| Project/Area Number |
15209003
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Drug development chemistry
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| Research Institution | Hokkaido University |
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Principal Investigator |
MATSUDA Akira Hokkaido Univ., Grad.School of Pharm.Sci., Prof., 大学院・薬学研究科, 教授 (90157313)
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| Co-Investigator(Kenkyū-buntansha) |
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 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥41,340,000 (Direct Cost: ¥31,800,000、Indirect Cost: ¥9,540,000)
Fiscal Year 2005: ¥10,660,000 (Direct Cost: ¥8,200,000、Indirect Cost: ¥2,460,000)
Fiscal Year 2004: ¥11,700,000 (Direct Cost: ¥9,000,000、Indirect Cost: ¥2,700,000)
Fiscal Year 2003: ¥18,980,000 (Direct Cost: ¥14,600,000、Indirect Cost: ¥4,380,000)
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| Keywords | thermal stability / nuclease stability / 4'-thonucleoside / 4'-thioRNA / aptamer / SELEX / 4'-thioDNA / KOD dash / T7 RNA polymerase / Rnase A耐性 / 分子認識 / 逆転写酵素 / 4-チオRNA / RNase A耐性 / 4-チオ核酸 |
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| Research Abstract |
We have prepared 4'-thioribonucleosides phosphoramidites for the chemical synthesis of 4'-thioRNA (thioRNA). Full modified thioRNAs were prepared. The Tm values of duplexes were determined by UV melting measurements. The resulting overall order of thermal stabilities for the duplexes was : thioRNA:thioRNA>>thioRNA:RNA>RNA:RNA>RNA:DNA>thioRNA:DNA. The stability of thioRNA in human serum was 600 times greater than that of natural RNA. Neither the RNA nor the thioRNA duplexes were digested under normal conditions. The synthesis of the triphosphates of 4'-thiouridine and 4'-thiocytidine, thioUTP and thioCTP, and their utility for SELEX have been carried out. Since SELEX requires both in vitro and reverse transcription, we examined the ability of thioUTP and thioCTP for SELEX by focusing on the two steps. Incorporation of thioUTP and thioCTP by T7 RNA polymerase to give thioRNA proceeded well and was superior to those of the two sets of frequently used modified NTP analogs for SELEX (2'-NH_
… More
2dUTP and 2'-NH_2dCTP, and 2'-FdUTP and 2'-FdCTP), when an adequate leader sequence of DNA template was selected. 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, and found a thioRNA aptamer with high binding affinity (Kd=4.6 nM).
We converted 4'-thioribonucleosides into 2'-deoxy-4'-thioribonucleosides via radical reduction of their corresponding 2'-bromo derivatives), which were then further converted into their phosphoramidite units and their 5'-triphosphates. Thermal and nuclease stabilities of thioDNA were also studied. 2'-Deoxy-4'-thioribonucleoside 5'-triphosphates were found to be a good substrates of DNA polymerases such as Therminator and KOD dash. Less
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