In vitro selection of functional RNAs using 4'-thioNTP

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，T7 RNA聚合酶掺入4 '-thioUTP和4'-thioCTP得到4 '-thioRNA（thioRNA）的效果上级两组常用的修饰NTP类似物（2'-NH_2dUTP和2 '-NH_2dCTP，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。

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).