, John D.
    Angewandte Chemie, International Edition (2017), 56 (23), 6563-6566CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Nonenzymic oligomerization of activated ribonucleotides leads to RNAs that contain a mixt. of 2',5'- and 3',5'-linkages, and overcoming this backbone heterogeneity has long been considered a major limitation to the prebiotic emergence of RNA. Here, we demonstrate nonenzymic chem. that progressively converts 2',5'-linkages into 3',5'-linkages through iterative degrdn. and repair. The energetic costs of this proofreading are met by the hydrolytic turnover of a phosphate activating agent and an acylating agent. With multiple rounds of this energy-dissipative recycling, we show that all-3',5'-linked duplex RNA can emerge from a backbone heterogeneous mixt., thereby delineating a route that could have driven RNA evolution on the early Earth.
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    (a) Covington, A. K.; Paabo, M.; Robinson, R. A.; Bates, R. G. Use of the Glass Electrode in Deuterium Oxide and the Relation Between the Standardized pD (paD) Scale and the Operational pH in Heavy Water. Anal. Chem. 1968, 40, 700– 706,  DOI: 10.1021/ac60260a013
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    Use of the glass electrode in deuterium oxide and the relation between the standardized pD (paD) scale and the operational pH in heavy water
    Covington, Arthur K.; Paabo, Maya; Robinson, Robert Anthony; Bates, Roger G.
    Analytical Chemistry (1968), 40 (4), 700-6CODEN: ANCHAM; ISSN:0003-2700.
    Both direct and indirect comparison of com. glass electrodes with the D gas electrode at 25° in buffered solns. of pD 1-13 confirmed that the glass electrode functions as well in heavy water as in ordinary water. The relation between the operational pH of a buffer soln. in heavy water, as obtained with a glass electrode standardized in an ordinary water buffer soln., and the corresponding pD value, as obtained from measurements on cells without liq. junction, is examd. and correction factors are detd. for both glass and the D gas electrode. The operational pH of buffer solns. in heavy water at 25°, which are measured with the glass electrode, can be converted into a pD value by adding 0.41 (molar scale) or 0.45 (molal scale), for pD 2-9. 26 references.
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    (b) Krȩżel, A.; Bal, W. A Formula for Correlating pKa Values Determined in D2O and H2O. J. Inorg. Biochem. 2004, 98, 161– 166,  DOI: 10.1016/j.jinorgbio.2003.10.001
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    A formula for correlating pKa values determined in D2O and H2O
    Krezel, Artur; Bal, Wojciech
    Journal of Inorganic Biochemistry (2004), 98 (1), 161-166CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier Science Inc.)
    A linear correlation between pH-meter readings in equiv. D2O and H2O solns., detd. exptl., leads to a novel equation, which allows for a direct recalcn. of pKa values measured in D2O into a H2O equiv.: pKH=0.929pKH*+0.42. The comparison of this equation with the previously used approach is discussed.
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    Yi, R.; Hongo, Y.; Fahrenbach, A. C. Synthesis of Imidazole-Activated Ribonucleotides Using Cyanogen Chloride. Chem. Commun. 2018, 54, 511– 514,  DOI: 10.1039/C7CC08489G
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    Synthesis of imidazole-activated ribonucleotides using cyanogen chloride
    Yi, Ruiqin; Hongo, Yayoi; Fahrenbach, Albert C.
    Chemical Communications (Cambridge, United Kingdom) (2018), 54 (5), 511-514CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    We report the syntheses of ribonucleoside 5'-monophosphates activated with imidazole, using a mechanism which relies on the in situ generation of cyanogen chloride from the reaction of cyanide anion with hypochlorous acid. Cyanogen chloride reacts rapidly with imidazole to form diimidazole imine as the major product, a species which affords the activation of ribonucleoside 5'-monophosphates to their 5'-phosphorimidazolides.
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    (a) Drey, C. N. C.; Fruton, J. S. Synthesis and Properties of a Bis-Imidazole. Biochemistry 1965, 4, 1– 5,  DOI: 10.1021/bi00877a001
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    Synthesis and properties of a bisimidazole
    Drey, Charles N. C.; Fruton, Joseph S.
    Biochemistry (1965), 4 (1), 1-5CODEN: BICHAW; ISSN:0006-2960.
    4,4'(5,5')-Bisimidazolylmethane (I) was synthesized from histidine Me ester by redn. with NaHg in the presence of KSCN, followed by desulfurization of the resulting 2-mercapto compd. with FeCl3 or HNO3. I has apparent ionization consts. of 5.3 and 7.0. It exhibits strong intermol. H bonding in Me2SO and no evidence was found for intramol. H bonding. Examn. of the catalytic effect of I on the hydrolysis of p-nitrophenylacetate showed that, on an equimolar basis, I was slightly less effective than imidazole itself. I had no catalytic effect on the hydrolysis of uridine 2',3'-phosphate.
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    (b) Tanford, C.; Wagner, M. L. The Consecutive Constants for the Association of Cadmium with Imidazole. J. Am. Chem. Soc. 1953, 75, 434– 435,  DOI: 10.1021/ja01098a052
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    41b
    Consecutive constants for the association of cadmium with imidazole
    Tanford, Charles; Wagner, Myron L.
    Journal of the American Chemical Society (1953), 75 (), 434-5CODEN: JACSAT; ISSN:0002-7863.
    Since it has been indicated that the principal binding sites for the combination of metals with serum albumin are the imidazole (I) groups (cf. C.A. 45, 7446e), the binding consts. for metals with I should be approx. the same as with albumin. The successive assocn. consts. for the Cd-I complex were detd. potentiometrically by the method of Bjerrum (C.A. 35, 6206.3); at 25°, log k1 = 2.80 ± 0.05, log k2 = 2.10 ± 0.08, log k3 = 1.55 ± 0.5, and log k4 = 1.13 ± 0.2. ΔH1 is estd. to be -5 ±2 kcal./mol. from detns. at other temps. Log k1 is the same as for the assocn. const. (log k = 2.8 ± 0.2) of Cd with the I groups in bovine serum albumin.
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    (c) Mickel, B. L.; Andrews, A. C. Stability of the Histamine Chelates1. J. Am. Chem. Soc. 1955, 77, 5291– 5292,  DOI: 10.1021/ja01625a025
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    41c
    Stability of the histamine chelates
    Mickel, B. L.; Andrews, A. C.
    Journal of the American Chemical Society (1955), 77 (), 5291-2CODEN: JACSAT; ISSN:0002-7863.
    cf. C.A. 49, 6018e. The sep. contributions to chelate stability of the imidazole and amino N atoms and the probable structures of histamine chelates are discussed in terms of some new consecutive formation consts. for reactions of histamine and related compds. with various cations.
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    (d) Koltun, W. L.; Dexter, R. N.; Clark, R. E.; Gurd, F. R. N. Coördination Complexes and Catalytic Properties of Proteins and Related Substances. I. Effect of Cupric and Zinc Ions on the Hydrolysis of p-Nitrophenyl Acetate by Imidazole. J. Am. Chem. Soc. 1958, 80, 4188– 4194,  DOI: 10.1021/ja01549a018
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    41d
    Co.ovrddot.ordination complexes and catalytic properties of proteins and related substances. I. Effect of cupric and zinc ions on the hydrolysis of p-nitrophenyl acetate by imidazole
    Koltun, Walter L.; Dexter, Richard N.; Clark, Richard E.; Gurd, Frank R. N.
    Journal of the American Chemical Society (1958), 80 (), 4188-94CODEN: JACSAT; ISSN:0002-7863.
    The advantages of combining different methods, particularly kinetic and equil. techniques, for assessing the reactivity of polar groups in proteins were described. The model system contg. cupric and Zn chlorides, imidazole and imidazolium chloride were studied simultaneously by 2 independent techniques that measure the concn. of free basic imidazole quantitatively. The 1st technique depended on the catalysis by imidazole of the hydrolysis of p-nitrophenyl acetate (NPA); the 2nd technique involved measurement of the pH of the soln. Over a wide range of conditions the 2 techniques of measurement did not interfere with one another. The p-nitrophenolate ion released on hydrolysis of NPA combined with Zn and cupric ions about 100 times less strongly than did imidazole. Previous observations on the rate of decompn. of N-acetylimidazole were confirmed, and Zn ions were shown to have no detectable effect on the rate of decompn. The method of Scatchard was applied to the computation of the successive assocn. consts. in the Cu(II)- and Zn(II)-imidazole systems, with results that differed only slightly from those reported previously.
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    (e) Li, N. C.; White, J. M.; Doody, E. Cadmium and Copper Complexes of Imidazole and 1-Methylimidazole. J. Am. Chem. Soc. 1954, 76, 6219– 6223,  DOI: 10.1021/ja01653a002
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    41e
    Cadmium and copper complexes of imidazole and 1-methylimidazole
    Li, Norman C.; White, James M.; Doody, Edward
    Journal of the American Chemical Society (1954), 76 (), 6219-23CODEN: JACSAT; ISSN:0002-7863.
    The formation consts. of Cd complexes of imidazole were detd. in water at 25° and in 18.8% EtOH at 0, 25, and 35°, and for Cu imidazole complexes at 25° in aq. medium. The solvent effect of EtOH on complex formation is negligible. Consts. for Cu and Cd 1-methylimidazole complexes were also detd. The results indicate that the site of binding on the imidazole mol. is the pyridine nitrogen rather than the pyrrole nitrogen.
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    (f) Martin, R. B.; Edsall, J. T. The Combination of Manganous and Cobaltous Ions with Imidazole. J. Am. Chem. Soc. 1958, 80, 5033– 5035,  DOI: 10.1021/ja01552a007
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    41f
    Combination of manganous and cobaltous ions with imidazole
    Martin, R. Bruce; Edsall, John T.
    Journal of the American Chemical Society (1958), 80 (), 5033-5CODEN: JACSAT; ISSN:0002-7863.
    cf. Nozaki, et al., C.A. 51, 12887e. Imidazole forms weak complexes with Mn(II) ion, log k1 = 1.65, log k2 = 1.25; and with Co(II) ion, log k1 = 2.42, log k2 = 1.95, log k3 = 1.58, log k4 = 1.2, all at 25° and ionic strength 0.16. The weakness of the binding does not permit a detn. of the higher assocn. consts. at this ionic strength. A correlation is described between the binding of a series of metal ions with NH3 and with imidazole.
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    (g) Edsall, J. T.; Felsenfeld, G.; Goodman, D. S.; Gurd, F. R. N. The Association of Imidazole with the Ions of Zinc and Cupric Copper. J. Am. Chem. Soc. 1954, 76, 3054– 3061,  DOI: 10.1021/ja01640a068
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    41g
    Association of imidazole with the ions of zinc and cupric copper
    Edsall, John T.; Felsenfeld, Gary; Goodman, DeWitt S.; Gurd, Frank R. N.
    Journal of the American Chemical Society (1954), 76 (), 3054-61CODEN: JACSAT; ISSN:0002-7863.
    The interaction of imidazole with Zn++ and Cu++ was detd. by pH measurements on solns. contg. imidazole, imidazolium ion, and either Cu++ or Zn++, in solns. contg. nitrate ion at ionic strength 0.16. In both cases the co.ovrddot.ordination no. of the ion for imidazole was 4. The successive logarithmic intrinsic assocn. consts. at 4.5° were for Cu++: 4.00, 3.63, 3.27, and 2.90; and for Zn++: 2.16, 2.32, 2.53, and 2.80; at 22.5° for Cu++: 3.76, 3.39, 3.03, and 2.66; at 24° for Zn++: 1.98, 2.19, 2.41, and 2.62. For the Cu-imidazole interaction, it was estd. that the standard heats of reaction for each successive step are approx. equal and neg.; under the conditions of this study ΔH° equals roughly -2200 cal./ mole and the successive standard entropy changes of roughly 13, 10, 6, and 3 cal. deg.-1 mole-1, resp. Cu-imidazole complexes showed marked absorption in the visible between 590 and 690 mμ and in the ultraviolet between 230 and 290 mμ. Absorption spectra of the Cu-histidine complex indicated that the imidazole residues of histidine were involved in the chelate linkages formed.
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    (h) Li, N. C.; Chu, T. L.; Fujii, C. T.; White, J. M. Association of Imidazole with Nickel(II) and Alkaline Earth Ions. J. Am. Chem. Soc. 1955, 77, 859– 861,  DOI: 10.1021/ja01609a010
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    41h
    Association of imidazole with nickel(II) and alkaline earth ions
    Li, Norman C.; Chu, Ting Li; Fujii, Charles T.; White, James M.
    Journal of the American Chemical Society (1955), 77 (), 859-61CODEN: JACSAT; ISSN:0002-7863.
    The interaction of Ni++ with imidazole (I) was detd. by polarographic and pH measurements at ionic strength 0.15. The co.ovrddot.ordination no. of the Ni was 6, the structure being octahedral and the bonds essentially ionic. At 25° the successive logarithmic formation consts. were 3.27, 2.68, 2.15, 1.65, 1.12, and 0.52. The molar extinction coeff. of NiI++ is about 3.3 l. mole-1 cm.-1 at 650 mμ, the same as that for NiNH3++ at the same wave length. Ba and Ca ions have only slight tendency to complex with I at 25 and 35°.
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  42. 42
    (a) Duan, W.; Satoh, K.; Sawada, K. Stability and Structure of Ethylenedinitrilopoly(methylphosphonate) Complexes of the Divalent Transition Metal Ions in Aqueous Solution. Bull. Chem. Soc. Jpn. 2001, 74, 487– 493,  DOI: 10.1246/bcsj.74.487
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    42a
    Stability and structure of ethylenedinitrilopoly(methylphosphonate) complexes of the divalent transition metal ions in aqueous solution
    Duan, Wubiao; Satoh, Keiichi; Sawada, Kiyoshi
    Bulletin of the Chemical Society of Japan (2001), 74 (3), 487-493CODEN: BCSJA8; ISSN:0009-2673. (Chemical Society of Japan)
    The formation and protonation of the complexes of a series of ethylenedinitrilopoly(methylphosphonic acids) (EDMP) [(Me)2-p(H2O3PCH2)pNC2H4N(Me)2-q(CH2PO3H2)q, p = 0-2, q = 1-2] with divalent transition metal ions (M = Mn2+, Co2+, Ni2+, Cu2+, Zn2+ or Cd2+) were investigated by means of potentiometry and 31P NMR spectroscopy at 25.0 °C. The complex formation consts. and protonation consts. of these complexes were detd. by pH titrn. The 31P NMR spectra of zinc and cadmium complexes were measured as a function of the pH, and the 31P NMR chem. shifts of each chem. species were evaluated by using the equil. consts. detd. by pH titrn. The results for EDMP complexes were compared with those of aminopoly(methylphosphonate) (NMP) [(CH3)3-rN(CH2PO3H2)r, r = 1 - 3] complexes and alk. earth metal EDMP complexes. The stability consts. of the metal complexes increase upon increasing the no. of the phosphonate groups and are around the same between the EDMP and NMP complexes of a given metal ion having the same total no. of coordinating atoms. The stabilities of the edtmp (p = q = 2) complexes are around the same as those of the medtmp (p = 1, q = 2) complexes. These results reveal the structures in which two nitrogen atoms of the ethylenedinitrilopoly(methylphosphonate) coordinate to the transition metal ion in any complex. These structures are different from those of the corresponding alk. earth metal complexes. The structures of the protonated complexes were estd. from the results of the protonation consts. and the 31P chem. shifts of the complexes.
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    (b) Irving, H.; Williams, R. J. P. 637. The Stability of Transition-Metal Complexes. J. Chem. Soc. 1953, 3192– 3210,  DOI: 10.1039/jr9530003192
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    42b
    Stability of transition metal complexes
    Irving, H. M.; Williams, R. J. P.
    Journal of the Chemical Society (1953), (), 3192-3210CODEN: JCSOA9; ISSN:0368-1769.
