site-specific incorporation of functional components into rna by an unnatural base pair transcription system特定站点的一个不自然的碱基对转录成rna功能组件的系统.pdf

Molecules 2012, 17, 2855-2876; doi:10.3390/molecule OPEN ACCESS molecules ISSN 1420-3049 /journal/molecules Article Site-Specific Incorporation of Functional Components into RNA by an Unnatural Base Pair Transcription System Nobuyuki Morohashi 1, Michiko Kimoto 1,2, Akira Sato 2, Rie Kawai 1,2 and Ichiro Hirao 1,2,* 1 TagCyx Biotechnologies, 1-6-126 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan; E-Mails: morohash@chem.meisei-u.ac.jp (N.M.); michikoh@riken.jp (M.K.); rkawai@you.jp (R.K.) 2 RIKEN Systems and Structural Biology Center (SSBC), 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan; E-Mail: asato@riken.jp * Author to whom correspondence should be addressed; E-Mail: ihirao@riken.jp; Tel.: +81-45-503-9644; Fax: +81-45-503-9645. Received: 16 December 2011; in revised form: 22 February 2012 / Accepted: 24 February 2012 / Published: 7 March 2012 Abstract: Toward the expansion of the genetic alphabet, an unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) functions as a third base pair in replication and transcription, and provides a useful tool for the site-specific, enzymatic incorporation of functional components into nucleic acids. We have synthesized several modified-Pa substrates, such as alkylamino-, biotin-, TAMRA-, FAM-, and digoxigenin-linked PaTPs, and examined their transcription by T7 RNA polymerase using Ds-containing DNA templates with various sequenc