title: Site-specific fluorescent labeling of RNA molecules by specific transcription using unnatural base pairs.
abstract: 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.
authors.affiliation: Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
authors.forename: Rie
authors.initials: R
authors.lastname: Kawai
authors.forename: Michiko
authors.initials: M
authors.lastname: Kimoto
authors.forename: Shuji
authors.initials: S
authors.lastname: Ikeda
authors.forename: Tsuneo
authors.initials: T
authors.lastname: Mitsui
authors.forename: Masayuki
authors.initials: M
authors.lastname: Endo
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
authors.forename: Ichiro
authors.initials: I
authors.lastname: Hirao
mesh_terms: 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
chemical_list: D052157:Aptamers, Nucleotide; D012313:RNA; D013806:Theophylline