    The published values of the stability consts. for the bivalent ions of Mn, Fe, Co, Ni, Cu, and Zn with ligands exhibiting NN, NO, and OO co.ovrddot.ordinating patterns are tabulated. In almost all cases the order of stability is Mn < Fe < Co < Ni < Cu > Zn irrespective of the nature of the ligand. This order is a consequence of the monotonic change in the 2nd ionization potentials and the reciprocal radii, the parameters serving as a guide to the magnitude of the covalent and electrostatic interactions, resp., of the ions involved. When other ions are inserted in the order of stability, the new order varies with the ligand because there is not generally the same relation for both factors throughout the series. Characteristic co.ovrddot.ordination no., stereochem. considerations, and entropy factors may, however, affect the series given here. Examples of the former two effects are the low value for k3 in the Cu++-(NH2CH2)2 system, the high values for the consts. for the Fe++-ο-phenanthroline system, and the low values for k2 in systems with (MeNHCH2)2. The entropy effect is such that the predominant factor, the entropy of hydration of M++, follows the order of stabilization and hence will not be a factor in exceptions.
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  43. 43
    Walton, T.; Szostak, J. W. A Highly Reactive Imidazolium-Bridged Dinucleotide Intermediate in Nonenzymatic RNA Primer Extension. J. Am. Chem. Soc. 2016, 138, 11996– 12002,  DOI: 10.1021/jacs.6b07977
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    43
    A Highly Reactive Imidazolium-Bridged Dinucleotide Intermediate in Nonenzymatic RNA Primer Extension
    Walton, Travis; Szostak, Jack W.
    Journal of the American Chemical Society (2016), 138 (36), 11996-12002CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Because of its importance for the origin of life, the nonenzymic copying of RNA templates has been the subject of intense study for several decades. Previous characterizations of template-directed primer extension using 5'-phosphoryl-2-methylimidazole-activated nucleotides (2-MeImpNs) as substrates have assumed a classical in-line nucleophilic substitution mechanism, in which the 3'-hydroxyl of the primer attacks the phosphate of the incoming monomer, displacing the 2-methylimidazole leaving group. However, we have found that the initial rate of primer extension depends on the pH and concn. at which the activated monomer is maintained prior to the primer extension reaction. These and other results suggest an alternative mechanism, in which two monomers react with each other to form an imidazolium-bridged dinucleotide intermediate, which then binds to the template. Subsequent attack of the 3'-hydroxyl of the primer displaces an activated nucleotide as the leaving group and results in extension of the primer by one nucleotide. Anal. of monomer solns. by NMR indicates formation of the proposed imidazolium-bridged dinucleotide in the expected pH-dependent manner. We have used synthetic methods to prep. material that is enriched in this proposed intermediate and show that it is a highly reactive substrate for primer extension. The formation of an imidazolium-bridged dinucleotide intermediate provides a mechanistic interpretation of previously obsd. catalysis by an activated nucleotide located downstream from the site of primer extension.
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  44. 44
    (a) Pinheiro, V. B.; Taylor, A. I.; Cozens, C.; Abramov, M.; Renders, M.; Zhang, S.; Chaput, J. C.; Wengel, J.; Peak-Chew, S. -Y.; McLaughlin, S. H.; Herdewijn, P.; Holliger, P. Synthetic Genetic Polymers Capable of Heredity and Evolution. Science 2012, 336, 341– 344,  DOI: 10.1126/science.1217622
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    44a
    Synthetic Genetic Polymers Capable of Heredity and Evolution
    Pinheiro, Vitor B.; Taylor, Alexander I.; Cozens, Christopher; Abramov, Mikhail; Renders, Marleen; Zhang, Su; Chaput, John C.; Wengel, Jesper; Peak-Chew, Sew-Yeu; McLaughlin, Stephen H.; Herdewijn, Piet; Holliger, Philipp
    Science (Washington, DC, United States) (2012), 336 (6079), 341-344CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Genetic information storage and processing rely on just two polymers, DNA and RNA, yet whether their role reflects evolutionary history or fundamental functional constraints is currently unknown. With the use of polymerase evolution and design, we show that genetic information can be stored in and recovered from six alternative genetic polymers based on simple nucleic acid architectures not found in nature [xeno-nucleic acids (XNAs)]. We also select XNA aptamers, which bind their targets with high affinity and specificity, demonstrating that beyond heredity, specific XNAs have the capacity for Darwinian evolution and folding into defined structures. Thus, heredity and evolution, two hallmarks of life, are not limited to DNA and RNA but are likely to be emergent properties of polymers capable of information storage.
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    (b) Yu, H.; Zhang, S.; Chaput, J. C. Darwinian Evolution of an Alternative Genetic System Provides Support for TNA as an RNA Progenitor. Nat. Chem. 2012, 4, 183– 187,  DOI: 10.1038/nchem.1241
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    44b
    Darwinian evolution of an alternative genetic system provides support for TNA as an RNA progenitor
    Yu, Hanyang; Zhang, Su; Chaput, John C.
    Nature Chemistry (2012), 4 (3), 183-187CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
    The pre-RNA world hypothesis postulates that RNA was preceded in the evolution of life by a simpler genetic material, but it is not known if such systems can fold into structures capable of eliciting a desired function. Presumably, whatever chem. gave rise to RNA would have produced other RNA analogs, some of which may have preceded or competed directly with RNA. Threose nucleic acid (TNA), a potentially natural deriv. of RNA, has received considerable interest as a possible RNA progenitor due to its chem. simplicity and ability to exchange genetic information with itself and RNA. Here, the authors applied Darwinian evolution methods to evolve, in vitro, a TNA receptor that binds to an arbitrary target with high affinity and specificity. This demonstration showed that TNA has the ability to fold into tertiary structures with sophisticated chem. functions, which provides evidence that TNA could have served as an ancestral genetic system during an early stage of life.
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    (c) Houlihan, G.; Arangundy-Franklin, S.; Holliger, P. Exploring the Chemistry of Genetic Information Storage and Propagation through Polymerase Engineering. Acc. Chem. Res. 2017, 50, 1079– 1087,  DOI: 10.1021/acs.accounts.7b00056
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    44c
    Exploring the Chemistry of Genetic Information Storage and Propagation through Polymerase Engineering
    Houlihan, Gillian; Arangundy-Franklin, Sebastian; Holliger, Philipp
    Accounts of Chemical Research (2017), 50 (4), 1079-1087CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
    A review. Nucleic acids are a distinct form of sequence-defined biopolymer. What sets them apart from other sequence-defined biopolymers such as polypeptides or polysaccharides is their unique capacity to encode, store, and propagate genetic information (mol. heredity). In Nature, just 2 closely related nucleic acids, DNA and RNA, function as repositories and carriers of genetic information. They therefore are the mol. embodiment of biol. information. This naturally leads to questions regarding to what degree variations from this seemingly ideal "Goldilocks" chem. of DNA and RNA would still be compatible with the fundamental property of mol. heredity. To address this question, chemists have created a panoply of synthetic nucleic acids comprising unnatural sugar ring congeners, backbone linkages, and nucleobases in order to establish the mol. parameters for encoding genetic information and its emergence at the origin of life. A deeper anal. of the potential of these synthetic genetic polymers for mol. heredity requires a means of replication and a detn. of the fidelity of information transfer. While nonenzymic synthesis is an increasingly powerful method, it currently remains restricted to short polymers. Here, we discuss efforts toward establishing enzymic synthesis, replication, and evolution of synthetic genetic polymers through the engineering of polymerase enzymes found in Nature. To endow natural polymerases with the ability to efficiently utilize noncognate nucleotide substrates, novel strategies for the screening and directed evolution of polymerase function have been realized. High throughput plate-based screens, phage display and water-in-oil emulsion technol. based methods have yielded a no. of polymerases, some of which can synthesize and reverse transcribe synthetic genetic polymers with good efficiency and fidelity. The inception of such polymerases demonstrates that, at a basic level at least, mol. heredity is not restricted to the natural nucleic acids, DNA and RNA, but may be found in a large (if likely finite) no. of synthetic genetic polymers, and has opened up these novel sequence spaces for exploration. Although, largely unexplored, the 1st tentative forays have yielded ligands (aptamers) against a range of different targets and several catalysts elaborated in a range of distinct chemistries. Finally, taking the lead from established DNA designs, simple polyhedron nanostructures have been described. We anticipate that further progress in this area will expand the range of synthetic genetic polymers that can be synthesized, replicated, and evolved providing access to a rich sequence, structure and phenotypic space. "Synthetic genetics", i.e., the exploration of these spaces will illuminate the chem. parameter range for encoding and decoding information, 3-dimensional folding and catalysis, and yield novel ligands, catalysts, and nanostructures and devices for applications in biotechnol. and medicine.
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    (d) Mei, H.; Liao, J. -Y.; Jimenez, R. M.; Wang, Y.; Bala, S.; McCloskey, C.; Switzer, C.; Chaput, J. C. Synthesis and Evolution of a Threose Nucleic Acid Aptamer Bearing 7-Deaza-7-Substituted Guanosine Residues. J. Am. Chem. Soc. 2018, 140, 5706– 5713,  DOI: 10.1021/jacs.7b13031
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    44d
    Synthesis and evolution of a threose nucleic acid aptamer bearing 7-Deaza-7-Substituted guanosine residues
    Mei, Hui; Liao, Jen-Yu; Jimenez, Randi M.; Wang, Yajun; Bala, Saikat; McCloskey, Cailen; Switzer, Christopher; Chaput, John C.
    Journal of the American Chemical Society (2018), 140 (17), 5706-5713CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    In vitro selection expts. carried out on artificial genetic polymers require robust and faithful methods for copying genetic information back and forth between DNA and xeno-nucleic acids (XNA). Previously, we have shown that Kod-RI, an engineered polymerase developed to transcribe DNA templates into threose nucleic acid (TNA), can function with high fidelity in the absence of manganese ions. However, the transcriptional efficiency of this enzyme diminishes greatly when individual templates are replaced with libraries of DNA sequences, indicating that manganese ions are still required for in vitro selection. Unfortunately, the presence of manganese ions in the transcription mixt. leads to the misincorporation of tGTP nucleotides opposite dG residues in the templating strand, which are detected as G-to-C transversions when the TNA is reverse transcribed back into DNA. Here we report the synthesis and fidelity of TNA replication using 7-deaza-7-modified guanosine base analogs in the DNA template and incoming TNA nucleoside triphosphate. Our findings reveal that tGTP misincorporation occurs via a Hoogsteen base pair in which the incoming tGTP residue adopts a syn conformation with respect to the sugar. Substitution of tGTP for 7-deaza-7-Ph tGTP enabled the synthesis of TNA polymers with >99% overall fidelity. A TNA library contg. the 7-deaza-7-Ph guanine analog was used to evolve a biol. stable TNA aptamer that binds to HIV reverse transcriptase with low nanomolar affinity.
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    (e) Rangel, A. E.; Chen, Z.; Ayele, T. M.; Heemstra, J. M. In Vitro Selection of an XNA Aptamer Capable of Small-Molecule Recognition. Nucleic Acid Res. 2018, 46, 8057– 8068,  DOI: 10.1093/nar/gky667
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    (f) Eremeeva, E.; Fikatas, A.; Margamuljana, L.; Abramov, M.; Schols, D.; Groaz, E.; Herdewijn, P. Highly Stable Hexitol Based XNA Aptamers Targeting the Vascular Endothelial Growth Factor. Nucleic Acid Res. 2019, 47, 4927– 4939,  DOI: 10.1093/nar/gkz252
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    There is no corresponding record for this reference.
    (g) Levi-Acobas, F.; Katolik, A.; Röthlisberger, P.; Cokelaer, T.; Sarac, I.; Damha, M. J.; Leumann, C. J.; Hollenstein, M. Compatibility of 5-Ethynyl-2’F-ANA UTP with In Vitro Selection for the Generation of Base-Modified Nuclease Resistant Aptamers. Org. Biomol. Chem. 2019, 17, 8083– 8087,  DOI: 10.1039/C9OB01515A
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    44g
    Compatibility of 5-ethynyl-2'F-ANA UTP with in vitro selection for the generation of base-modified, nuclease resistant aptamers
    Levi-Acobas, Fabienne; Katolik, Adam; Rothlisberger, Pascal; Cokelaer, Thomas; Sarac, Ivo; Damha, Masad J.; Leumann, Christian J.; Hollenstein, Marcel
    Organic & Biomolecular Chemistry (2019), 17 (35), 8083-8087CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    A modified nucleoside triphosphate bearing two modifications based on a 2'-deoxy-2'-fluoro-arabinofuranose sugar and a uracil nucleobase equipped with a C5-ethynyl moiety (5-ethynyl-2'F-ANA UTP) was synthesized. This nucleotide analog could enzymically be incorporated into DNA oligonucleotides by primer extension and reverse transcribed to unmodified DNA. This nucleotide could be used in SELEX for the identification of high binding affinity and nuclease resistant aptamers.
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  1. Andrea C. Bardales, Joseph R. Mills, Dmitry M. Kolpashchikov. DNA Nanostructures as Catalysts: Double Crossover Tile-Assisted 5′ to 5′ and 3′ to 3′ Chemical Ligation of Oligonucleotides. Bioconjugate Chemistry 2024, 35 (1) , 28-33. https://doi.org/10.1021/acs.bioconjchem.3c00502
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  • Abstract

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    Figure 1

    Figure 1. (a) Chemical structures of DNA and L-aTNA. (b) Schematic illustration of the chemical ligation reaction by CNIm and a divalent metal cation. (c) Sequences of L-aTNA and DNA used for chemical ligation. (d) Denaturing PAGE analysis of chemical ligation of 8-mer L-aTNA fragments (T8A/T8B-3′p) on the 16-mer template (T16t) in the presence of CNIm and chloride salt of an indicated divalent metal cation (MCl2). Reaction conditions: 0.9 μM T8A, 1.1 μM T8B-3′p, 1.0 μM T16t, 100 mM NaCl, 5 mM MCl2, 20 mM CNIm, 25 °C. PAGE conditions: 20% acrylamide, 8 M urea, 1 × TBE, 4 W at room temperature for 1.5 or 2 h. Negative control (lane 1) included only T8A and NaCl. (e) Yield as a function of time for ligation of T8A to T8B-3′p on the T16t template (left) and of D16A-3′p to D16B on the D32t template (right) in the presence of indicated divalent metal cations. The graphs are based on data shown from panel d and Figure S1. (f) Calculated kobs values for chemical ligation of indicated oligomers in the presence of indicated metal ions. Reaction conditions: 0.9 μM fragment A, 1.1 μM fragment B, 1.0 μM template, 100 mM NaCl, 5 mM MCl2, 20 mM CNIm, 25 °C. kobs values were calculated from linearized plots of -ln([Fragment A]/[Fragment A]0) assuming a pseudo-first-order reaction.

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    Figure 2

    Figure 2. (a) Scheme of reaction of PhP and CNIm in the presence of Cd2+. (b) 1H NMR time course analysis. 1H NMR peaks colored brown, red, and green were assigned to PhP, CNIm, and Im, respectively. Reaction conditions: 5 mM PhP, CdCl2, and CNIm, 25 °C. (c) Possible reaction scheme and intermediates (i) suggested by NMR and (ii) not observed. (d) Normalized peak intensity of CNIm (red) and Im (green) calculated from NMR spectra.

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    Figure 3. (a) Plot of -ln([Fragment A]/[Fragment A]0) as a function of time over a range of CNIm concentrations. Reaction conditions: 0.9 μM T8A, 1.1 μM T8B-3′p, 1.0 μM T16t, 5 mM CdCl2, 1, 5, 10, or 20 mM CNIm, 25 °C. (b) Apparent k1 obtained assuming a pseudo-first-order reaction. k1,app of T8A/T8B-3′p/T16t and the other k1,apps were calculated from plots shown in Figures 3a and S11–S15, respectively. Reaction conditions: 0.9 μM fragment A, 1.1 μM fragment B, 1.0 μM template, 5 mM CdCl2, 1, 5, 10, or 20 mM CNIm, 25 °C. (c) Plots of apparent k1 values for each component versus CNIm concentration based on the Ostwald isolation method. (d) Energy-minimized structures of T8B-3′p and template (top) and T8A-1′p and template (bottom) illustrating interactions of phosphate with the neighboring amide group. (e) Correlation between the concentration of Cd2+ ions and the rate constant k1. (f) Schematic illustration of the predicted mechanism involving divalent metal cations in activation of the phosphate group and rates for steps 1 and 2 and the overall reaction.

