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Nucleic Acids Research Advance Access originally published online on August 9, 2007
Nucleic Acids Research 2007 35(16):5360-5369; doi:10.1093/nar/gkm508
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Nucleic Acids Research, 2007, Vol. 35, No. 16 5360-5369
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

RNA

Fluorescent probing for RNA molecules by an unnatural base-pair system

Michiko Kimoto1, Tsuneo Mitsui1, Yoko Harada1, Akira Sato1, Shigeyuki Yokoyama1,2,3 and Ichiro Hirao1,*

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

*To whom correspondence should be addressed. Tel: +81 45 503 9644; Fax: +81 45 503 9645; Email: ihirao{at}riken.jp

Correspondence may also be addressed to Shigeyuki Yokoyama. Tel: +81 3 5841 4413; Fax: +81 3 5841 8057; Email: yokoyama{at}biochem.s.u-tokyo.ac.jp

Received May 9, 2007. Revised June 11, 2007. Accepted June 11, 2007.

Fluorescent labeling of nucleic acids is widely used in basic research and medical applications. We describe the efficient site-specific incorporation of a fluorescent base analog, 2-amino-6-(2-thienyl)purine (s), into RNA by transcription mediated by an unnatural base pair between s and pyrrole-2-carbaldehyde (Pa). The ribonucleoside 5'-triphosphate of s was site-specifically incorporated into RNA, by T7 RNA polymerase, opposite Pa in DNA templates. The fluorescent intensity of s in RNA molecules changes according to the structural environment. The site-specific s labeling of RNA hairpins and tRNA molecules provided characteristic fluorescent profiles, depending on the labeling sites, temperature and Mg2+ concentration. The Pa-containing DNA templates can be amplified by PCR using 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds), another pairing partner of Pa. This site-specific fluorescent probing by the unnatural pair system including the s-Pa and Ds-Pa pairs provides a powerful tool for studying the dynamics of the local structural features of 3D RNA molecules and their intra- and intermolecular interactions.


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