Basic Studies of Molecular Recognition by 4'-Thionucleic acids

我们制备了4‘-硫代核苷亚磷酰胺，用于4’-硫代核糖核酸(ThioRNA)的化学合成。制备了完全修饰的硫代RNA。用紫外熔融法测定了双链的Tm值。由此得到的双链的热稳定性的总体顺序为：thioRNA:thioRNA&gt；&gt；thioRNA:RNA&gt；RNA:RNA&gt；RNA:DNA&gt；thioRNA:DNA.硫代RNA在人血清中的稳定性是天然RNA的600倍。在正常条件下，RNA和硫代RNA双链都不能被消化。合成了4‘-硫代尿苷和4’-硫代胞苷、硫代UTP和硫代CTP的三磷酸盐及其在SELEX中的应用。由于SELEX既需要体外转录，也需要逆转录，我们通过这两个步骤检测了thioUTP和thioCTP对SELEX的作用能力。T7RNA聚合酶掺入硫代UTP和硫代CTP得到硫代RNA的进展顺利，优于SELEX的两套常用的改良型NTP类似物(2‘-NH_…2dUTP和2‘-NH_2dCTP，以及2’-FdUTP和2‘-FdCTP)。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，我们尝试了对人α-凝血酶的硫代RNA适配子的选择，发现了一个高结合亲和力的硫代RNA适配子(Kd=4.6nM)。我们通过相应的2‘-溴衍生物的自由基还原将4’-硫代核苷转化为2‘-脱氧-4’-硫代核苷，然后再转化为它们的亚磷酰胺单元和5‘-三磷酸。对硫代DNA的热稳定性和核酸酶稳定性进行了研究。2‘-脱氧-4’-硫代核苷5‘-三磷酸是Therminator和KOD-DASH等DNA聚合酶的良好底物。较少

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