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    Figure 4

    Figure 4. (a and b) Schematic illustrations of elongation via chemical ligation of an L-aTNA primer in (a) 3′ → 1′ direction and (b) 1′ → 3′ direction. (c) Denaturing PAGE of L-aTNA elongation reaction products in the presence of Cd2+ with ligation in the 1′ → 3′ direction. (d) Comparison of yields of full-length elongation products in different conditions. Reaction conditions: 0.9 μM primer, 100 μM T3Bmix, 1.0 μM template, 100 mM NaCl, 5 mM CdCl2 or 20 mM MnCl2, 20 mM CNIm, 4 °C for 6 h in the presence of Cd2+ or for 24 h in the presence of Mn2+. PAGE conditions: 20% acrylamide, 8 M urea, 1 × TBE, 3 h, 4 °C, 4 W. Negative control (lane 1) included only primer, NaCl, and divalent metal cation salt (Lane 1). Markers were prepared by the reaction using complementary fragments (Figure S23).

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    Figure 5

    Figure 5. (a) Schematic illustration of chemical primer extension on 23-mer and 29-mer L-aTNA templates with optimized conditions. (b) Denaturing PAGE of the reaction with T23t as the template (left) and T29t as the template (right). Reaction conditions: 0.45 μM Rev-T8A-3′p, 200 μM T3Bmix for T23t or 400 μM T3Bmix for T29t, 1.0 μM T23t or T29t, 100 mM NaCl, 5 mM CdCl2, 20 mM CNIm, 4 °C for 4 h. PAGE conditions: 20% acrylamide, 8 M urea, 1 × TBE, 3 h with T23t as the template or 3.5 h with T29t as the template, 15 °C, 4 W. Negative controls (lanes 1 and 6) included only Rev-T8A-3′p, NaCl, and CdCl2. (c) MALDI-TOF MS analyses of products of the elongation reaction for 2 h with T23t as the template (left) and for 4 h with T29t as the template (right).

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  • References

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    This article references 44 other publications.

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      Cold Spring Harbor Perspectives in Biology (2012), 4 (7), a006742/1-a006742/5CODEN: CSHPEU; ISSN:1943-0264. (Cold Spring Harbor Laboratory Press)
      A review. There are two RNA worlds. The first is the primordial RNA world, a hypothetical era when RNA served as both information and function, both genotype and phenotype. The second RNA world is that of today's biol. systems, where RNA plays active roles in catalyzing biochem. reactions, in translating mRNA into proteins, in regulating gene expression, and in the const. battle between infectious agents trying to subvert host defense systems and host cells protecting themselves from infection. This second RNA world is not at all hypothetical, and although we do not have all the answers about how it works, we have the tools to continue our interrogation of this world and refine our understanding. The fun comes when we try to use our secure knowledge of the modern RNA world to infer what the primordial RNAworld might have looked like.
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      Cold Spring Harbor Perspectives in Biology (2012), 4 (5), a003608, 22 pp.CODEN: CSHPEU; ISSN:1943-0264. (Cold Spring Harbor Laboratory Press)
      A review. The general notion of an "RNA World" is that, in the early development of life on the Earth, genetic continuity was assured by the replication of RNA and genetically encoded proteins were not involved as catalysts. There is now strong evidence indicating that an RNA World did indeed exist before DNA- and protein-based life. However, arguments regarding whether life on Earth began with RNA are more tenuous. It might be imagined that all of the components of RNA were available in some prebiotic pool and that these components assembled into replicating, evolving polynucleotides without the prior existence of any evolved macromols. A thorough consideration of this "RNA-first" view of the origin of life must reconcile concerns regarding the intractable mixts. that are obtained in expts. designed to simulate the chem. of the primitive Earth. Perhaps these concerns will eventually be resolved and recent exptl. findings provide some reason for optimism. However, the problem of the origin of the RNA World is far from being solved and it is fruitful to consider the alternative possibility that RNA was preceded by some other replicating, evolving mol., just as DNA and proteins were preceded by RNA.
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      McCall, M. J.; Hendry, P.; Jennings, P. A. Minimal Sequence Requirements for Ribozyme Activity. Proc. Natl. Acad. Sci. U. S. A. 1992, 89, 5710– 5714,  DOI: 10.1073/pnas.89.13.5710
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      Minimal sequence requirements for ribozyme activity
      McCall, Maxine J.; Hendry, Philip; Jennings, Philip A.
      Proceedings of the National Academy of Sciences of the United States of America (1992), 89 (13), 5710-14CODEN: PNASA6; ISSN:0027-8424.
      The hammerhead ribozyme, as engineered by J. Haseloff and W. L. Gerlach (1988), is an RNA mol. contg. 2 regions of conserved nucleotides, a double helix (helix II), which connects the 2 conserved regions, and flanking arms of variable sequence, which hydridize the ribozyme to its specific target. Here, it is shown that this ribozyme may be reduced in size and still retain cleavage activity by replacing helix II with just a few nucleotides that cannot form Watson-Crick base pairs between themselves. Furthermore, the nucleotides replacing helix II and the nucleotides in the flanking arms may be substituted with DNA, and this small, DNA-contg. ribozyme was fully as active as the original, full-size ribozyme. The cleavage activity of the minimized ribozyme (minizyme) depended on the no. and sequence of the few nucleotides that replaced helix II; optimal activity, thus far, was achieved by 4 or 5 deoxyribopyrimidines. The minizyme was active as a monomer, as shown by its nearly const. activity over a concn. range varying 25,000-fold, by the mobility of the minizyme-substrate complex in nondenaturing polyacrylamide gels as compared with other nucleic acid mols. of known size, and by other observations. These minizymes provide an excellent model system for studying the structure and mechanism of catalytic RNA, and they may also be useful in a variety of biol. applications.
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      Becker, S.; Feldmann, J.; Wiedemann, S.; Okamura, H.; Schneider, C.; Iwan, K.; Crisp, A.; Rossa, M.; Amatov, T.; Carell, T. Unified Prebiotically Plausible Synthesis of Pyrimidine and Purine RNA Ribonucleotides. Science 2019, 366, 76– 82,  DOI: 10.1126/science.aax2747
      4
      Unified prebiotically plausible synthesis of pyrimidine and purine RNA ribonucleotides
      Becker, Sidney; Feldmann, Jonas; Wiedemann, Stefan; Okamura, Hidenori; Schneider, Christina; Iwan, Katharina; Crisp, Antony; Rossa, Martin; Amatov, Tynchtyk; Carell, Thomas
      Science (Washington, DC, United States) (2019), 366 (6461), 76-82CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
      Theories about the origin of life require chem. pathways that allow formation of life's key building blocks under prebiotically plausible conditions. Complex mols. like RNA must have originated from small mols. whose reactivity was guided by physico-chem. processes. RNA is constructed from purine and pyrimidine nucleosides, both of which are required for accurate information transfer, and thus Darwinian evolution. Sep. pathways to purines and pyrimidines have been reported, but their concurrent syntheses remain a challenge. We report the synthesis of the pyrimidine nucleosides from small mols. and ribose, driven solely by wet-dry cycles. In the presence of phosphate-contg. minerals, 5'-mono- and diphosphates also form selectively in one-pot reactions. The pathway is compatible with purine synthesis, allowing the concurrent formation of all Watson-Crick bases.
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      5a
      Substituent control of the poly(C)-directed oligomerization of guanosine 5'-phosphoroimidazolide
      Inoue, Tan; Orgel, Leslie E.
      Journal of the American Chemical Society (1981), 103 (25), 7666-7CODEN: JACSAT; ISSN:0002-7863.
      The poly(C)-directed oligomerization of closely-related guanosine 5'-phosphorimidazolides demonstrates the extreme sensitivity of the efficiency and regioselectivity of the reactions to minor changes in the nature of the imidazole moiety. The reaction of the 2-methylimidazole deriv. generates 89% of oligomers 4 or more units long and the product is predominantly 3'-5'-linked, while related alkyl derivs. give much lower yields of mixed 2'-5'- and 3'-5'-linked isomers.
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      (b) Tam, C. P.; Zhou, L.; Fahrenbach, A. C.; Zhang, W.; Walton, T.; Szostak, J. W. Synthesis of a Nonhydrolyzable Nucleotide Phosphoroimidazolide Analogue that Catalyzes Nonenzymatic RNA Primer Extension. J. Am. Chem. Soc. 2018, 140, 783– 792,  DOI: 10.1021/jacs.7b11623
      5b
      Synthesis of a Non-hydrolyzable Nucleotide Phosphoroimidazolide Analogue That Catalyzes Nonenzymatic RNA Primer Extension
      Tam, Chun Pong; Zhou, Lijun; Fahrenbach, Albert C.; Zhang, Wen; Walton, Travis; Szostak, Jack W.
      Journal of the American Chemical Society (2018), 140 (2), 783-792CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      We report the synthesis of guanosine 5'-(4-methylimidazolyl)phosphonate (ICG), the third member of a series of nonhydrolyzable nucleoside 5'-phosphoro-2-methylimidazolide (2-MeImpN) analogs designed for mechanistic studies of nonenzymic RNA primer extension. The addn. of a 2-MeImpN monomer to a primer is catalyzed by the presence of a downstream activated monomer, yet the three nonhydrolyzable analogs do not show catalytic effects under std. mildly basic primer extension conditions. Surprisingly, ICG, which has a pKa similar to that of 2-MeImpG, is a modest catalyst of nonenzymic primer extension at acidic pH. Here we show that ICG reacts with 2-MeImpC to form a stable 5'-5'-imidazole-bridged guanosine-cytosine dinucleotide, with both a labile nitrogen-phosphorus and a stable carbon-phosphorus linkage flanking the central imidazole bridge. Cognate RNA primer-template complexes react with this GC-dinucleotide by attack of the primer 3'-hydroxyl on the activated N-P side of the 5'-5'-imidazole bridge. These observations support the hypothesis that 5'-5'-imidazole-bridged dinucleotides can bind to cognate RNA primer-template duplexes and adopt appropriate conformations for subsequent phosphodiester bond formation, consistent with our recent mechanistic proposal that the formation of activated 5'-5'-imidazolium-bridged dinucleotides is responsible for 2-MeImpN-driven primer extension.
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    6. 6
      Li, L.; Prywes, N.; Tam, C. P.; O’Flaherty, D. K.; Lelyveld, V. S.; Izgu, E. C.; Pal, A.; Szostak, J. W. Enhanced Nonenzymatic RNA Copying with 2-Aminoimidazole Activated Nucleotides. J. Am. Chem. Soc. 2017, 139, 1810– 1813,  DOI: 10.1021/jacs.6b13148
      6
      Enhanced nonenzymatic RNA copying with 2-aminoimidazole activated nucleotides
      Li, Li; Prywes, Noam; Tam, Chun Pong; O'Flaherty, Derek K.; Lelyveld, Victor S.; Izgu, Enver Cagri; Pal, Ayan; Szostak, Jack W.
      Journal of the American Chemical Society (2017), 139 (5), 1810-1813CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Achieving efficient nonenzymic replication of RNA is an important step toward the synthesis of self-replicating protocells that may mimic early forms of life. Despite recent progress, the nonenzymic copying of templates contg. mixed sequences remains slow and inefficient. Here we demonstrate that activating nucleotides with 2-aminoimidazole results in superior reaction kinetics and improved yields of primer extension reaction products. This new leaving group significantly accelerates monomer addn. as well as trimer-assisted RNA primer extension, allowing efficient copying of a variety of short RNA templates with mixed sequences.
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    7. 7
      Zhang, S. J.; Duzdevich, D.; Szostak, J. W. Potentially Prebiotic Activation Chemistry Compatible with Nonenzymatic RNA Copying. J. Am. Chem. Soc. 2020, 142, 14810– 14813,  DOI: 10.1021/jacs.0c05300
      7
      Potentially Prebiotic Activation Chemistry Compatible with Nonenzymatic RNA Copying
      Zhang, Stephanie J.; Duzdevich, Daniel; Szostak, Jack W.
      Journal of the American Chemical Society (2020), 142 (35), 14810-14813CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The nonenzymic replication of RNA may have enabled the propagation of genetic information during the origin of life. RNA copying can be initiated in the lab. with chem. activated nucleotides, but continued copying requires a source of chem. energy for in situ nucleotide activation. Recent work has illuminated a potentially prebiotic cyanosulfidic chem. that activates nucleotides, but its application to nonenzymic RNA copying had not been demonstrated. Here, we report a novel pathway that activates RNA nucleotides in a manner compatible with template-directed nonenzymic copying. We show that this pathway, which we refer to as bridge-forming activation, selectively yields the reactive imidazolium-bridged dinucleotide intermediate required for copying. Our results will enable more realistic simulations of RNA propagation based on continuous in situ nucleotide activation.
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    8. 8
      Izgu, E. C.; Oh, S. S.; Szostak, J. W. Synthesis of Activated 3’-Amino-3’-Deoxy-2-Thio-Thymidine, a Superior Substrate for the Nonenzymatic Copying of Nucleic Acid Templates. Chem. Commun. 2016, 52, 3684– 3686,  DOI: 10.1039/C5CC10317G
      8
      Synthesis of activated 3'-amino-3'-deoxy-2-thio-thymidine, a superior substrate for the non-enzymatic copying of nucleic acid templates
      Izgu, Enver Cagri; Oh, Seung Soo; Szostak, Jack W.
      Chemical Communications (Cambridge, United Kingdom) (2016), 52 (18), 3684-3686CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      We present a scalable synthesis of 3'-amino-3'-deoxy-2-thio-thymidine-5'-phosphoro-2-methylimidazolide, an activated monomer that can copy adenosine residues in nucleic acid templates rapidly without a polymerase. The sulfur atom substitution enhances the rate of template copying by 5-fold compared with the 3'-amino-3'-deoxy-T monomer, while the 3'-amino monomers exhibit a 2- to 30-fold enhancement compared with their ribonucleotide counterparts.
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    9. 9
      Ding, D.; Zhou, L.; Giurgiu, C.; Szostak, J. W. Kinetic Explanations for the Sequence Biases Observed in the Nonenzymatic Copying of RNA Templates. Nucleic Acids Res. 2022, 50, 35– 45,  DOI: 10.1093/nar/gkab1202
      9
      Kinetic explanations for the sequence biases observed in the nonenzymatic copying of RNA templates
      Ding, Dian; Zhou, Lijun; Giurgiu, Constantin; Szostak, Jack W.
      Nucleic Acids Research (2022), 50 (1), 35-45CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      The identification of nonenzymic pathways for nucleic acid replication is a key challenge in understanding the origin of life. We have previously shown that nonenzymic RNA primer extension using 2-aminoimidazole (2AI) activated nucleotides occurs primarily through an imidazolium-bridged dinucleotide intermediate. The reactive nature and preorganized structure of the intermediate increase the efficiency of primer extension but remain insufficient to drive extensive copying of RNA templates contg. all four canonical nucleotides. To understand the factors that limit RNA copying, we synthesized all ten 2AI-bridged dinucleotide intermediates and measured the kinetics of primer extension in a model system. The affinities of the ten dinucleotides for the primer/template/helper complexes vary by over 7,000-fold, consistent with nearest neighbor energetic predictions. Surprisingly, the reaction rates at satg. intermediate concns. still vary by over 15-fold, with the most weakly binding dinucleotides exhibiting a lower maximal reaction rate. Certain noncanonical nucleotides can decrease sequence dependent differences in affinity and primer extension rate, while monomers bridged to short oligonucleotides exhibit enhanced binding and reaction rates. We suggest that more uniform binding and reactivity of imidazolium-bridged intermediates may lead to the ability to copy arbitrary template sequences under prebiotically plausible conditions.
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    10. 10
      Orgel, L. E. Prebiotic Chemistry and the Origin of the RNA World. Crit. Rev. Biochem. Mol. Biol. 2004, 39, 99– 123,  DOI: 10.1080/10409230490460765
      10
      Prebiotic chemistry and the origin of the RNA world
      Orgel, Leslie E.
      Critical Reviews in Biochemistry and Molecular Biology (2004), 39 (2), 99-123CODEN: CRBBEJ; ISSN:1040-9238. (Taylor & Francis, Inc.)
