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Nucleic Acids Research 2005 33(9):2942-2951; doi:10.1093/nar/gki578
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Published online 24 May 2005

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

New NTP analogs: the synthesis of 4'-thioUTP and 4'-thioCTP and their utility for SELEX

Yuka Kato1,3, Noriaki Minakawa1, Yasuo Komatsu2, Hiroyuki Kamiya1, Naoki Ogawa3, Hideyoshi Harashima1 and Akira Matsuda1,*

1Graduate School of Pharmaceutical Sciences, Hokkaido University Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan 2Institute for Biological Resources and Functions and Fellow Research Group, National Institute of Advanced Industrial Science, Technology (AIST, Hokkaido) 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan 3GeneticLab Co., Ltd Kita-27, Nishi-6, Kita-ku, Sapporo 001-0027, Japan

*To whom correspondence should be addressed. Tel: +81 11 706 3228; Fax: +81 11 706 4980; Email: matuda{at}pharm.hokudai.ac.jp

Received April 5, 2005. Revised April 27, 2005. Accepted April 27, 2005.

The synthesis of the triphosphates of 4'-thiouridine and 4'-thiocytidine, 4'-thioUTP (7; thioUTP) and 4'-thioCTP (8; thioCTP), and their utility for SELEX (systematic evolution of ligands by exponential enrichment) is described. The new nucleoside triphosphate (NTP) analogs 7 and 8 were prepared from appropriately protected 4'-thiouridine and -cytidine derivatives using the one-pot method reported by J. Ludwig and F. Eckstein [(1989) J. Org. Chem., 54, 631–635]. Because SELEX requires both in vitro transcription and reverse transcription, we examined the ability of 7 and 8 for SELEX by focusing on the two steps. Incorporation of 7 and 8 by T7 RNA polymerase to give 4'-thioRNA (thioRNA) proceeded well and was superior to those of the two sets of frequently used modified NTP analogs for SELEX (2'-NH2dUTP and 2'-NH2dCTP; 2'-FdUTP and 2'-FdCTP), when an adequate leader sequence of DNA template was selected. We revealed that a leader sequence of about +15 of DNA template is important for the effective incorporation of modified NTP analogs by T7 RNA polymerase. In addition, reverse transcription of the resulting thioRNA into the complementary DNA in the presence of 2'-deoxynucleoside triphosphates (dNTPs) also proceeded smoothly and precisely. The stability of the thioRNA toward RNase A was 50 times greater than that of the corresponding natural RNA. With these successful results in hand, we attempted the selection of thioRNA aptamers to human {alpha}-thrombin using thioUTP and thioCTP, and found a thioRNA aptamer with high binding affinity (Kd = 4.7 nM).

Correspondence may also be addressed to Noriaki Minakawa. Tel: +81 11 706 3230; Fax: +81 11 706 4980; Email: noriaki{at}pharm.hokudai.ac.jp


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