New NTP analogs:: the synthesis of 4′-thioUTP and 4′-thioCTP and their utility for SELEX
New NTP analogs:: the synthesis of 4′-thioUTP and 4′-thioCTP and their utility for SELEX
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DOI：
10.1093/nar/gki578
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发表时间：
2005-01-01
影响因子：
14.9
通讯作者：
Matsuda, A
中科院分区：
文献类型：
--
作者：
Kato, Y;Minakawa, N;Matsuda, A
介绍了4′-硫脲和4′-硫代胞苷三磷酸盐、4′-硫outp （7; thioUTP）和4′-硫octp （8; thioCTP）的合成及其在SELEX（通过指数富集的配体系统进化）中的应用。新的三磷酸核苷（NTP）类似物7和8由适当保护的4'-硫脲和-胞苷衍生物采用一锅法制备[J. Ludwig和F. Eckstein [1989] J. Org]。化学。[j].农业科学，2004,18(4):631 - 635。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7和8对SELEX的能力。7和8被T7 RNA聚合酶结合得到4'-硫核糖核酸（thioRNA）进展良好，优于常用的两组修饰的SELEX NTP类似物(2'- nh2 (2)dUTP和2'- nh2 (2)dCTP)；2'-FdUTP和20-FdCTP)，当选择合适的DNA模板先导序列时。我们发现约+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类似物很重要。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，我们尝试用thioUTP和thioCTP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（K-d = 4.7 nM）的硫核糖核酸适配体。
The synthesis of the triphosphates of 4'-thiouridine and 4'-thiocytidine, 4'-thioUTP ( 7; thioUTP) and 4'-thioCTP ( 8; thioCTP), and their utility for SELEX ( systematic evolution of ligands by exponential enrichment) is described. The new nucleoside triphosphate ( NTP) analogs 7 and 8 were prepared from appropriately protected 4'-thiouridine and - cytidine derivatives using the one-pot method reported by J. Ludwig and F. Eckstein [( 1989) J. Org. Chem., 54, 631 - 635]. Because SELEX requires both in vitro transcription and reverse transcription, we examined the ability of 7 and 8 for SELEX by focusing on the two steps. Incorporation of 7 and 8 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 SELEX (2'- NH(2)dUTP and 2'-NH(2)dCTP; 2'-FdUTP and 20-FdCTP), when an adequate leader sequence of DNA template was selected. We revealed that a leader sequence of about +15 of DNA template is important for the 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, we attempted the selection of thioRNA aptamers to human alpha-thrombin using thioUTP and thioCTP, and found a thioRNA aptamer with high binding affinity (K-d = 4.7 nM).