      A review and discussion. The demonstration that ribosomal peptide synthesis is a ribozyme-catalyzed reaction makes it almost certain that there was once an RNA World. The central problem for origin-of-life studies, therefore, is to understand how a protein-free RNA World became established on the primitive Earth. The author 1st reviews the literature on the prebiotic synthesis of nucleotides, nonenzymic synthesis, the copying of polynucleotides, and the selection of ribozyme catalysts of a kind that might have facilitated polynucleotide replication. This leads to a brief outline of the "mol. biologists' dream" (MBD), an optimistic scenario for the origin of the RNA World. In the 2nd part of the review, the author points out the many unresolved problems presented by the MBD. This in turn leads to a discussion of genetic systems simpler than RNA that might have "invented" RNA. Finally, studies of prebiotic membrane formation are reviewed.
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    11. 11
      Schöning, K. -U.; Scholz, P.; Guntha, S.; Wu, X.; Krishnamurthy, R.; Eschenmoser, A. Chemical Etiology of Nucleic Acid Structure: The α-Threofuranosyl-(3′→2′) Oligonucleotide System. Science 2000, 290, 1347– 1351,  DOI: 10.1126/science.290.5495.1347
      11
      Chemical etiology of nucleic acid structure: The α-threofuranosyl-(3'→2') oligonucleotide system
      Schoning, K.-U.; Scholz, P.; Guntha, S.; Wu, X.; Krishnamurthy, R.; Eschenmoser, A.
      Science (Washington, D. C.) (2000), 290 (5495), 1347-1351CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      TNAs [(L)-α-threofuranosyl oligonucleotides] contg. vicinally connected (3'→2') phosphodiester bridges undergo informational base pairing in antiparallel strand orientation and are capable of cross-pairing with RNA and DNA. Being derived from a sugar contg. only four carbons, TNA is structurally the simplest of all potentially natural oligonucleotide-type nucleic acid alternatives studied thus far. This, along with the base-pairing properties of TNA, warrants close scrutiny of the system in the context of the problem of RNA's origin.
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      Hendrix, C.; Rosemeyer, H.; Verheggen, I.; Seela, F.; Aerschot, A. V.; Herdewijn, P. 1′,5′-Anhydrohexitol Oligonucleotides: Synthesis, Base Pairing and Recognition by Regular Oligodeoxyribonucleotides and Oligoribonucleotides. Chem. – Eur. J. 1997, 3, 110– 120,  DOI: 10.1002/chem.19970030118
      12
      1',5'-Anhydrohexitol oligonucleotides: synthesis, base pairing and recognition by regular oligodeoxyribonucleotides and oligoribonucleotides
      Hendrix, Chris; Rosemeyer, Helmut; Verheggen, Ilse; Seela, Frank; Van Aerschot, Arthur; Herdewijn, Piet
      Chemistry - A European Journal (1997), 3 (1), 110-120CODEN: CEUJED; ISSN:0947-6539. (VCH)
      Oligonucleotides constructed of 1',5'-anhydrohexitol nucleoside building blocks (hexitol nucleic acids, HNA) are completely stable towards 3'-exonuclease and form very stable self-complementary duplexes as well as sequence-selective stable duplexes with the natural DNA and RNA. Triple-helix formation has also been obsd. These hybridization characteristics are highly dependent on the base sequence and the exptl. conditions. When using a phosphate buffer contg. 0.1 M NaCl, a homopurine HNA dodecamer gives a δTm of +1.3°C/base pair with DNA as complement and a ΔTm of +3.0°C/base pair with RNA as complement. These oligomers may therefore be of considerable interest as antisense constructs.
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    13. 13
      (a) Zhang, L.; Peritz, A.; Meggers, E. A Simple Glycol Nucleic Acid. J. Am. Chem. Soc. 2005, 127, 4174– 4175,  DOI: 10.1021/ja042564z
      13a
      A Simple Glycol Nucleic Acid
      Zhang, Lilu; Peritz, Adam; Meggers, Eric
      Journal of the American Chemical Society (2005), 127 (12), 4174-4175CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A glycol nucleic acid (GNA) with an acyclic propylene glycol phosphodiester backbone forms stable antiparallel duplexes following the Watson-Crick base pairing rules.
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      (b) Meggers, E.; Zhang, L. Synthesis and Properties of the Simplified Nucleic Acid Glycol Nucleic Acid. Acc. Chem. Res. 2010, 43, 1092– 1102,  DOI: 10.1021/ar900292q
      13b
      Synthesis and Properties of the Simplified Nucleic Acid Glycol Nucleic Acid
      Meggers, Eric; Zhang, Lilu
      Accounts of Chemical Research (2010), 43 (8), 1092-1102CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
      In the present review, the nucleosides of glycol nucleic acid (GNA), with the backbone comprising just the three carbons and one stereocenter of propylene glycol (1,2-propanediol), probably constitute the simplest possible building blocks for a chem. stable nucleic acid that contains phosphodiester bonds is analyzed. However, it was not until 2005 that the astonishing duplex formation properties of GNA homoduplexes were discovered in our lab. The R- and S-enantiomers of GNA, (R)-GNA and (S)-GNA, pair in like-sym. combinations to form highly stable antiparallel duplexes in a Watson-Crick fashion, with thermal and thermodn. stabilities exceeding those of analogous duplexes of DNA and RNA. Interestingly, (R)-GNA and (S)-GNA do not significantly cross-pair with each other, either in a parallel or antiparallel fashion. GNA discriminates strongly in favor of the Watson-Crick base-pairing scheme, with only slightly lower fidelity than DNA. Two (S)-GNA homoduplex structures recently detd. by X-ray crystallog., one a brominated 6-mer duplex and the other an 8-mer duplex contg. two copper(II) ions, reveal that the overall GNA double helix is distinct from canonical A- and B-form nucleic acids. The structure is perhaps best described as a helical ribbon loosely wrapped around the helix axis. Within the backbone, the propylene glycol nucleotides adopt two different conformations, gauche and anti, with respect to the torsional angles between the vicinal C3'-O and C2'-O bonds. A strikingly large backbone-base inclination results in extensive zipper-like interstrand and reduced intrastrand base-base interactions. This strong backbone-base inclination might explain the observation that neither the R- nor S-enantiomer of GNA cross-pairs with DNA, whereas (S)-GNA can interact with RNA strands that are devoid of G:C base pairs. Given the combination of structural simplicity, straightforward synthetic accessibility, and high duplex stability of GNA duplexes, GNA affords a promising nucleic acid scaffold for biotechnol. and nanotechnol. Along these lines, we describe the functionalization of GNA duplexes through the incorporation of metal-ion-mediated base pairs. Finally, the properties of GNA discussed here reinforce its candidacy as one of the initial genetic mols. formed during the origins of life on Earth.
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    14. 14
      (a) Leumann, C. J. DNA Analogues: From Supramolecular Principles to Biological Properties. Bioorg. Med. Chem. 2002, 10, 841– 854,  DOI: 10.1016/S0968-0896(01)00348-0
      14a
      DNA analogues: From supramolecular principles to biological properties
      Leumann, Christian J.
      Bioorganic & Medicinal Chemistry (2002), 10 (4), 841-854CODEN: BMECEP; ISSN:0968-0896. (Elsevier Science Ltd.)
      A review, with refs. Mainly driven by the needs of antisense research, a large no. of oligonucleotide analogs have been prepd. and evaluated over the last 15 yr. Besides minor structural modifications of the building blocks of DNA and RNA itself, a considerable effort has been devoted to the de novo design of nucleoside analogs with improved binding properties. A particularly successful concept turned out to be that of conformational restriction. This review focuses on recent advances in this area and tries to summarize scope and limitations of this design principle.
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      (b) Petersen, M.; Wengel, J. LNA: a Versatile Tool for Therapeutics and Genomics. Trends Biotechnol. 2003, 21, 74– 81,  DOI: 10.1016/S0167-7799(02)00038-0
      14b
      LNA: a versatile tool for therapeutics and genomics
      Petersen, Michael; Wengel, Jesper
      Trends in Biotechnology (2003), 21 (2), 74-81CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Science Ltd.)
      A review. Locked nucleic acid (LNA) is a nucleic acid analog that displays unprecedented hybridization affinity towards complementary DNA and RNA. Structural studies have shown LNA to be an RNA mimic, fitting seamlessly into an A-type duplex geometry. Several reports have revealed LNA as a most promising mol. for the development of oligonucleotide-based therapeutics. For example, Tat-dependent transcription and telomerase activity have been efficiently suppressed by LNA oligomers, and efficient cleavage of highly structured RNA has been achieved using LNA-modified DNAzymes ('LNAzyme'). Furthermore, convincing examples of the application of LNA to nucleic acid diagnostics have been reported, including high capturing efficiencies and unambiguous scoring of single-nucleotide polymorphisms.
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      (c) Zhang, S.; Switzer, C.; Chaput, J. C. The Resurgence of Acyclic Nucleic Acids. Chem. Biodiversity 2010, 7, 245– 258,  DOI: 10.1002/cbdv.200900281
      14c
      The Resurgence of Acyclic Nucleic Acids
      Zhang, Su; Switzer, Christopher; Chaput, John C.
      Chemistry & Biodiversity (2010), 7 (2), 245-258CODEN: CBHIAM; ISSN:1612-1872. (Verlag Helvetica Chimica Acta)
      A review, examg. acyclic nucleoside analogs as therapeutic agents, potential progenitor candidates to RNA, and novel building blocks for nucleic-acid nanotechnol. Together, these areas of research provide new insights into the structural and functional properties of nucleic acids and suggest new paradigms for nucleic acid self-assembly.
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      (d) Pinheiro, V. B.; Holliger, P. The XNA World: Progress Towards Replication and Evolution of Synthetic Genetic Polymers. Curr. Opin. Chem. Biol. 2012, 16, 245– 252,  DOI: 10.1016/j.cbpa.2012.05.198
      14d
      The XNA world: progress towards replication and evolution of synthetic genetic polymers
      Pinheiro, Vitor B.; Holliger, Philipp
      Current Opinion in Chemical Biology (2012), 16 (3-4), 245-252CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
      A review. Life's diversity is built on the wide range of properties and functions that can be encoded in natural biopolymers such as polypeptides and nucleic acids. However, despite their versatility, the range of chem. functionalities is limited, particularly in the case of nucleic acids. Chem. modification of nucleic acids can greatly increase their functional diversity but access to the full phenotypic potential of such polymers requires a system of replication. Here we review progress in the chem. and enzymic synthesis, replication and evolution of unnatural nucleic acid polymers, which promises to enable the exploration of a vast sequence space not accessible to nature and deliver ligands, catalysts and materials based on this new class of biopolymers.
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      (e) Chaput, J. C.; Herdewijn, P. What Is XNA?. Angew. Chem., Int. Ed. 2019, 58, 11570– 11572,  DOI: 10.1002/anie.201905999
      14e
      What Is XNA?
      Chaput, John C.; Herdewijn, Piet
      Angewandte Chemie, International Edition (2019), 58 (34), 11570-11572CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. The term "xeno-nucleic acids", abbreviated XNA, has grown in popularity to the point that it has become a catch-all phrase for almost any unnatural nucleic acid, raising the question: what is XNA and how does it differ from chem. modified DNA.
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      (f) Murayama, K.; Asanuma, H. Design and Hybridization Properties of Acyclic Xeno Nucleic Acid Oligomers. ChemBioChem 2021, 22, 2507– 2515,  DOI: 10.1002/cbic.202100184
      14f
      Design and Hybridization Properties of Acyclic Xeno Nucleic Acid Oligomers
      Murayama, Keiji; Asanuma, Hiroyuki
      ChemBioChem (2021), 22 (15), 2507-2515CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. Xeno nucleic acids (XNAs) are analogs of DNA and RNA that have a non-ribose artificial scaffold. XNAs are possible prebiotic genetic carriers as well as alternative genetic systems in artificial life. In addn., XNA oligomers can be used as biol. tools. Acyclic XNAs, which do not have cyclic scaffolds, are attractive due to facile their synthesis and remarkably high nuclease resistance. To maximize the performance of XNAs, a neg. charged backbone is preferable to provide sufficient water soly.; however, acyclic XNAs contg. polyanionic backbones suffer from high entropy cost upon duplex formation, because of the high flexibility of the acyclic nature. Herein, we review the relationships between the structure and duplex hybridization properties of various acyclic XNA oligomers with polyanion backbones.
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    15. 15
      Taylor, A. I.; Houlihan, G.; Holliger, P. Beyond DNA and RNA: The Expanding Toolbox of Synthetic Genetics. Cold Spring Harbor Perspect. Biol. 2019, 11, a032490  DOI: 10.1101/cshperspect.a032490
      15
      Beyond DNA and RNA: the expanding toolbox of synthetic genetics
      Taylor, Alexander I.; Houlihan, Gillian; Holliger, Philipp
      Cold Spring Harbor Perspectives in Biology (2019), 11 (6), a032490CODEN: CSHPEU; ISSN:1943-0264. (Cold Spring Harbor Laboratory Press)
      The remarkable physicochem. properties of the natural nucleic acids, DNA and RNA, define modern biol. at the mol. level and are widely believed to have been central to life's origins. However, their ability to form repositories of information as well as functional structures such as ligands (aptamers) and catalysts (ribozymes/DNAzymes) is not unique. A range of nonnatural alternatives, collectively termed xeno nucleic acids (XNAs), are also capable of supporting genetic information storage and propagation as well as evolution. This gives rise to a new field of "synthetic genetics," which seeks to expand the nucleic acid chem. toolbox for applications in both biotechnol. and mol. medicine. In this review, we outline XNA polymerase and reverse transcriptase engineering as a key enabling technol. and summarize the application of "synthetic genetics" to the development of aptamers, enzymes, and nanostructures.
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    16. 16
      (a) Bhowmik, S.; Krishnamurthy, R. The Role of Sugar-Backbone Heterogeneity and Chimeras in the Simultaneous Emergence of RNA and DNA. Nat. Chem. 2019, 11, 1009– 1018,  DOI: 10.1038/s41557-019-0322-x
      16a
      The role of sugar-backbone heterogeneity and chimeras in the simultaneous emergence of RNA and DNA
      Bhowmik, Subhendu; Krishnamurthy, Ramanarayanan
      Nature Chemistry (2019), 11 (11), 1009-1018CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)
      Hypotheses of the origins of RNA and DNA are generally centered on the prebiotic synthesis of a pristine system (pre-RNA or RNA), which gives rise to its descendent. However, a lack of specificity in the synthesis of genetic polymers would probably result in chimeric sequences; the roles and fate of such sequences are unknown. Here, we show that chimeras, exemplified by mixed threose nucleic acid (TNA)-RNA and RNA-DNA oligonucleotides, preferentially bind to, and act as templates for, homogeneous TNA, RNA and DNA ligands. The chimeric templates can act as a catalyst that mediates the ligation of oligomers to give homogeneous backbone sequences, and the regeneration of the chimeric templates potentiates a scenario for a possible cross-catalytic cycle with amplification. This process provides a proof-of-principle demonstration of a heterogeneity-to-homogeneity scenario and also gives credence to the idea that DNA could appear concurrently with RNA, instead of being its later descendent.
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      (b) Kim, S. C.; O’Flaherty, D. K.; Giurgiu, C.; Zhou, L.; Szostak, J. W. The Emergence of RNA from the Heterogeneous Products of Prebiotic Nucleotide Synthesis. J. Am. Chem. Soc. 2021, 143, 3267– 3279,  DOI: 10.1021/jacs.0c12955
      16b
      The Emergence of RNA from the Heterogeneous Products of Prebiotic Nucleotide Synthesis
      Kim, Seohyun Chris; O'Flaherty, Derek K.; Giurgiu, Constantin; Zhou, Lijun; Szostak, Jack W.
      Journal of the American Chemical Society (2021), 143 (9), 3267-3279CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A review. Recent advances in prebiotic chem. are beginning to outline plausible pathways for the synthesis of the canonical ribonucleotides and their assembly into oligoribonucleotides. However, these reaction pathways suggest that many noncanonical nucleotides are likely to have been generated alongside the std. ribonucleotides. Thus, the oligomerization of prebiotically synthesized nucleotides is likely to have led to a highly heterogeneous collection of oligonucleotides comprised of a wide range of types of nucleotides connected by a variety of backbone linkages. How then did relatively homogeneous RNA emerge from this primordial heterogeneity. Here we focus on nonenzymic template-directed primer extension as a process that would have strongly enriched for homogeneous RNA over the course of multiple cycles of replication. We review the effects on copying the kinetics of nucleotides with altered nucleobase and sugar moieties, when they are present as activated monomers and when they are incorporated into primer and template oligonucleotides. We also discuss three variations in backbone connectivity, all of which are nonheritable and regenerate native RNA upon being copied. The kinetic superiority of RNA synthesis suggests that nonenzymic copying served as a chem. selection mechanism that allowed relatively homogeneous RNA to emerge from a complex mixt. of prebiotically synthesized nucleotides and oligonucleotides.
