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Nucleic Acids Research 2005 33(15):e129; doi:10.1093/nar/gni128
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Published online 19 August 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Methods Online

Site-specific biotinylation of RNA molecules by transcription using unnatural base pairs

Kei Moriyama1, Michiko Kimoto2, Tsuneo Mitsui1, Shigeyuki Yokoyama2,3,4 and Ichiro Hirao1,2,*

1Research Center for Advanced Science and Technology, The University of Tokyo 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan 2Protein Research Group, RIKEN Genomic Sciences Center 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan 3Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan 4RIKEN Harima Institute at SPring-8 1-1-1 Kohto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan

*To whom correspondence should be addressed at Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan. Tel: +81 3 5452 5442; Fax: +81 3 5452 5442; Email: ihirao{at}riken.jp

Received May 16, 2005. Revised July 29, 2005. Accepted July 29, 2005.

Direct site-specific biotinylation of RNA molecules was achieved by specific transcription mediated by unnatural base pairs. Unnatural base pairs between 2-amino-6-(2-thienyl)purine (denoted by s) and 2-oxo(1H)pyridine (denoted by y), or 2-amino-6-(2-thiazolyl)purine (denoted as v) and y specifically function in T7 transcription. Using these unnatural base pairs, the substrate of biotinylated-y (Bio-yTP) was selectively incorporated into RNA, opposite s or v in the DNA templates, by T7 RNA polymerase. This method was applied to the immobilization of an RNA aptamer on sensor chips, and the aptamer accurately recognized its target protein. This direct site-specific biotinylation will provide a tool for RNA-based biotechnologies.

Correspondence may also be addressed to S. Yokoyama, Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel: +81 3 5841 4413; Fax: +81 3 5841 8057; Email: yokoyama{at}biochem.s.u-tokyo.ac.jp


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