Basic Studies of Molecular Recognition by 4'-Thionucleic acids

我们已经制备了4 '-硫代核糖核苷亚磷酰胺，用于化学合成4'-硫代RNA（thioRNA）。制备完全修饰的硫代RNA。通过UV熔解测量确定双链体的Tm值。所得双链体的热稳定性的总体顺序为：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thiRNA双链体均不被消化。合成了4 '-硫代尿苷和4'-硫代胞苷的三磷酸盐、thioUTP和thioCTP，并将其用于SELEX。由于SELEX需要体外转录和逆转录，我们通过关注这两个步骤来检查thioUTP和thioCTP用于SELEX的能力。通过T7 RNA聚合酶掺入thioUTP和thioCTP以产生thioRNA进行良好，并且优于用于SELEX的两组经常使用的修饰的NTP类似物（2 '-NH_2）的那些上级。 关于我们 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，我们尝试筛选与人α-凝血酶具有高亲和力（Kd=4.6 nM）的thioRNA适体，并将4 '-硫代核糖核苷通过自由基还原成2'-脱氧-4 '-硫代核糖核苷（2'-溴代衍生物），然后进一步转化为亚磷酰胺单元和5 '-三磷酸。还研究了硫代DNA的热稳定性和核酸酶稳定性。发现2 '-脱氧-4'-硫代核糖核苷5 '-三磷酸是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