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    17. 17
      Chen, J. J.; Cai, X.; Szostak, J. W. N2’→P3’ Phosphoramidate Glycerol Nucleic Acid as a Potential Alternative Genetic System. J. Am. Chem. Soc. 2009, 131, 2119– 2121,  DOI: 10.1021/ja809069b
      17
      N2' → P3' phosphoramidate glycerol nucleic acid as a potential alternative genetic system
      Chen, Jesse J.; Cai, Xin; Szostak, Jack W.
      Journal of the American Chemical Society (2009), 131 (6), 2119-2121CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Glycerol nucleic acid (GNA) is an interesting base-pairing system with an acyclic, three-carbon backbone. In the present study, GNA analogs with N2' → P3' phosphoramidate linkages (npGNA) have been synthesized and their base-pairing properties examd. Thermal denaturation and CD studies show that npGNA can form stable duplexes with itself and with GNA. Furthermore, we show that npGNA can be assembled by template-directed ligation of 3'-imidazole-activated-2'-amino GNA dinucleotides. These results suggest that npGNA is a potential candidate for a self-replicating system based upon phosphoramidate linkages.
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    18. 18
      Brudno, Y.; Birnbaum, M. E.; Kleiner, R. E.; Liu, D. R. An In Vitro Translation, Selection and Amplification System for Peptide Nucleic Acids. Nat. Chem. Biol. 2010, 6, 148– 155,  DOI: 10.1038/nchembio.280
      18
      An in vitro translation, selection and amplification system for peptide nucleic acids
      Brudno, Yevgeny; Birnbaum, Michael E.; Kleiner, Ralph E.; Liu, David R.
      Nature Chemical Biology (2010), 6 (2), 148-155CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      Methods to evolve synthetic, rather than biol., polymers could significantly expand the functional potential of polymers that emerge from in vitro evolution. Requirements for synthetic polymer evolution include (i) sequence-specific polymn. of synthetic building blocks on an amplifiable template, (ii) display of the newly translated polymer strand in a manner that allows it to adopt folded structures, (iii) selection of synthetic polymer libraries for desired binding or catalytic properties and (iv) amplification of template sequences that survive selection in a manner that allows subsequent translation. Here we report the development of such a system for peptide nucleic acids (PNAs) using a set of 12 PNA pentamer building blocks. We validated the system by performing six iterated cycles of translation, selection and amplification on a library of 4.3 × 108 PNA-encoding DNA templates and obsd. >1,000,000-fold overall enrichment of a template encoding a biotinylated (streptavidin-binding) PNA. These results collectively provide an exptl. foundation for PNA evolution in the lab.
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    19. 19
      Murayama, K.; Kashida, H.; Asanuma, H. Acyclic L-Threoninol Nucleic Acid (L-aTNA) with Suitable Structural Rigidity Cross-Pairs with DNA and RNA. Chem. Commun. 2015, 51, 6500– 6503,  DOI: 10.1039/C4CC09244A
      19
      Acyclic L-threoninol nucleic acid (L-aTNA) with suitable structural rigidity cross-pairs with DNA and RNA
      Murayama, Keiji; Kashida, Hiromu; Asanuma, Hiroyuki
      Chemical Communications (Cambridge, United Kingdom) (2015), 51 (30), 6500-6503CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      We report the hybridization properties of a novel artificial nucleic acid: acyclic L-threoninol nucleic acid (L-aTNA). L-ATNA formed a more stable duplex with DNA and RNA than either D-aTNA or serinol nucleic acid (SNA) as the rigidity of the L-form was more optimal for interaction with natural nucleic acids.
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    20. 20
      Murayama, K.; Okita, H.; Kuriki, T.; Asanuma, H. Nonenzymatic Polymerase-Like Template-Directed Synthesis of Acyclic L-threoninol Nucleic Acid. Nat. Commun. 2021, 12, 804,  DOI: 10.1038/s41467-021-21128-0
      20
      Nonenzymatic polymerase-like template-directed synthesis of acyclic L-threoninol nucleic acid
      Murayama, Keiji; Okita, Hikari; Kuriki, Takumi; Asanuma, Hiroyuki
      Nature Communications (2021), 12 (1), 804CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Evolution of xeno nucleic acid (XNA) world essentially requires template-directed synthesis of XNA polymers. In this study, we demonstrate template-directed synthesis of an acyclic XNA, acyclic L-threoninol nucleic acid (L-aTNA), via chem. ligation mediated by N-cyanoimidazole. The ligation of an L-aTNA fragment on an L-aTNA template is significantly faster and occurs in considerably higher yield than DNA ligation. Both L-aTNA ligation on a DNA template and DNA ligation on an L-aTNA template are also obsd. High efficiency ligation of trimer L-aTNA fragments to a template-bound primer is achieved. Furthermore, a pseudo primer extension reaction is demonstrated using a pool of random L-aTNA trimers as substrates. To the best of our knowledge, this is the first example of polymerase-like primer extension of XNA with all four nucleobases, generating phosphodiester bonding without any special modification. This technique paves the way for a genetic system of the L-aTNA world.
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    21. 21
      (a) Kramer, M.; Richert, C. Enzyme-Free Ligation of 5’-Phosphorylated Oligodeoxynucleotides in a DNA Nanostructure. Chem. Biodiversity 2017, 14, e1700315  DOI: 10.1002/cbdv.201700315
      There is no corresponding record for this reference.
      (b) Weizenmann, N.; Scheidgen-Kleyboldt, G.; Ye, J.; Krause, C. B.; Kauert, D.; Helmi, S.; Rouillon, C.; Seidel, R. Chemical Ligation of an Entire DNA Origami Nanostructure. Nanoscale 2021, 13, 17556– 17565,  DOI: 10.1039/D1NR04225D
      21b
      Chemical ligation of an entire DNA origami nanostructure
      Weizenmann, Nicole; Scheidgen-Kleyboldt, Gerda; Ye, Jingjing; Krause, Cordula B.; Kauert, Dominik; Helmi, Seham; Rouillon, Christophe; Seidel, Ralf
      Nanoscale (2021), 13 (41), 17556-17565CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
      Within the field of DNA nanotechnol., numerous methods were developed to produce complex two- and three-dimensional DNA nanostructures for many different emerging applications. These structures typically suffer from a low tolerance against non-optimal environmental conditions including elevated temps. Here, we apply a chem. ligation method to covalently seal the nicks between adjacent 5 phosphorylated and 3 amine-modified strands within the DNA nanostructures. Using a cost-effective enzymic strand modification procedure, we are able to batch-modify all DNA strands even of large DNA objects, such as origami nanostructures. The covalent strand linkage increases the temp. stability of the structures by ~ 10 K. Generally, our method also allows a 'surgical' introduction of covalent strand linkages at preselected positions. It can also be used to map the strand ligation into chains throughout the whole nanostructure and identify assembly defects. We expect that our method can be applied to a large variety of DNA nanostructures, in particular when full control over the introduced covalent linkages and the absence of side adducts and DNA damages are required.
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    22. 22
      (a) Prakash, G.; Kool, E. T. Structural Effects in the Recognition of DNA by Circular Oligonucleotides. J. Am. Chem. Soc. 1992, 114, 3523– 3527,  DOI: 10.1021/ja00035a056
      22a
      Structural effects in the recognition of DNA by circular oligonucleotides
      Prakash, Gautam; Kool, Eric T.
      Journal of the American Chemical Society (1992), 114 (9), 3523-7CODEN: JACSAT; ISSN:0002-7863.
      It was recently reported that certain pyrimidine-rich circular DNA oligomers can bind strongly and specifically to purine-rich DNA or RNA strands by forming bimol. triple helical complexes. In this study, the effects of structural variations on the strength of binding for this new class of nucleotide-binding ligand are investigated. The no. of loop nucleotides (nt) optimal for bridging the two binding domains of a circle is examd. Comparing loop sizes of 3, 4, 5, 6, and 10 nt, the optimum no. of nucleotides in a loop is 5 for the sequences studied. To test the method of construction and the ability of these compds. to bind sites of varied length, circles of varied size were synthesized. Circles over the size range 24-46 nt were successfully constructed. Varying the target site length shows that oligomers of 4, 8, 12, and 18 nucleotides can be complexed strongly by circles, with melting temps. (Tm) 17° to >33° higher at pH 7.0 than the corresponding Watson-Crick duplexes of the same length. Also studied is the effect of the covalently closed circular structure in comparison to linear oligomers having the same sequence; a covalently closed circle has considerably higher binding affinity than do three different nicked circles (linear oligomers) which contain the same bases. The high binding affinities of these circles are thus attributed to the entropic benefit of preorganization. Finally, the ability of such circles to bind to complementary sites within longer oligomers, the ends of which must pass beyond the loops of a circle, is confirmed by melting studies with synthetic target strands 36 bases in length.
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      (b) Petkovic, S.; Müller, S. RNA Circularization Strategies In Vivo and In Vitro. Nucleic Acids Res. 2015, 43, 2454– 2465,  DOI: 10.1093/nar/gkv045
      22b
      RNA circularization strategies in vivo and in vitro
      Petkovic, Sonja; Mueller, Sabine
      Nucleic Acids Research (2015), 43 (4), 2454-2465CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      A review. In the plenitude of naturally occurring RNAs, circular RNAs (circRNAs) and their biol. role were underestimated for years. However, circRNAs are ubiquitous in all domains of life, including eukaryotes, archaea, bacteria and viruses, where they can fulfill diverse biol. functions. Some of those functions, as for example playing a role in the life cycle of viral and viroid genomes or in the maturation of tRNA genes, have been elucidated; other putative functions still remain elusive. Due to the resistance to exonucleases, circRNAs are promising tools for in vivo application as aptamers, trans-cleaving ribozymes or siRNAs. How are circRNAs generated in vivo and what approaches do exist to produce ring-shaped RNAs in vitro. In this review we illustrate the occurrence and mechanisms of RNA circularization in vivo, survey methods for the generation of circRNA in vitro and provide appropriate protocols.
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      (c) Liang, X.; Chen, H.; Li, L.; An, R.; Komiyama, M. Ring-Structured DNA and RNA as Key Players In Vivo and In Vitro. Bull. Chem. Soc. Jpn. 2021, 94, 141– 157,  DOI: 10.1246/bcsj.20200235
      22c
      Ring-Structured DNA and RNA as Key Players In Vivo and In Vitro
      Liang, Xingguo; Chen, Hui; Li, Lin; An, Ran; Komiyama, Makoto
      Bulletin of the Chemical Society of Japan (2021), 94 (1), 141-157CODEN: BCSJA8; ISSN:0009-2673. (Chemical Society of Japan)
      A review. Ring-structured DNA and RNA exhibit a variety of unique features in chem., biol., medicine, material science, and so on, which cannot be accomplished by their non-cyclic counterparts. In this review, both naturally occurring DNA/RNA rings and artificially synthesized ones have been comprehensively covered, mainly to bridge these two growing fields. In the first part, the structures and functions of naturally occurring DNA/RNA rings (extrachromosomal circular DNA, circulating cell-free DNAs, cyclic RNAs, and others) are described. Their roles as biomarkers for disease diagnosis are esp. noteworthy. The second part mainly presents recent methods to synthesize DNA/RNA rings selectively and efficiently from oligonucleotide fragments. DNA/RNA rings of desired sequences and sizes are successfully prepd. in large amts. for versatile applications. Prodn. of RNA rings in cells using autocatalytic transcripts is also described. Lastly, practical applications of DNA/RNA rings are briefly reviewed. Crit. significance of the cooperation of these two areas for further developments, as well as strong potential for interdisciplinary studies, have been emphasized.
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      (d) Silverman, A. P.; Kool, E. T. Detecting RNA and DNA with Templated Chemical Reactions. Chem. Rev. 2006, 106, 3775– 3789,  DOI: 10.1021/cr050057+
      22d
      Detecting RNA and DNA with templated chemical reactions
      Silverman, Adam P.; Kool, Eric T.
      Chemical Reviews (Washington, DC, United States) (2006), 106 (9), 3775-3789CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. Recent advances in the development of chem. reactions that are triggered by hybridization of an oligonucleotide (a synthetic short strand of DNA) to a target RNA or DNA strand are summarized. A description is given of how these reactions are being used, or could be used, in detecting and identifying such targets in a biol. sample or cell. The basic principles of DNA and RNA mol. recognition underlying this research are discussed. The historical development of such templated reactions is outlined and then the current state of the art is examd. Some exciting new technologies that are under development in this field and future areas of special promise are mentioned.
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    23. 23
      O’Reilly, R. K.; Turberfield, A. J.; Wilks, T. R. The Evolution of DNA-Templated Synthesis as a Tool for Materials Discovery. Acc. Chem. Res. 2017, 50, 2496– 2509,  DOI: 10.1021/acs.accounts.7b00280
      23
      The Evolution of DNA-Templated Synthesis as a Tool for Materials Discovery
      O'Reilly, Rachel K.; Turberfield, Andrew J.; Wilks, Thomas R.
      Accounts of Chemical Research (2017), 50 (10), 2496-2509CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
      A review on the processes involved in DTS (DNA templated synthesis) and highlights the challenges that remain in creating a general system for mol. discovery by evolution.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsV2iurnJ&md5=4225db9723265c4f5c91a8a779dae461
    24. 24
      Shabarova, Z. A.; Dolinnaya, N. G.; Turkin, S. I.; Gromova, E. S. DNA-like Duplexes with Repetitions. I. Properties of Concatemer Duplexes Formed by d(T-G-C-A-C-A-T-G). Nucleic Acids Res. 1980, 8, 2413– 2430,  DOI: 10.1093/nar/8.11.2413
      24
      DNA-like duplexes with repetitions. I. Properties of concatemer duplexes formed by d(T-G-C-A-C-A-T-G)
      Shabarova, Z. A.; Dolinnaya, N. G.; Turkin, S. I.; Gromova, E. S.
      Nucleic Acids Research (1980), 8 (11), 2413-29CODEN: NARHAD; ISSN:0305-1048.
      A new class of synthetic DNA duplexes contg. repeating oligonucleotide sequences, double-helical concatemers, is characterized. The UV absorption and CD of a concatemer formed in self-assocn. of d(T-G-C-A-C-T-G) were studied. The thermodn. parameters of complex formation are the following: ΔH10 = -9.2 kcal/mol, ΔS10 = -27 entropy units. The data show that pseudopolymeric duplexes having structures that are similar to DNA-B-type helixes are formed in solns. of d(T-G-C-A-C-A-T-G). Polymn. of 32P-labeled d(pT-G-C-A-C-A-T-G) induced by water-sol. carbodiimide was carried out under the conditions of concatemer stability. The yield of the dimer, a 16-member oligonucleotide, was 13%.
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    25. 25
      Kukwikila, M.; Gale, N.; El-Sagheer, A. H.; Brown, T.; Tavassoli, A. Assembly of a Biocompatible Triazole-Linked Gene by One-pot Click-DNA Ligation. Nat. Chem. 2017, 9, 1089– 1098,  DOI: 10.1038/nchem.2850
      25
      Assembly of a biocompatible triazole-linked gene by one-pot click-DNA ligation
      Kukwikila, Mikiembo; Gale, Nittaya; El-Sagheer, Afaf H.; Brown, Tom; Tavassoli, Ali
      Nature Chemistry (2017), 9 (11), 1089-1098CODEN: NCAHBB; ISSN:1755-4330. (Nature Research)
      The chem. synthesis of oligonucleotides and their enzyme-mediated assembly into genes and genomes has significantly advanced multiple scientific disciplines. However, these approaches are not without their shortcomings; enzymic amplification and ligation of oligonucleotides into genes and genomes makes automation challenging, and site-specific incorporation of epigenetic information and/or modified bases into large constructs is not feasible. Here we present a fully chem. one-pot method for the assembly of oligonucleotides into a gene by click-DNA ligation. We synthesize the 335 base-pair gene that encodes the green fluorescent protein iLOV from ten functionalized oligonucleotides that contain 5'-azide and 3'-alkyne units. The resulting click-linked iLOV gene contains eight triazoles at the sites of chem. ligation, and yet is fully biocompatible; it is replicated by DNA polymerases in vitro and encodes a functional iLOV protein in Escherichia coli. We demonstrate the power and potential of our one-pot gene-assembly method by prepg. an epigenetically modified variant of the iLOV gene.
