Basic Studies of Molecular Recognition by 4'-Thionucleic acids

我们制备了4'-硫代核糖核苷亚磷酰胺，用于化学合成4'-硫代RNA（thioRNA）。制备了完全修饰的thioRNA。双链体的 Tm 值通过 UV 熔化测量来确定。所得双链体热稳定性总体顺序为：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 thioRNA 双链体都不会被消化。 4'-硫尿苷和4'-硫胞苷三磷酸、thioUTP和thioCTP的合成及其在SELEX中的应用已经完成。由于SELEX需要体外转录和逆转录，因此我们通过关注这两个步骤来检查thioUTP和thioCTP对SELEX的能力。当选择足够的 DNA 模板前导序列时，T7 RNA 聚合酶掺入 thioUTP 和 thioCTP 生成 thioRNA 的过程进展顺利，并且优于 SELEX 中常用的两组修饰 NTP 类似物（2'-NH_…更多 2dUTP 和 2'-NH_2dCTP，以及 2'-FdUTP 和 2'-FdCTP）。此外，在 2'-脱氧核苷三磷酸 (dNTP) 存在下，所得 thioRNA 逆转录成互补 DNA 也顺利且精确地进行。 thioRNA 对 RNase A 的稳定性比相应的天然 RNA 高 50 倍。有了这些成功的结果，我们尝试筛选人α-凝血酶的thioRNA适体，发现了具有高结合亲和力（Kd=4.6 nM）的thioRNA适体。我们通过自由基还原其相应的2'-溴衍生物，将4'-硫代核糖核苷转化为2'-脱氧-4'-硫代核糖核苷，然后将其进一步转化为其亚磷酰胺单元及其5'-三磷酸盐。还研究了硫代 DNA 的热稳定性和核酸酶稳定性。 2'-脱氧-4'-硫代核糖核苷 5'-三磷酸被发现是 DNA 聚合酶（例如 Therminator 和 KOD dash）的良好底物。较少的

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