In vitro selection of functional RNAs using 4'-thioNTP

使用 4-thioNTP 体外选择功能性 RNA

• 批准号: 15510169
• 负责人: MINAKAWA Noriaki
• 金额: $ 2.43万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15510169/
• 关键词: nucleoside; nucleotide; 4'-thioUTP; 4'-thioCTP; in vitro selection; RNA; aptamer; thrombin; 4'-thioRNA

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).

体外选择（或SELEX）是一种生物技术方法，用于从随机的RNA组合文库中分离功能性RNA分子，如核酶和适体。这项工作的目的是开发新的NTP类似物用于体外选择。合成了4 ′-硫代核糖核苷的三磷酸，得到4 ′-硫代UTP、-CTP、-ATP、-GTP。当选择了合适的DNA模板前导序列时，通过T7 RNA聚合酶掺入4 '-thioUTP和4'-thioCTP得到4 '-thioRNA（thioRNA）进行良好，并且上级优于两组常用的用于体外选择的修饰的NTP类似物（2'-NH_2dUTP和2 '-NH_2dCTP，以及2'-FdUTP和2 '-FdCTP）。我揭示了约+15 DNA模板的前导序列对于通过T7 RNA聚合酶有效掺入修饰的NTP类似物是重要的。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，本论文尝试了用4 '-thioUTP和4'-thioCTP筛选与人α-凝血酶结合的thioRNA适体，发现了一个与人α-凝血酶具有高亲和力（Kd =4.7nM）的适体。

项目成果

4'-Thionucleic Acids : Chemistry, Properties, and Applications for Developing Functional Oligonucleotides.

4-硫代核酸：化学、性质和开发功能性寡核苷酸的应用。

• 期刊: Frontiers in Organic Chemistry (in press)
• 作者: Kato;Y. et al.;N.Minakawa et al.;S.Hoshika et al.;N.Minakawa et al.
• 通讯作者: N.Minakawa et al.

N.Minakawa et al.: "New Base Paring Motifs. The Synthesis and Thermal Stability of Oligodeoxynucleotides Containing Imidazopyridopyrimidine Nucleosides with the Ability to Form Four Hydrogen Bonds"J.Am.Chem.Soc.. 125. 9970-9982 (2003)

N.Minakawa 等：“新碱基配对基序。含有能够形成四个氢键的咪唑并吡啶并嘧啶核苷的寡脱氧核苷酸的合成和热稳定性”J.Am.Chem.Soc.. 125. 9970-9982 (2003)

In vitro selection of human α-thrombin RNA aptamer using 4'-thioUTP and 4'-thioCTP

使用 4-thioUTP 和 4-thioCTP 体外选择人 α-凝血酶 RNA 适体

• 发表时间: 2004
• 期刊: Nucleic Acids Res.Suppl. 48
• 作者: N.Minakawa et al.;Y.Kato et al.;S.Hoshika et al.;Y.Kato et al.
• 通讯作者: Y.Kato et al.

Investigation of physical and physiological properties of 4'-thioribonucleotide (4'-thioRNA).

4-硫代核糖核苷酸 (4-thioRNA) 的物理和生理特性的研究。

• 发表时间: 2003
• 期刊: Nucleic Acids Res.Suppl. 3
• 作者: Kato;Y. et al.;N.Minakawa et al.;S.Hoshika et al.
• 通讯作者: S.Hoshika et al.

N.Minakawa et al.: "A Versatile Modification of On-Column Oligodeoxynucleotides Using a Copper-Catalyzed Oxidative Acetylenic Coupling Reaction"J.Am.Chem.Soc.. 125. 11545-11525 (2003)

N.Minakawa 等人：“使用铜催化氧化乙炔偶联反应对柱上寡脱氧核苷酸进行多功能修饰”J.Am.Chem.Soc.. 125. 11545-11525 (2003)