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    26. 26
      Naylor, R.; Gilham, P. T. Studies on Some Interactions and Reactions of Oligonucleotides in Aqueous Solution. Biochemistry 1966, 5, 2722– 2728,  DOI: 10.1021/bi00872a032
      26
      Studies on some interactions and reactions of oligonucleotides in aqueous solution
      Naylor, R.; Gilham, P. T.
      Biochemistry (1966), 5 (8), 2722-8CODEN: BICHAW; ISSN:0006-2960.
      The interactions between thymidine and deoxyadenosine oligonucleotides of various chain lengths as measured by percentage hypochromicity have been studied in salt solns. at 0°. The amt. of complexing was found to be dependent on the chain lengths of the 2 interacting species, esp. with those with chain lengths of <8 nucleotides. However, no interaction could be detected with thymidine and deoxyadenosine oligonucleotides of chain lengths <5 and 4 nucleotides, resp. The amt. and types of interaction between thymidine penta- and hexanucleotides with poly(adenylic acid) have been investigated with a view to using the complexes formed to direct the synthesis of internucleotide linkages in aq. soln. Water-sol. carbodiimides have been studied as reagents for the activation of terminal phosphate groups of oligonucleotides in aq. soln. In model expts., these reagents were shown to be capable of effecting the rapid cyclization of nucleoside 2',3'-phosphates at pH 6, and to be capable of converting adenosine 5'-phosphate to its Et ester and to adenylyl-(5' → 5')-adenosine on reaction with the appropriate hydroxylic component. By using the complexes formed with poly(adenylic acid) to achieve favorable orientations of the oligonucleotides and by employing a water-sol. carbodiimide as an activating agent, thymidine penta- and hexanucleotides have been converted in aq. soln. to thymidine deca- and dodecanucleotides in yields of 3 and 5%, resp.
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    27. 27
      Uesugi, S.; Ts’o, P. O. P. Chemical Polymerization of Oligonucleotides Directed by a Complementary Polynucleotide. Preparation and Polymerization of Oligo(2’-O-methylinosine 3′-phosphate). Biochemistry 1974, 13, 3142– 3152,  DOI: 10.1021/bi00712a022
      27
      Chemical polymerization of oligonucleotides directed by a complementary polynucleotide. Preparation and polymerization of oligo(2'-O-methylinosine 3'-phosphate)
      Uesugi, S.; Tso, P. O. P.
      Biochemistry (1974), 13 (15), 3142-52CODEN: BICHAW; ISSN:0006-2960.
      Substantial quantities of oligo(2'-O-methylinosinates) with 3'-terminal phosphates and defined chain lengths (n = 2-12) were prepd. by controlled hydrolysis of poly(2'-O-methylinosinate) with micrococcal nuclease, followed by DEAE-cellulose column chromatog. Two oligonucleotide fractions, hexa(2'-O-methylinosine 3'-phosphate) and penta(2'-O-methylinosine 3'-phosphate), were used as starting materials in a polymn. reaction directed by a poly(C) template. These reactions were carried out in aq. soln. at low temp. (0 or -15°) with a H2O-sol. carbodiimide as the activating agent. The abs. overall yield was 38-61%, and the relative overall yield based on the recovered material was 43-71%; the yield of the 30-mer fraction (product with 5-6 linkages) can be ≤15%. The stability of the 1:1 oligo(2'-O-methylinosine)-poly(C) complex was an important factor in detg. the extent of the polymn. and the chain length of the product.
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    28. 28
      Shabarova, Z. A.; Dolinnaya, N. G.; Drutsa, V. L.; Melnikova, N. P.; Purmal, A. A. DNA like Duplexes with Repetitions. III. Efficient Template-Guided Chemical Polymerization of d(TGGCCAAGCTp). Nucleic Acids Res. 1981, 9, 5747– 5761,  DOI: 10.1093/nar/9.21.5747
      28
      DNA-like duplexes with repetitions. III. Efficient template-guided chemical polymerization of d(TGGCCAAGCTp)
      Shabarova, Z. A.; Dolinnaya, N. G.; Druca, V.; Mel'nikova, N. P.; Purmalis, A.
      Nucleic Acids Research (1981), 9 (21), 5747-61CODEN: NARHAD; ISSN:0305-1048.
      Self-assocn. of a decanucleotide d(TGGCCAAGCTp) in an aq. soln. is shown by UV and CD spectroscopy and sedimentation anal. to yield a pseudopolymeric (concatemeric) duplex having a geometry similar to that of DNA B-type. Under conditions where the concatemeric duplex is stable, a water-sol. carbodiimide induces efficient polymn. of the 3'- or 5'-phosphorylated decanucleotide, and the resulting polymers d(TGGCCAAGCTp)2-10 contain only natural phosphodiester bonds. In conditions optimal for template-guided polymn. of d(TGGCCAAGCTp), the overall yield of 20-100-member polynucleotides is >90%. The polymeric duplexes are cleaved by restriction endonuclease AluI, BsuRI, and HindIII to corresponding decamers which were isolated and sequenced.
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    29. 29
      Edeleva, E.; Salditt, A.; Stamp, J.; Schwintek, P.; Boekhoven, J.; Braun, D. Continuous Nonenzymatic Cross-Replication of DNA Strands with In Situ Activated DNA Oligonucleotides. Chem. Sci. 2019, 10, 5807– 5814,  DOI: 10.1039/C9SC00770A
      29
      Continuous nonenzymatic cross-replication of DNA strands with in situ activated DNA oligonucleotides
      Edeleva, Evgeniia; Salditt, Annalena; Stamp, Julian; Schwintek, Philipp; Boekhoven, Job; Braun, Dieter
      Chemical Science (2019), 10 (22), 5807-5814CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      Continuous enzyme-free replication of oligonucleotides is central for open-ended evolution expts. that mimic the origin of life. Here, we studied a reaction system, whereby two 24mer DNA templates cross-catalyzed each other's synthesis from four 12mer DNA fragments, two of which were in situ activated with the condensing agent 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC). We circumvented the problem of product inhibition by melting the stable product duplexes for their reuse as templates in the following ligation step. The system reproduced itself through ligation/melting cycles and survived exponential diln. We quantified EDC-induced side reactions in a detailed kinetic model. The model allowed us to analyze the effects of various reaction rates on the system's kinetics and confirmed maximal replication under the chosen conditions. The presented system enables us to study nonenzymic open-ended evolution expts. starting from diverse sequence pools.
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    30. 30
      Obianyor, C.; Newnam, G.; Clifton, B. E.; Grover, M. A.; Hud, N. V. Towards Efficient Nonenzymatic DNA Ligation: Comparing Key Parameters for Maximizing Ligation Rates and Yields with Carbodiimide Activation. ChemBioChem 2020, 21, 3359– 3370,  DOI: 10.1002/cbic.202000335
      30
      Towards Efficient Nonenzymatic DNA Ligation: Comparing Key Parameters for Maximizing Ligation Rates and Yields with Carbodiimide Activation
      Obianyor, Chiamaka; Newnam, Gary; Clifton, Bryce E.; Grover, Martha A.; Hud, Nicholas V.
      ChemBioChem (2020), 21 (23), 3359-3370CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Chem. ligation is an important tool for the generation of synthetic DNA structures, which are used for a wide range of applications. Surprisingly, reported chem. ligation yields can range from 30 to 95% for the same chem. activating agent and comparable DNA structures. We report a systematic study of DNA ligation by using a well-defined bimol. test system and a water-sol. carbodiimide (EDC) as a phosphate-activating agent. Our results emphasize the interplay between template-substrate complex stability and the rates of the chem. steps of ligation, with 3' phosphate substrates providing yields near 100% after 24 h for particularly favorable reaction conditions. Ligation rates are also shown to be sensitive to the identity of the base pairs flanking a nick site, with as much as threefold variation. Finally, the observation that DNA substrates are modified by EDC at rates that can be comparable with ligation rates emphasizes the importance of considering side reactions when designing protocols to maximize ligation yields.
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    31. 31
      (a) Sokolova, N. I.; Ashirbekova, D. T.; Dolinnaya, N. G.; Shabarova, Z. A. Chemical Reactions within DNA Duplexes Cyanogen Bromide as an Effective Oligodeoxyribonucleotide Coupling Agent. FEBS Lett. 1988, 232, 153– 155,  DOI: 10.1016/0014-5793(88)80406-X
      31a
      Chemical reactions within DNA duplexes. Cyanogen bromide as an effective oligodeoxyribonucleotide coupling agent
      Sokolova, N. I.; Ashirbekova, D. T.; Dolinnaya, N. G.; Shabarova, Z. A.
      FEBS Letters (1988), 232 (1), 153-5CODEN: FEBLAL; ISSN:0014-5793.
      CNBr condensed oligodeoxyribonucleotides on a complementary template in aq. soln. Optimum conditions for this vigorous and effective reaction were developed. CNBr proved to be useful for incorporation of phosphoramidate or pyrophosphate internucleotide bonds in DNA duplexes.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXkvV2ht7k%253D&md5=73db71941cbcd267f049bbba6bde35db
      (b) Fedorova, O. A.; Gottikh, M. B.; Oretskaya, T. S.; Shabarova, Z. A. Cyanogen Bromide-Induced Chemical Ligation: Mechanism and Op-timization of the Reaction Conditions. Nucleosides, Nucleotides Nucleic Acids 1996, 15, 1137– 1147,  DOI: 10.1080/07328319608007382
      There is no corresponding record for this reference.
    32. 32
      Vogel, H.; Gerlach, C.; Richert, C. Reactions of Buffers in Cyanogen Bromine-Induced Ligations. Nucleosides, Nucleotides Nucleic Acids 2013, 32, 17– 27,  DOI: 10.1080/15257770.2012.744036
      32
      Reactions of Buffers in Cyanogen Bromide-Induced Ligations
      Vogel, Heike; Gerlach, Claudia; Richert, Clemens
      Nucleosides, Nucleotides & Nucleic Acids (2013), 32 (1), 17-27CODEN: NNNAFY; ISSN:1525-7770. (Taylor & Francis Ltd.)
      Rapid, template-directed ligation reactions between a phosphate-terminated oligonucleotide and an un-phosphorylated reaction partner may be induced by cyanogen bromide (BrCN). Frequently, however, the reaction is low yielding, and even a large excess of the condensing agent can fail to induce quant. conversions. In this study, we used BrCN to induce chem. primer extension reactions. Here, we report that buffers contg. hydroxyl groups react with short oligodeoxynucleotides in the presence of BrCN. One stable adduct between HEPBS buffer and cytosine was characterized by mass spectrometry and NMR after HPLC purifn., indicating that a side reaction occurred at this nucleobase. Further, a first example of a primer extension reaction between an unmodified oligodeoxynucleotide as primer and dGMP is reported. Together, our results shed light on the potency, as well as the drawbacks of BrCN as a highly reactive condensing reagent for the ligation of unmodified nucleic acids.
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    33. 33
      Kanaya, E.; Yanagawa, H. Template-Directed Polymerization of Oligoadenylates Using Cyanogen Bomide. Biochemistry 1986, 25, 7423– 7430,  DOI: 10.1021/bi00371a026
      33
      Template-directed polymerization of oligoadenylates using cyanogen bromide
      Kanaya, Eiko; Yanagawa, Hiroshi
      Biochemistry (1986), 25 (23), 7423-30CODEN: BICHAW; ISSN:0006-2960.
      BrCN condensed oligoadenylates [oligo(A)] on a poly(uridylic acid) [poly(U)] template in an aq. soln. Imidazole and divalent metal ions such as Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Mg2+, and Fe2+ were required for the condensation. Chain length of oligo(A) and reaction temp. affected the coupling yield. Hexaadenylate [(pA)6] was converted to (pA)12, (pA)18, (pA)24, (pA)30, (pA)36, (pA)42, and (pA)48 in a 68% overall yield for 20 h at 25°. The coupling yield increased with increases in the poly(U) concn. Five to seven-fold molar excess of uridylyl residues of poly(U) to adenylyl residues of oligo(A) gave the best yield (68%). Metal ions affected the formation of linkage isomers of the phosphate bonds: The 2',5'- and 3',5'-phosphodiester bonds were predominant in the presence of Co2+, Zn2+, and Ni2+, and the 5',5'-pyrophosphate bond was predominant in the presence of Mn2+. In particular, Ni2+ gave the highest ratio of the 3',5'-phosphodiester bond (30%). n-Cyanoimidazole (I), N,N'-iminodiimidazole (II), and N-carboxamidoimidazole (III) were formed in a reaction of imidazole with BrCN in an aq. soln. I and II had much the same condensing activity for the polymn. of adenylates as BrCN. A reaction pathway was proposed in which I and II are not only intermediates for the prodn. of III but also the true condensing agent in the coupling reaction of oligo(A). Phosphorimidazolide deriv. was detected in a reaction of 5'-AMP with either I or II. The condensation would proceed by way of N-cyanoimidazole-phosphate adduct, or the phosphorimidazolide deriv., or both.
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    34. 34
      Luebke, K. J.; Dervan, P. B. Nonenzymatic Ligation of Oligodeoxyribonucleotides on a Duplex DNA Template by Triple-Helix Formation. J. Am. Chem. Soc. 1989, 111, 8733– 8735,  DOI: 10.1021/ja00205a033
      34
      Nonenzymatic ligation of oligodeoxyribonucleotides on a duplex DNA template by triple-helix formation
      Luebke, Kevin J.; Dervan, Peter B.
      Journal of the American Chemical Society (1989), 111 (23), 8733-5CODEN: JACSAT; ISSN:0002-7863.
      A double-stranded DNA template can direct the sequence-specific formation of a phosphodiester linkage between pyrimidine oligodeoxynucleotides in aq. soln. by juxtaposing the oligonucleotide termini head-to-tail in a triple helical complex. Within the context of the development of chem. systems for macromol. information transfer, triple helix-directed ligation can create sequences that are neither identical nor complementary in a Watson-Crick sense to the template, but rather new sequences of nucleic acids.
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    35. 35
      Ferris, J. P.; Huang, C. -H.; Hagan, W. J. N-Cyanoimidazole and Diimidazole Imine: Water-Soluble Condensing Agents for the Formation of the Phosphodiester Bond. Nucleosides Nucleotides 1989, 8, 407– 414,  DOI: 10.1080/07328318908054184
      35
      N-cyanoimidazole and diimidazole imine: water-soluble condensing agents for the formation of the phospho diester bond
      Ferris, James P.; Huang, Chun Hsien; Hagan, William J., Jr.
      Nucleosides & Nucleotides (1989), 8 (3), 407-14CODEN: NUNUD5; ISSN:0732-8311.
      The reaction of BrCN with imidazole results in the formation of N-cyanoimidazole and diimidazole imine. These compds. were shown to be useful condensing agents for the formation of the phosphodiester bond in aq. soln.
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    36. 36
      (a) Li, T.; Weinstein, D. S.; Nicolaou, K. C. The Chemical End-Ligation of Homopyrimidine Oligodeoxyribonucleotides within a DNA Triple Helix. Chem. Biol. 1997, 4, 209– 214,  DOI: 10.1016/S1074-5521(97)90290-8
      36a
      The chemical end-ligation of homopyrimidine oligodeoxyribonucleotides within a DNA triple helix
      Li, Tianhu; Weinstein, David A.; Nicolaou, K. C.
