Basic Studies of Molecular Recognition by 4'-Thionucleic acids

制备了用于化学合成4′-硫核糖核酸（thioRNA）的4′-硫核糖核苷类磷酰胺。制备了完全修饰的硫原子nas。用紫外熔融法测定了双相化合物的Tm值。得到的双链物的总体热稳定性顺序为：thioRNA:thioRNA>>thioRNA:RNA>RNA:RNA>RNA:DNA>thioRNA:DNA。人血清中硫核糖核酸的稳定性是天然RNA的600倍。在正常条件下，RNA和硫核糖核酸双链都没有被消化。研究了4′-硫脲和4′-硫胞苷三磷酸、硫outp和硫octp的合成及其在SELEX中的应用。由于SELEX需要体外和逆转录，我们通过关注这两个步骤来检测thioUTP和thioCTP对SELEX的能力。当选择合适的DNA模板先导序列时，T7 RNA聚合酶结合thioUTP和thioCTP生成thioRNA的效果较好，优于常用的两组修饰的SELEX NTP类似物（2'-NH_…More 2dUTP和2'-NH_2dCTP，以及2'-FdUTP和2'-FdCTP）。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，我们尝试选择人α-凝血酶的硫核糖核酸适配体，并找到了一个高结合亲和力（Kd=4.6 nM）的硫核糖核酸适配体。我们通过对相应的2'-溴衍生物进行自由基还原，将4'-硫核糖核苷转化为2'-脱氧-4'-硫核糖核苷，然后进一步转化为它们的酰胺磷酸基和5'-三磷酸基。研究了硫代脱氧核糖核酸的热稳定性和核酸酶稳定性。2'-脱氧-4'-硫核糖核苷5'-三磷酸被发现是DNA聚合酶如Therminator和KOD dash的良好底物。少

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