In vitro selection of functional RNAs using 4'-thioNTP

体外选择（SELEX）是一种生物技术方法，用于从随机的RNA组合文库中分离出功能性RNA分子，如核酶和适体。这项工作的目的是开发新的NTP类似物体外选择。合成了4′-硫核糖核苷的三磷酸，得到了4′-硫核糖核苷、-CTP、-ATP、-GTP。T7 RNA聚合酶将4'-thioUTP和4'-thioCTP结合得到4'-thioRNA (thioRNA)，当选择合适的DNA模板先导序列时，其效果优于常用的两组修饰NTP类似物（2'-NH_2dUTP和2'-NH_2dCTP，以及2'-FdUTP和2'-FdCTP）。我发现大约+15个DNA模板的先导序列对于T7 RNA聚合酶有效结合修饰的NTP类似物是重要的。此外，在2'-脱氧核苷三磷酸（dNTPs）存在的情况下，所得到的硫核糖核酸（thioRNA）逆转录到互补DNA中也顺利而精确地进行。thioRNA对RNase A的稳定性是相应天然RNA的50倍。有了这些成功的结果，我尝试用4'-thioUTP和4'-thioCTP选择人α-凝血酶的thioRNA适配体，并找到了一个高结合亲和力（K_d=4.7nM）的thioRNA适配体。

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).