      Chemistry & Biology (1997), 4 (3), 209-214CODEN: CBOLE2; ISSN:1074-5521. (Current Biology)
      Triple-helical nucleic acids, first reported in the late 1950s, are receiving attention for their possible involvement in controlling gene expression. Certain sequences of DNA are believed to form local triple-helical structures (H-form DNA), although this has not been directly obsd. in vivo. Studies carried out in our labs. have suggested that self-replicating oligonucleotides could have been involved in chem. evolution via triple-helical intermediates. In addn. to self-replication mechanisms, elucidating processes for the nonenzymic elongation of biol. relevant polymers remains an important challenge in understanding the origin of life. To this end, we have studied a novel ligation of oligodeoxyribonucleotides that lie within a triple helix. The chem. end-ligation of homopyrimidine oligodeoxyribonucleotides on a triple helix is reported. This selective process, induced by cyanoimidazole, is facilitated by a template effect of the DNA aggregate and occurs between the 3' end (hydroxyl) of the third minor-groove-bound strand and the 5' end (phosphate) of the antiparallel oligopyrimidine strand. Double-helical homopurine/homopyrimidine DNA can serve as a template for the elongation of oligonucleotides in a manner that has not been described previously. The end-ligation of homopyrimidine oligomers, a nonenzymic process, proceeds via a requisite triple-helical intermediate and constitutes an efficient and selective method for the template-directed elongation of nucleic acids. Such a process could conceivably have been involved in the elongation of primordial information-bearing biopolymers.
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      (b) Li, T.; Liu, D.; Chen, J.; Lee, A. H. F.; Qi, J.; Chan, A. S. C. Construction of Circular Oligodeoxyribonucleotides on the New Structural Basis of i-Motif. J. Am. Chem. Soc. 2001, 123, 12901– 12902,  DOI: 10.1021/ja011401x
      36b
      Construction of Circular Oligodeoxyribonucleotides on the New Structural Basis of i-Motif
      Li, Tianhu; Liu, Dongsheng; Chen, Jian; Lee, Alex H. F.; Qi, Jianying; Chan, Albert S. C.
      Journal of the American Chemical Society (2001), 123 (51), 12901-12902CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      We now report for the first time that beyond the scope of the previous duplex and triplex strategies, the i-motif, a four stranded assembly, can direct the sequence-specific formation of a phosphodiester linkage and thus represents a new type of structural template for constructing circular oligonucleotides.
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    37. 37
      Chen, H.; Du, F.; Chen, G.; Streckenbach, F.; Yasmeen, A.; Zhao, Y.; Tang, Z. Template-Directed Chemical Ligation to Obtain 3′-3′ and 5′-5’ Phosphodiester DNA Linkages. Sci. Rep. 2014, 4, 4595,  DOI: 10.1038/srep04595
      37
      Template-directed Chemical Ligation to Obtain 3'-3' and 5'-5' Phosphodiester DNA Linkages
      Chen, Haodong; Du, Feng; Chen, Gangyi; Streckenbach, Frank; Yasmeen, Afshan; Zhao, Yun; Tang, Zhuo
      Scientific Reports (2014), 4 (), 4595/1-4595/6CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
      Up to now, the direct ligation of two DNA fragments with opposite directions to obtain 3'-3' or 5'-5' phosphate ester bonds is still challenging. The only way to obtain DNA oligonucleotides contg. a 3'-3' or 5'-5' inversion of polarity sites is based on professional DNA chem. synthesis. Herein, we demonstrate a convenient template-directed chem. ligation that enables 3'-3' and 5'-5' linkages of two DNA oligonucleotides. This method is based on the assembly of two oligonucleotides on a template in opposite directions through forming antiparallel and parallel duplexes simultaneously, followed by coupling with [ital: null]-Cyanoimidazole under mild condition. Moreover, on the basis of DNA oligonucleotides with 5'-5' linkage obtained through our template-directed chem. ligation, we developed a new cDNA display technique for [ital: null] selection of functional polypeptides.
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    38. 38
      Mariani, A.; Sutherland, J. D. Non-Enzymatic RNA Backbone Proofreading through Energy-Dissipative Recycling. Angew. Chem., Int. Ed. 2017, 56, 6563– 6566,  DOI: 10.1002/anie.201703169
      38
      Non-enzymatic RNA backbone proofreading through energy-dissipative recycling
      Mariani, Angelica; Sutherland, John D.
      Angewandte Chemie, International Edition (2017), 56 (23), 6563-6566CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Nonenzymic oligomerization of activated ribonucleotides leads to RNAs that contain a mixt. of 2',5'- and 3',5'-linkages, and overcoming this backbone heterogeneity has long been considered a major limitation to the prebiotic emergence of RNA. Here, we demonstrate nonenzymic chem. that progressively converts 2',5'-linkages into 3',5'-linkages through iterative degrdn. and repair. The energetic costs of this proofreading are met by the hydrolytic turnover of a phosphate activating agent and an acylating agent. With multiple rounds of this energy-dissipative recycling, we show that all-3',5'-linked duplex RNA can emerge from a backbone heterogeneous mixt., thereby delineating a route that could have driven RNA evolution on the early Earth.
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    39. 39
      (a) Covington, A. K.; Paabo, M.; Robinson, R. A.; Bates, R. G. Use of the Glass Electrode in Deuterium Oxide and the Relation Between the Standardized pD (paD) Scale and the Operational pH in Heavy Water. Anal. Chem. 1968, 40, 700– 706,  DOI: 10.1021/ac60260a013
      39a
      Use of the glass electrode in deuterium oxide and the relation between the standardized pD (paD) scale and the operational pH in heavy water
      Covington, Arthur K.; Paabo, Maya; Robinson, Robert Anthony; Bates, Roger G.
      Analytical Chemistry (1968), 40 (4), 700-6CODEN: ANCHAM; ISSN:0003-2700.
      Both direct and indirect comparison of com. glass electrodes with the D gas electrode at 25° in buffered solns. of pD 1-13 confirmed that the glass electrode functions as well in heavy water as in ordinary water. The relation between the operational pH of a buffer soln. in heavy water, as obtained with a glass electrode standardized in an ordinary water buffer soln., and the corresponding pD value, as obtained from measurements on cells without liq. junction, is examd. and correction factors are detd. for both glass and the D gas electrode. The operational pH of buffer solns. in heavy water at 25°, which are measured with the glass electrode, can be converted into a pD value by adding 0.41 (molar scale) or 0.45 (molal scale), for pD 2-9. 26 references.
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      (b) Krȩżel, A.; Bal, W. A Formula for Correlating pKa Values Determined in D2O and H2O. J. Inorg. Biochem. 2004, 98, 161– 166,  DOI: 10.1016/j.jinorgbio.2003.10.001
      39b
      A formula for correlating pKa values determined in D2O and H2O
      Krezel, Artur; Bal, Wojciech
      Journal of Inorganic Biochemistry (2004), 98 (1), 161-166CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier Science Inc.)
      A linear correlation between pH-meter readings in equiv. D2O and H2O solns., detd. exptl., leads to a novel equation, which allows for a direct recalcn. of pKa values measured in D2O into a H2O equiv.: pKH=0.929pKH*+0.42. The comparison of this equation with the previously used approach is discussed.
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    40. 40
      Yi, R.; Hongo, Y.; Fahrenbach, A. C. Synthesis of Imidazole-Activated Ribonucleotides Using Cyanogen Chloride. Chem. Commun. 2018, 54, 511– 514,  DOI: 10.1039/C7CC08489G
      40
      Synthesis of imidazole-activated ribonucleotides using cyanogen chloride
      Yi, Ruiqin; Hongo, Yayoi; Fahrenbach, Albert C.
      Chemical Communications (Cambridge, United Kingdom) (2018), 54 (5), 511-514CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      We report the syntheses of ribonucleoside 5'-monophosphates activated with imidazole, using a mechanism which relies on the in situ generation of cyanogen chloride from the reaction of cyanide anion with hypochlorous acid. Cyanogen chloride reacts rapidly with imidazole to form diimidazole imine as the major product, a species which affords the activation of ribonucleoside 5'-monophosphates to their 5'-phosphorimidazolides.
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    41. 41
      (a) Drey, C. N. C.; Fruton, J. S. Synthesis and Properties of a Bis-Imidazole. Biochemistry 1965, 4, 1– 5,  DOI: 10.1021/bi00877a001
      41a
      Synthesis and properties of a bisimidazole
      Drey, Charles N. C.; Fruton, Joseph S.
      Biochemistry (1965), 4 (1), 1-5CODEN: BICHAW; ISSN:0006-2960.
      4,4'(5,5')-Bisimidazolylmethane (I) was synthesized from histidine Me ester by redn. with NaHg in the presence of KSCN, followed by desulfurization of the resulting 2-mercapto compd. with FeCl3 or HNO3. I has apparent ionization consts. of 5.3 and 7.0. It exhibits strong intermol. H bonding in Me2SO and no evidence was found for intramol. H bonding. Examn. of the catalytic effect of I on the hydrolysis of p-nitrophenylacetate showed that, on an equimolar basis, I was slightly less effective than imidazole itself. I had no catalytic effect on the hydrolysis of uridine 2',3'-phosphate.
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      (b) Tanford, C.; Wagner, M. L. The Consecutive Constants for the Association of Cadmium with Imidazole. J. Am. Chem. Soc. 1953, 75, 434– 435,  DOI: 10.1021/ja01098a052
      41b
      Consecutive constants for the association of cadmium with imidazole
      Tanford, Charles; Wagner, Myron L.
      Journal of the American Chemical Society (1953), 75 (), 434-5CODEN: JACSAT; ISSN:0002-7863.
      Since it has been indicated that the principal binding sites for the combination of metals with serum albumin are the imidazole (I) groups (cf. C.A. 45, 7446e), the binding consts. for metals with I should be approx. the same as with albumin. The successive assocn. consts. for the Cd-I complex were detd. potentiometrically by the method of Bjerrum (C.A. 35, 6206.3); at 25°, log k1 = 2.80 ± 0.05, log k2 = 2.10 ± 0.08, log k3 = 1.55 ± 0.5, and log k4 = 1.13 ± 0.2. ΔH1 is estd. to be -5 ±2 kcal./mol. from detns. at other temps. Log k1 is the same as for the assocn. const. (log k = 2.8 ± 0.2) of Cd with the I groups in bovine serum albumin.
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      (c) Mickel, B. L.; Andrews, A. C. Stability of the Histamine Chelates1. J. Am. Chem. Soc. 1955, 77, 5291– 5292,  DOI: 10.1021/ja01625a025
      41c
      Stability of the histamine chelates
      Mickel, B. L.; Andrews, A. C.
      Journal of the American Chemical Society (1955), 77 (), 5291-2CODEN: JACSAT; ISSN:0002-7863.
      cf. C.A. 49, 6018e. The sep. contributions to chelate stability of the imidazole and amino N atoms and the probable structures of histamine chelates are discussed in terms of some new consecutive formation consts. for reactions of histamine and related compds. with various cations.
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      (d) Koltun, W. L.; Dexter, R. N.; Clark, R. E.; Gurd, F. R. N. Coördination Complexes and Catalytic Properties of Proteins and Related Substances. I. Effect of Cupric and Zinc Ions on the Hydrolysis of p-Nitrophenyl Acetate by Imidazole. J. Am. Chem. Soc. 1958, 80, 4188– 4194,  DOI: 10.1021/ja01549a018
      41d
      Co.ovrddot.ordination complexes and catalytic properties of proteins and related substances. I. Effect of cupric and zinc ions on the hydrolysis of p-nitrophenyl acetate by imidazole
      Koltun, Walter L.; Dexter, Richard N.; Clark, Richard E.; Gurd, Frank R. N.
      Journal of the American Chemical Society (1958), 80 (), 4188-94CODEN: JACSAT; ISSN:0002-7863.
      The advantages of combining different methods, particularly kinetic and equil. techniques, for assessing the reactivity of polar groups in proteins were described. The model system contg. cupric and Zn chlorides, imidazole and imidazolium chloride were studied simultaneously by 2 independent techniques that measure the concn. of free basic imidazole quantitatively. The 1st technique depended on the catalysis by imidazole of the hydrolysis of p-nitrophenyl acetate (NPA); the 2nd technique involved measurement of the pH of the soln. Over a wide range of conditions the 2 techniques of measurement did not interfere with one another. The p-nitrophenolate ion released on hydrolysis of NPA combined with Zn and cupric ions about 100 times less strongly than did imidazole. Previous observations on the rate of decompn. of N-acetylimidazole were confirmed, and Zn ions were shown to have no detectable effect on the rate of decompn. The method of Scatchard was applied to the computation of the successive assocn. consts. in the Cu(II)- and Zn(II)-imidazole systems, with results that differed only slightly from those reported previously.
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      (e) Li, N. C.; White, J. M.; Doody, E. Cadmium and Copper Complexes of Imidazole and 1-Methylimidazole. J. Am. Chem. Soc. 1954, 76, 6219– 6223,  DOI: 10.1021/ja01653a002
      41e
      Cadmium and copper complexes of imidazole and 1-methylimidazole
      Li, Norman C.; White, James M.; Doody, Edward
      Journal of the American Chemical Society (1954), 76 (), 6219-23CODEN: JACSAT; ISSN:0002-7863.
      The formation consts. of Cd complexes of imidazole were detd. in water at 25° and in 18.8% EtOH at 0, 25, and 35°, and for Cu imidazole complexes at 25° in aq. medium. The solvent effect of EtOH on complex formation is negligible. Consts. for Cu and Cd 1-methylimidazole complexes were also detd. The results indicate that the site of binding on the imidazole mol. is the pyridine nitrogen rather than the pyrrole nitrogen.
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      (f) Martin, R. B.; Edsall, J. T. The Combination of Manganous and Cobaltous Ions with Imidazole. J. Am. Chem. Soc. 1958, 80, 5033– 5035,  DOI: 10.1021/ja01552a007
      41f
      Combination of manganous and cobaltous ions with imidazole
      Martin, R. Bruce; Edsall, John T.
      Journal of the American Chemical Society (1958), 80 (), 5033-5CODEN: JACSAT; ISSN:0002-7863.
      cf. Nozaki, et al., C.A. 51, 12887e. Imidazole forms weak complexes with Mn(II) ion, log k1 = 1.65, log k2 = 1.25; and with Co(II) ion, log k1 = 2.42, log k2 = 1.95, log k3 = 1.58, log k4 = 1.2, all at 25° and ionic strength 0.16. The weakness of the binding does not permit a detn. of the higher assocn. consts. at this ionic strength. A correlation is described between the binding of a series of metal ions with NH3 and with imidazole.
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      (g) Edsall, J. T.; Felsenfeld, G.; Goodman, D. S.; Gurd, F. R. N. The Association of Imidazole with the Ions of Zinc and Cupric Copper. J. Am. Chem. Soc. 1954, 76, 3054– 3061,  DOI: 10.1021/ja01640a068
      41g
      Association of imidazole with the ions of zinc and cupric copper
      Edsall, John T.; Felsenfeld, Gary; Goodman, DeWitt S.; Gurd, Frank R. N.
      Journal of the American Chemical Society (1954), 76 (), 3054-61CODEN: JACSAT; ISSN:0002-7863.
      The interaction of imidazole with Zn++ and Cu++ was detd. by pH measurements on solns. contg. imidazole, imidazolium ion, and either Cu++ or Zn++, in solns. contg. nitrate ion at ionic strength 0.16. In both cases the co.ovrddot.ordination no. of the ion for imidazole was 4. The successive logarithmic intrinsic assocn. consts. at 4.5° were for Cu++: 4.00, 3.63, 3.27, and 2.90; and for Zn++: 2.16, 2.32, 2.53, and 2.80; at 22.5° for Cu++: 3.76, 3.39, 3.03, and 2.66; at 24° for Zn++: 1.98, 2.19, 2.41, and 2.62. For the Cu-imidazole interaction, it was estd. that the standard heats of reaction for each successive step are approx. equal and neg.; under the conditions of this study ΔH° equals roughly -2200 cal./ mole and the successive standard entropy changes of roughly 13, 10, 6, and 3 cal. deg.-1 mole-1, resp. Cu-imidazole complexes showed marked absorption in the visible between 590 and 690 mμ and in the ultraviolet between 230 and 290 mμ. Absorption spectra of the Cu-histidine complex indicated that the imidazole residues of histidine were involved in the chelate linkages formed.
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      (h) Li, N. C.; Chu, T. L.; Fujii, C. T.; White, J. M. Association of Imidazole with Nickel(II) and Alkaline Earth Ions. J. Am. Chem. Soc. 1955, 77, 859– 861,  DOI: 10.1021/ja01609a010
      41h
      Association of imidazole with nickel(II) and alkaline earth ions
      Li, Norman C.; Chu, Ting Li; Fujii, Charles T.; White, James M.
      Journal of the American Chemical Society (1955), 77 (), 859-61CODEN: JACSAT; ISSN:0002-7863.
      The interaction of Ni++ with imidazole (I) was detd. by polarographic and pH measurements at ionic strength 0.15. The co.ovrddot.ordination no. of the Ni was 6, the structure being octahedral and the bonds essentially ionic. At 25° the successive logarithmic formation consts. were 3.27, 2.68, 2.15, 1.65, 1.12, and 0.52. The molar extinction coeff. of NiI++ is about 3.3 l. mole-1 cm.-1 at 650 mμ, the same as that for NiNH3++ at the same wave length. Ba and Ca ions have only slight tendency to complex with I at 25 and 35°.
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    42. 42
      (a) Duan, W.; Satoh, K.; Sawada, K. Stability and Structure of Ethylenedinitrilopoly(methylphosphonate) Complexes of the Divalent Transition Metal Ions in Aqueous Solution. Bull. Chem. Soc. Jpn. 2001, 74, 487– 493,  DOI: 10.1246/bcsj.74.487
      42a
      Stability and structure of ethylenedinitrilopoly(methylphosphonate) complexes of the divalent transition metal ions in aqueous solution
      Duan, Wubiao; Satoh, Keiichi; Sawada, Kiyoshi
      Bulletin of the Chemical Society of Japan (2001), 74 (3), 487-493CODEN: BCSJA8; ISSN:0009-2673. (Chemical Society of Japan)
      The formation and protonation of the complexes of a series of ethylenedinitrilopoly(methylphosphonic acids) (EDMP) [(Me)2-p(H2O3PCH2)pNC2H4N(Me)2-q(CH2PO3H2)q, p = 0-2, q = 1-2] with divalent transition metal ions (M = Mn2+, Co2+, Ni2+, Cu2+, Zn2+ or Cd2+) were investigated by means of potentiometry and 31P NMR spectroscopy at 25.0 °C. The complex formation consts. and protonation consts. of these complexes were detd. by pH titrn. The 31P NMR spectra of zinc and cadmium complexes were measured as a function of the pH, and the 31P NMR chem. shifts of each chem. species were evaluated by using the equil. consts. detd. by pH titrn. The results for EDMP complexes were compared with those of aminopoly(methylphosphonate) (NMP) [(CH3)3-rN(CH2PO3H2)r, r = 1 - 3] complexes and alk. earth metal EDMP complexes. The stability consts. of the metal complexes increase upon increasing the no. of the phosphonate groups and are around the same between the EDMP and NMP complexes of a given metal ion having the same total no. of coordinating atoms. The stabilities of the edtmp (p = q = 2) complexes are around the same as those of the medtmp (p = 1, q = 2) complexes. These results reveal the structures in which two nitrogen atoms of the ethylenedinitrilopoly(methylphosphonate) coordinate to the transition metal ion in any complex. These structures are different from those of the corresponding alk. earth metal complexes. The structures of the protonated complexes were estd. from the results of the protonation consts. and the 31P chem. shifts of the complexes.
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      (b) Irving, H.; Williams, R. J. P. 637. The Stability of Transition-Metal Complexes. J. Chem. Soc. 1953, 3192– 3210,  DOI: 10.1039/jr9530003192
      42b
      Stability of transition metal complexes
      Irving, H. M.; Williams, R. J. P.
      Journal of the Chemical Society (1953), (), 3192-3210CODEN: JCSOA9; ISSN:0368-1769.
      The published values of the stability consts. for the bivalent ions of Mn, Fe, Co, Ni, Cu, and Zn with ligands exhibiting NN, NO, and OO co.ovrddot.ordinating patterns are tabulated. In almost all cases the order of stability is Mn < Fe < Co < Ni < Cu > Zn irrespective of the nature of the ligand. This order is a consequence of the monotonic change in the 2nd ionization potentials and the reciprocal radii, the parameters serving as a guide to the magnitude of the covalent and electrostatic interactions, resp., of the ions involved. When other ions are inserted in the order of stability, the new order varies with the ligand because there is not generally the same relation for both factors throughout the series. Characteristic co.ovrddot.ordination no., stereochem. considerations, and entropy factors may, however, affect the series given here. Examples of the former two effects are the low value for k3 in the Cu++-(NH2CH2)2 system, the high values for the consts. for the Fe++-ο-phenanthroline system, and the low values for k2 in systems with (MeNHCH2)2. The entropy effect is such that the predominant factor, the entropy of hydration of M++, follows the order of stabilization and hence will not be a factor in exceptions.
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    43. 43
      Walton, T.; Szostak, J. W. A Highly Reactive Imidazolium-Bridged Dinucleotide Intermediate in Nonenzymatic RNA Primer Extension. J. Am. Chem. Soc. 2016, 138, 11996– 12002,  DOI: 10.1021/jacs.6b07977
      43
      A Highly Reactive Imidazolium-Bridged Dinucleotide Intermediate in Nonenzymatic RNA Primer Extension
      Walton, Travis; Szostak, Jack W.
      Journal of the American Chemical Society (2016), 138 (36), 11996-12002CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Because of its importance for the origin of life, the nonenzymic copying of RNA templates has been the subject of intense study for several decades. Previous characterizations of template-directed primer extension using 5'-phosphoryl-2-methylimidazole-activated nucleotides (2-MeImpNs) as substrates have assumed a classical in-line nucleophilic substitution mechanism, in which the 3'-hydroxyl of the primer attacks the phosphate of the incoming monomer, displacing the 2-methylimidazole leaving group. However, we have found that the initial rate of primer extension depends on the pH and concn. at which the activated monomer is maintained prior to the primer extension reaction. These and other results suggest an alternative mechanism, in which two monomers react with each other to form an imidazolium-bridged dinucleotide intermediate, which then binds to the template. Subsequent attack of the 3'-hydroxyl of the primer displaces an activated nucleotide as the leaving group and results in extension of the primer by one nucleotide. Anal. of monomer solns. by NMR indicates formation of the proposed imidazolium-bridged dinucleotide in the expected pH-dependent manner. We have used synthetic methods to prep. material that is enriched in this proposed intermediate and show that it is a highly reactive substrate for primer extension. The formation of an imidazolium-bridged dinucleotide intermediate provides a mechanistic interpretation of previously obsd. catalysis by an activated nucleotide located downstream from the site of primer extension.
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    44. 44
      (a) Pinheiro, V. B.; Taylor, A. I.; Cozens, C.; Abramov, M.; Renders, M.; Zhang, S.; Chaput, J. C.; Wengel, J.; Peak-Chew, S. -Y.; McLaughlin, S. H.; Herdewijn, P.; Holliger, P. Synthetic Genetic Polymers Capable of Heredity and Evolution. Science 2012, 336, 341– 344,  DOI: 10.1126/science.1217622
      44a
      Synthetic Genetic Polymers Capable of Heredity and Evolution
      Pinheiro, Vitor B.; Taylor, Alexander I.; Cozens, Christopher; Abramov, Mikhail; Renders, Marleen; Zhang, Su; Chaput, John C.; Wengel, Jesper; Peak-Chew, Sew-Yeu; McLaughlin, Stephen H.; Herdewijn, Piet; Holliger, Philipp
      Science (Washington, DC, United States) (2012), 336 (6079), 341-344CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Genetic information storage and processing rely on just two polymers, DNA and RNA, yet whether their role reflects evolutionary history or fundamental functional constraints is currently unknown. With the use of polymerase evolution and design, we show that genetic information can be stored in and recovered from six alternative genetic polymers based on simple nucleic acid architectures not found in nature [xeno-nucleic acids (XNAs)]. We also select XNA aptamers, which bind their targets with high affinity and specificity, demonstrating that beyond heredity, specific XNAs have the capacity for Darwinian evolution and folding into defined structures. Thus, heredity and evolution, two hallmarks of life, are not limited to DNA and RNA but are likely to be emergent properties of polymers capable of information storage.
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      (b) Yu, H.; Zhang, S.; Chaput, J. C. Darwinian Evolution of an Alternative Genetic System Provides Support for TNA as an RNA Progenitor. Nat. Chem. 2012, 4, 183– 187,  DOI: 10.1038/nchem.1241
      44b
      Darwinian evolution of an alternative genetic system provides support for TNA as an RNA progenitor
      Yu, Hanyang; Zhang, Su; Chaput, John C.
      Nature Chemistry (2012), 4 (3), 183-187CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)
      The pre-RNA world hypothesis postulates that RNA was preceded in the evolution of life by a simpler genetic material, but it is not known if such systems can fold into structures capable of eliciting a desired function. Presumably, whatever chem. gave rise to RNA would have produced other RNA analogs, some of which may have preceded or competed directly with RNA. Threose nucleic acid (TNA), a potentially natural deriv. of RNA, has received considerable interest as a possible RNA progenitor due to its chem. simplicity and ability to exchange genetic information with itself and RNA. Here, the authors applied Darwinian evolution methods to evolve, in vitro, a TNA receptor that binds to an arbitrary target with high affinity and specificity. This demonstration showed that TNA has the ability to fold into tertiary structures with sophisticated chem. functions, which provides evidence that TNA could have served as an ancestral genetic system during an early stage of life.
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      (c) Houlihan, G.; Arangundy-Franklin, S.; Holliger, P. Exploring the Chemistry of Genetic Information Storage and Propagation through Polymerase Engineering. Acc. Chem. Res. 2017, 50, 1079– 1087,  DOI: 10.1021/acs.accounts.7b00056
      44c
      Exploring the Chemistry of Genetic Information Storage and Propagation through Polymerase Engineering
      Houlihan, Gillian; Arangundy-Franklin, Sebastian; Holliger, Philipp
      Accounts of Chemical Research (2017), 50 (4), 1079-1087CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
      A review. Nucleic acids are a distinct form of sequence-defined biopolymer. What sets them apart from other sequence-defined biopolymers such as polypeptides or polysaccharides is their unique capacity to encode, store, and propagate genetic information (mol. heredity). In Nature, just 2 closely related nucleic acids, DNA and RNA, function as repositories and carriers of genetic information. They therefore are the mol. embodiment of biol. information. This naturally leads to questions regarding to what degree variations from this seemingly ideal "Goldilocks" chem. of DNA and RNA would still be compatible with the fundamental property of mol. heredity. To address this question, chemists have created a panoply of synthetic nucleic acids comprising unnatural sugar ring congeners, backbone linkages, and nucleobases in order to establish the mol. parameters for encoding genetic information and its emergence at the origin of life. A deeper anal. of the potential of these synthetic genetic polymers for mol. heredity requires a means of replication and a detn. of the fidelity of information transfer. While nonenzymic synthesis is an increasingly powerful method, it currently remains restricted to short polymers. Here, we discuss efforts toward establishing enzymic synthesis, replication, and evolution of synthetic genetic polymers through the engineering of polymerase enzymes found in Nature. To endow natural polymerases with the ability to efficiently utilize noncognate nucleotide substrates, novel strategies for the screening and directed evolution of polymerase function have been realized. High throughput plate-based screens, phage display and water-in-oil emulsion technol. based methods have yielded a no. of polymerases, some of which can synthesize and reverse transcribe synthetic genetic polymers with good efficiency and fidelity. The inception of such polymerases demonstrates that, at a basic level at least, mol. heredity is not restricted to the natural nucleic acids, DNA and RNA, but may be found in a large (if likely finite) no. of synthetic genetic polymers, and has opened up these novel sequence spaces for exploration. Although, largely unexplored, the 1st tentative forays have yielded ligands (aptamers) against a range of different targets and several catalysts elaborated in a range of distinct chemistries. Finally, taking the lead from established DNA designs, simple polyhedron nanostructures have been described. We anticipate that further progress in this area will expand the range of synthetic genetic polymers that can be synthesized, replicated, and evolved providing access to a rich sequence, structure and phenotypic space. "Synthetic genetics", i.e., the exploration of these spaces will illuminate the chem. parameter range for encoding and decoding information, 3-dimensional folding and catalysis, and yield novel ligands, catalysts, and nanostructures and devices for applications in biotechnol. and medicine.
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      (d) Mei, H.; Liao, J. -Y.; Jimenez, R. M.; Wang, Y.; Bala, S.; McCloskey, C.; Switzer, C.; Chaput, J. C. Synthesis and Evolution of a Threose Nucleic Acid Aptamer Bearing 7-Deaza-7-Substituted Guanosine Residues. J. Am. Chem. Soc. 2018, 140, 5706– 5713,  DOI: 10.1021/jacs.7b13031
      44d
      Synthesis and evolution of a threose nucleic acid aptamer bearing 7-Deaza-7-Substituted guanosine residues
      Mei, Hui; Liao, Jen-Yu; Jimenez, Randi M.; Wang, Yajun; Bala, Saikat; McCloskey, Cailen; Switzer, Christopher; Chaput, John C.
      Journal of the American Chemical Society (2018), 140 (17), 5706-5713CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      In vitro selection expts. carried out on artificial genetic polymers require robust and faithful methods for copying genetic information back and forth between DNA and xeno-nucleic acids (XNA). Previously, we have shown that Kod-RI, an engineered polymerase developed to transcribe DNA templates into threose nucleic acid (TNA), can function with high fidelity in the absence of manganese ions. However, the transcriptional efficiency of this enzyme diminishes greatly when individual templates are replaced with libraries of DNA sequences, indicating that manganese ions are still required for in vitro selection. Unfortunately, the presence of manganese ions in the transcription mixt. leads to the misincorporation of tGTP nucleotides opposite dG residues in the templating strand, which are detected as G-to-C transversions when the TNA is reverse transcribed back into DNA. Here we report the synthesis and fidelity of TNA replication using 7-deaza-7-modified guanosine base analogs in the DNA template and incoming TNA nucleoside triphosphate. Our findings reveal that tGTP misincorporation occurs via a Hoogsteen base pair in which the incoming tGTP residue adopts a syn conformation with respect to the sugar. Substitution of tGTP for 7-deaza-7-Ph tGTP enabled the synthesis of TNA polymers with >99% overall fidelity. A TNA library contg. the 7-deaza-7-Ph guanine analog was used to evolve a biol. stable TNA aptamer that binds to HIV reverse transcriptase with low nanomolar affinity.
      >> More from SciFinder ®
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      (e) Rangel, A. E.; Chen, Z.; Ayele, T. M.; Heemstra, J. M. In Vitro Selection of an XNA Aptamer Capable of Small-Molecule Recognition. Nucleic Acid Res. 2018, 46, 8057– 8068,  DOI: 10.1093/nar/gky667
      There is no corresponding record for this reference.
      (f) Eremeeva, E.; Fikatas, A.; Margamuljana, L.; Abramov, M.; Schols, D.; Groaz, E.; Herdewijn, P. Highly Stable Hexitol Based XNA Aptamers Targeting the Vascular Endothelial Growth Factor. Nucleic Acid Res. 2019, 47, 4927– 4939,  DOI: 10.1093/nar/gkz252
      There is no corresponding record for this reference.
      (g) Levi-Acobas, F.; Katolik, A.; Röthlisberger, P.; Cokelaer, T.; Sarac, I.; Damha, M. J.; Leumann, C. J.; Hollenstein, M. Compatibility of 5-Ethynyl-2’F-ANA UTP with In Vitro Selection for the Generation of Base-Modified Nuclease Resistant Aptamers. Org. Biomol. Chem. 2019, 17, 8083– 8087,  DOI: 10.1039/C9OB01515A
      44g
      Compatibility of 5-ethynyl-2'F-ANA UTP with in vitro selection for the generation of base-modified, nuclease resistant aptamers
      Levi-Acobas, Fabienne; Katolik, Adam; Rothlisberger, Pascal; Cokelaer, Thomas; Sarac, Ivo; Damha, Masad J.; Leumann, Christian J.; Hollenstein, Marcel
      Organic & Biomolecular Chemistry (2019), 17 (35), 8083-8087CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      A modified nucleoside triphosphate bearing two modifications based on a 2'-deoxy-2'-fluoro-arabinofuranose sugar and a uracil nucleobase equipped with a C5-ethynyl moiety (5-ethynyl-2'F-ANA UTP) was synthesized. This nucleotide analog could enzymically be incorporated into DNA oligonucleotides by primer extension and reverse transcribed to unmodified DNA. This nucleotide could be used in SELEX for the identification of high binding affinity and nuclease resistant aptamers.
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