In vitro selection of functional RNAs using 4'-thioNTP
| Project/Area Number |
15510169
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Living organism molecular science
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
MINAKAWA Noriaki Hokkaido Univ., Grad.School of Pharm.Sci., Asso.Prof., 大学院・薬学研究科, 助教授 (40209820)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2004: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2003: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | nucleoside / nucleotide / 4'-thioUTP / 4'-thioCTP / in vitro selection / RNA / aptamer / thrombin / 4'-thioRNA |
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| Research Abstract |
In vitro selection (or SELEX) is a biotechnological method used to isolate functional RNA molecules such as ribozymes and aptamers from a random pool of RNA combinatorial libraries. The aim of this work is to develop new NTP analogs for in vitro selection. The synthesis of the triphosphates of 4'-thioribonucleoside was achieved to give 4'-thioUTP, -CTP, -ATP, -GTP. Incorporation of 4'-thioUTP and 4'-thioCTP 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 in vitro selection (2'-NH_2dUTP and 2'-NH_2dCTP, and 2'-FdUTP and 2'-FdCTP), when an adequate leader sequence of DNA template was selected. I revealed that a leader sequence of about +15 of DNA template is important for 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, I attempted the selection of thioRNA aptamers to human α-thrombin using 4'-thioUTP and 4'-thioCTP, and found a thioRNA aptamer with high binding affinity (K_d=4.7nM).
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Report
(3 results)
Research Products
(19 results)
