Site-specific fluorescent labeling of RNA molecules by specific transcription using unnatural base pairs.
Site-specific fluorescent labeling of RNA molecules was achieved by specific transcription using an unnatural base pair system. The unnatural base pairs between 2-amino-6-(2-thienyl)purine (s) and 2-oxo(1H)pyridine (y), and 2-amino-6-(2-thiazolyl)purine (v) and y function in transcription, and the substrates of y and 5-modified y bases can be site-specifically incorporated into RNA, opposite s or v in DNA templates, by T7 RNA polymerase. Ribonucleoside 5'-triphosphates of 5-fluorophore-linked y bases were chemically synthesized from the nucleoside of y. These fluorescent substrates were site-specifically incorporated into RNA by transcription mediated by the s-y and v-y pairs. By using this fluorescent labeling method, specific positions of Raf-binding and theophylline-binding RNA aptamers were fluorescently labeled, and the specific binding to their target molecules was detected by their fluorescent intensities. This site-specific labeling method using an unnatural base pair system will be useful for analyzing conformational changes of RNA molecules and for detecting interactions between RNA and its binding species.
Kawai
Rie
R
Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
Kimoto
Michiko
M
Ikeda
Shuji
S
Mitsui
Tsuneo
T
Endo
Masayuki
M
Yokoyama
Shigeyuki
S
Hirao
Ichiro
I
D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000378:metabolism; D020029:Base Pairing; D001483:Base Sequence; D005453:Fluorescence; D008958:Models, Molecular; D012313:RNA / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D013194:Staining and Labeling / Q000379:methods*; D013806:Theophylline / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic
D052157:Aptamers, Nucleotide; D012313:RNA; D013806:Theophylline