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Nucleic Acids Research Advance Access originally published online on June 12, 2007
Nucleic Acids Research 2007 35(12):4186-4194; doi:10.1093/nar/gkm438
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Nucleic Acids Research, 2007, Vol. 35, No. 12 4186-4194
© 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

In vitro selection of a 5'-purine ribonucleotide transferase ribozyme

Taek Jin Kang1 and Hiroaki Suga1,2,*

1Research Center for Advanced Science and Technology, The University of Tokyo, 153-8904 Tokyo, Japan and 2Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 113-8656 Tokyo, Japan

*To whom correspondence should be addressed. Tel: +81-3-5452-5495; Fax: +81-3-5452-5495; Email: hsuga{at}rcast.u-tokyo.ac.jp.

Received March 13, 2007. Revised May 16, 2007. Accepted May 16, 2007.

Here we report in vitro selection of a novel ribozyme that catalyzes the 5'-nucleotidyl transfer reaction forming the 2'–5' phosphodiester bond. This ribozyme was retrieved as a sole sequence in the pool enriched for the 5'-triphosphate-dependent activities in incorporating ATP-{gamma}S. The originally selected ribozyme consisting of 109-nucleotide (nt) was miniaturized to 45-nt M4 ribozyme via a series of mutation studies, and based on this mini-ribozyme a trans-acting system was constructed. One of the most challenging tasks in our study was to determine the chemistry occurring at the 5'-ppp site. We utilized various analytical methods including MALDI-TOF analysis of the product generated by the trans-acting system and elucidated the chemistry to be 3'->5' mononucleotide extension forming the 2'–5' phosphodiester bond. Interestingly, M4 ribozyme promiscuously accepts a variety of purine nucleotides bearing 5'-mono-, di- and triphosphates as substrates. This remarkable ability of M4 ribozyme would lead us to the development of a new tool for the 5'-modification of RNAs with unique chemical groups.


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