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pubmed:abstractText |
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.
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pubmed:affiliation |
Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
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