Fluorescent probing for RNA molecules by an unnatural base-pair system.
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.
Kimoto
Michiko
M
Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
Mitsui
Tsuneo
T
Harada
Yoko
Y
Sato
Akira
A
Yokoyama
Shigeyuki
S
Hirao
Ichiro
I
D020029:Base Pairing; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D011687:Purines / Q000737:chemistry*; D011758:Pyrroles / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*; D012343:RNA, Transfer / Q000737:chemistry; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism
C435686:2-amino-6-(2-thienyl)purine; D005456:Fluorescent Dyes; D011687:Purines; D011758:Pyrroles; D014764:Viral Proteins; C051266:pyrrole-2-carboxaldehyde; D012313:RNA; D012343:RNA, Transfer; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases
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