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PMID: 17693436 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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

Nucleic acids research ·Vol. 35 ·No. 16 ·2007-00-00 ·Pages 5360-9

Kimoto M, Mitsui T, Harada Y, Sato A, Yokoyama S, Hirao I

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.

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
Chemicals
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
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Kimoto Michiko
Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
Mitsui Tsuneo
Harada Yoko
Sato Akira
Yokoyama Shigeyuki
Hirao Ichiro
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
1362-4962
DOI
10.1093/nar/gkm508
Published
2007-00-00
Epub
2007-00-09
Pages
5360-9
Language
English
Region
England
NLM ID
0411011
PMCID
PMC2018647
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/gkm508 PMC Full Text View journal details
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