| Abstract
| - Site-specific fluorescent labeling of RNA molecules was achieved by specific transcription usingan 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 ofy 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 chemicallysynthesized from the nucleoside of y. These fluorescent substrates were site-specifically incorporated intoRNA by transcription mediated by the s−y and v−y pairs. By using this fluorescent labeling method, specificpositions of Raf-binding and theophylline-binding RNA aptamers were fluorescently labeled, and the specificbinding to their target molecules was detected by their fluorescent intensities. This site-specific labelingmethod using an unnatural base pair system will be useful for analyzing conformational changes of RNAmolecules and for detecting interactions between RNA and its binding species.
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