Basic Studies of Molecular Recognition by 4'-Thionucleic acids

4-硫代核酸分子识别的基础研究

• 批准号: 15209003
• 负责人: MATSUDA Akira
• 金额: $ 26.46万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15209003/
• 关键词: 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

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

我们已经制备了4 '-硫代核糖核苷亚磷酰胺，用于化学合成4'-硫代RNA（thioRNA）。制备完全修饰的硫代RNA。通过UV熔解测量确定双链体的Tm值。所得双链体的热稳定性的总体顺序为：thioRNA：thioRNA>>thioRNA：RNA>RNA：RNA>RNA：DNA>thioRNA：DNA。thioRNA在人血清中的稳定性是天然RNA的600倍。在正常条件下，RNA和thiRNA双链体均不被消化。合成了4 '-硫代尿苷和4'-硫代胞苷的三磷酸盐、thioUTP和thioCTP，并将其用于SELEX。由于SELEX需要体外转录和逆转录，我们通过关注这两个步骤来检查thioUTP和thioCTP用于SELEX的能力。通过T7 RNA聚合酶掺入thioUTP和thioCTP以产生thioRNA进行良好，并且优于用于SELEX的两组经常使用的修饰的NTP类似物（2 '-NH_2）的那些上级。 ...更多信息 2dUTP和2’-NH_2dCTP，以及2’-FdUTP和2’-FdCTP）。此外，在2 '-脱氧核苷三磷酸（dNTPs）存在下，将所得硫代RNA逆转录成互补DNA也顺利且精确地进行。硫代RNA对RNase A的稳定性是相应天然RNA的50倍。在此基础上，我们尝试筛选与人α-凝血酶具有高亲和力（Kd=4.6 nM）的thioRNA适体，并将4 '-硫代核糖核苷通过自由基还原成2'-脱氧-4 '-硫代核糖核苷（2'-溴代衍生物），然后进一步转化为亚磷酰胺单元和5 '-三磷酸。还研究了硫代DNA的热稳定性和核酸酶稳定性。发现2 '-脱氧-4'-硫代核糖核苷5 '-三磷酸是DNA聚合酶如Therminator和KOD dash的良好底物。少

项目成果

Minakawa, N., Kato, Y., Uetake, K., Kaga, D., Matsuda, A.: "An improved large scale synthesis of 1,4-anhydro-4-thio-D-ribitol"Tetrahedron. 59. 1699-1702 (2003)

Minakawa, N.、Kato, Y.、Uetake, K.、Kaga, D.、Matsuda, A.：“1,4-脱水-4-硫代-D-核糖醇的改进大规模合成”四面体。

RNA interference induced by siRNA modified with 4'-thioribonucleosides in cultured niamnmalian cells.

在培养的 niamnmalian 细胞中，用 4-硫代核糖核苷修饰的 siRNA 诱导 RNA 干扰。

• 发表时间: 2005
• 期刊: FEBS Lett. 579
• 作者: Hoshika;S.;et al.
• 通讯作者: et al.

RNA interference induced by siRNAs modified with 4'-thioribonucleosides.

由 4-硫代核糖核苷修饰的 siRNA 诱导的 RNA 干扰。

• 发表时间: 2005
• 期刊: Nucleic Acids Sympo. Ser. 49
• 作者: Hoshika;S.;et al.
• 通讯作者: et al.

Frontiers in Organic Chemistry

有机化学前沿

• 发表时间: 2005
• 作者: Minakawa;N.;et al.
• 通讯作者: et al.

Synthesis of 2'-C-methyl-4'-thiocytidine : Unexpected anomerization of the 2'-keto-4'-thionucleoside precursor.

2-C-甲基-4-硫代胞苷的合成：2-酮-4-硫代核苷前体发生意外的异构化。

• 发表时间: 2005
• 期刊: Nucleosides, Nucleotides Nucleic Acids 24
• 作者: Kaga;D.;et al.
• 通讯作者: et al.