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组合文库中分离核酶和适体等功能性RNA分子。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‘-硫代核苷的三磷酸盐，得到了4’-硫代UTP、-CTP、-ATP、-GTP。在选择合适的DNA模板前导序列的情况下，T7RNA聚合酶将4‘-thioUTP和4’-thioCTP掺入，得到4‘-thioRNA(ThioRNA)，其体外筛选效果优于2’-NH_2dUTP和2‘-NH_2dCTP，以及2’-FdUTP和2‘-FdCTP。我揭示了大约+15的DNA模板的前导序列对于T7RNA聚合酶有效地掺入修饰的NTP类似物是重要的。此外，在2‘-脱氧核苷三磷酸盐(DNTPs)存在下，硫代RNA反转录为互补DNA的过程也进行得很顺利和准确。硫代RNA对核糖核酸酶A的稳定性比相应的天然RNA高50倍。有了这些成功的结果，我尝试用4‘-硫代UTP和4’-硫代CTP筛选人α-凝血酶的硫代RNA适配子，发现了一个高结合亲和力的硫代RNA适配子(K_d=4.7 nM)。

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).