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Nucleic Acids Research 2005 33(12):e108; doi:10.1093/nar/gni108
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Published online 7 July 2005

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

A novel method of screening thrombin-inhibiting DNA aptamers using an evolution-mimicking algorithm

Kazunori Ikebukuro*, Yuji Okumura1, Koichi Sumikura2 and Isao Karube3

Department of Biotechnology and Life Science, Tokyo University of Agriculture & Technology 2-24-16 Naka-machi, Koganei, Tokyo 184-8588, Japan 1Research Center for Advanced Science and Technology, The University of Tokyo 4-6-1 Komaba, Meguro-ku Tokyo 153-8904, Japan 2National Graduate Institute for Policy Studies 7-22-1 Roppongi, Minato-ku, Tokyo 106-8677, Japan 3School of Bionics, Tokyo University of Technology 1404-1 Katakura, Hachioji, Tokyo 192-0982, Japan

*To whom correspondence should be addressed. Tel/Fax: +81 42 388 7030; Email: ikebu{at}cc.tuat.ac.jp

Received May 20, 2005. Revised June 21, 2005. Accepted June 21, 2005.

Thrombin-inhibiting DNA aptamers have already been obtained through the systematic evolution of ligands by exponential enrichment (SELEX). However, SELEX is a method that screens DNA aptamers that bind to their target molecules, and it sometimes fails to screen good inhibitors. Therefore, it is necessary to develop a method of screening DNA aptamers based on their inhibitory effects on the target molecules. We developed a novel method of detecting aptamers using an evolution-mimicking algorithm, and we applied it to the search of new aptamers which inhibit thrombin. First, we randomly designed and synthesized ten 15mer oligonucleotides presumed to form G-quartet structures, and then measured their thrombin-inhibiting activities. The aptamers showing high inhibitory activity were selected, and we shuffled and mutated those sequences in silico to generate 10 new sequences of next-generation aptamers. After repeating the cycle five times, we successfully obtained the same aptamers reported previously, and they showed high inhibitory activity. In addition, we added 8mer oligonucleotides to both the 5' and the 3' end of the selected 15mer aptamers, and then repeated the evolution in silico. After two cycles, we were able to obtain aptamers with higher inhibitory activity than that of the 15mer aptamers.


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