Site-specific incorporation of fluorescent probes into RNA by specific transcription using unnatural base pairs.
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.
Kimoto
Michiko
M
Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
Kawai
Rie
R
Mitsui
Tsuneo
T
Harada
Yoko
Y
Sato
Akira
A
Yokoyama
Shigeyuki
S
Hirao
Ichiro
I
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
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