Fluorescent probing for RNA molecules by an unnatural base-pair system

Nucleic Acids Res. 2007;35(16):5360-9. doi: 10.1093/nar/gkm508. Epub 2007 Aug 9.

Abstract

Fluorescent labeling of nucleic acids is widely used in basic research and medical applications. We describe the efficient site-specific incorporation of a fluorescent base analog, 2-amino-6-(2-thienyl)purine (s), into RNA by transcription mediated by an unnatural base pair between s and pyrrole-2-carbaldehyde (Pa). The ribonucleoside 5'-triphosphate of s was site-specifically incorporated into RNA, by T7 RNA polymerase, opposite Pa in DNA templates. The fluorescent intensity of s in RNA molecules changes according to the structural environment. The site-specific s labeling of RNA hairpins and tRNA molecules provided characteristic fluorescent profiles, depending on the labeling sites, temperature and Mg2+ concentration. The Pa-containing DNA templates can be amplified by PCR using 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds), another pairing partner of Pa. This site-specific fluorescent probing by the unnatural pair system including the s-Pa and Ds-Pa pairs provides a powerful tool for studying the dynamics of the local structural features of 3D RNA molecules and their intra- and intermolecular interactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Pairing
  • DNA-Directed RNA Polymerases / metabolism
  • Fluorescent Dyes / chemistry*
  • Purines / chemistry*
  • Pyrroles / chemistry*
  • RNA / chemistry*
  • RNA, Transfer / chemistry
  • Transcription, Genetic
  • Viral Proteins / metabolism

Substances

  • 2-amino-6-(2-thienyl)purine
  • Fluorescent Dyes
  • Purines
  • Pyrroles
  • Viral Proteins
  • pyrrole-2-carboxaldehyde
  • RNA
  • RNA, Transfer
  • bacteriophage T7 RNA polymerase
  • DNA-Directed RNA Polymerases