title: Site-specific incorporation of fluorescent probes into RNA by specific transcription using unnatural base pairs.
abstract: To analyze the local conformational changes of RNA molecules, we developed a site-specific fluorescent labeling method for RNA fragments by T7 transcription, using unnatural base pairs between 2-amino-6-(2-thienyl)purine (s) and 2-oxo(1H)pyridine (y) and between 2-amino-6-(2-thiazolyl)purine (v) and y. Ribonucleoside 5'-triphosphates of 5-fluorescence-linked y derivatives can be site-specifically incorporated into RNA, opposite s or v in DNA templates, by T7 RNA polymerase. Using this specific transcription, the substrate of a fluorescein-linked y was introduced into a theophylline-binding RNA aptamer. The replacement of U6 by the fluorescein-linked y maintained both the binding ability and selectivity of the aptamer to theophylline. Furthermore, the fluorescence intensity was increased upon theophylline binding, but was not changed by the addition of caffeine.
authors.affiliation: Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
authors.forename: Michiko
authors.initials: M
authors.lastname: Kimoto
authors.forename: Rie
authors.initials: R
authors.lastname: Kawai
authors.forename: Tsuneo
authors.initials: T
authors.lastname: Mitsui
authors.forename: Yoko
authors.initials: Y
authors.lastname: Harada
authors.forename: Akira
authors.initials: A
authors.lastname: Sato
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*; D020029:Base Pairing; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011728:Pyridones / Q000737:chemistry*; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D005456:Fluorescent Dyes; D011728:Pyridones; D012265:Ribonucleotides; D014764:Viral Proteins; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases