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

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Title: New NTP analogs: the synthesis of 4'-thioUTP and 4'-thioCTP and their utility for SELEX
Authors: Kato, Yuka Browse this author
Minakawa, Noriaki Browse this author
Komatsu, Yasuo Browse this author
Kamiya, Hiroyuki Browse this author
Ogawa, Naoki Browse this author
Harashima, Hideyoshi Browse this author →KAKEN DB
Matsuda, Akira Browse this author →KAKEN DB
Issue Date: May-2005
Publisher: Oxford University Press
Journal Title: Nucleic Acids Research
Volume: 33
Issue: 9
Start Page: 2942
End Page: 2951
Publisher DOI: 10.1093/nar/gki578
PMID: 15914669
Abstract: 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'-NH_{2}dUTP and 2'-NH_{2}dCTP; 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 α-thrombin using thioUTP and thioCTP, and found a thioRNA aptamer with high binding affinity (Kd = 4.7 nM).
Description: Nucleic Acids Research (c) Oxford University Press 2004. All rights reserved.
Type: article
Appears in Collections:薬学研究院 (Faculty of Pharmaceutical Sciences) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 南川 典昭

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