Institute of Organic Chemistry, University of Stuttgart, 70569 Stuttgart, Germany.
Nucleic Acids Res. 2019 May 7;47(8):3836-3845. doi: 10.1093/nar/gkz160.
The template-directed formation of phosphodiester bonds between two nucleic acid components is a pivotal process in biology. To induce such a reaction in the absence of enzymes is a challenge. This challenge has been met for the extension of a primer with mononucleotides, but the ligation of short oligonucleotides (dimers or trimers) has proven difficult. Here we report a method for ligating dimers and trimers of ribonucleotides using in situ activation in aqueous buffer. All 16 different dimers and two trimers were tested. Binding studies by NMR showed low millimolar dissociation constants for complexes between representative dimers and hairpins mimicking primer-template duplexes, confirming that a weak template effect is not the cause of the poor ligating properties of these short oligomers. Rather, cyclization was found to compete with ligation, with up to 90% of dimer being converted to the cyclic form during the course of an assay. This side reaction is strongly sequence dependent and more pronounced for dimers than for trimers. Under optimized reaction conditions, high yields were observed with strongly pairing purines at the 3'-terminus. These results show that short oligomers of ribonucleotides are competent reactants in enzyme-free copying.
模板指导下的两个核酸组分之间磷酸二酯键的形成是生物学中的一个关键过程。在没有酶的情况下诱导这种反应是一个挑战。人们已经成功地实现了用单核苷酸延伸引物，但短寡核苷酸（二聚体或三聚体）的连接一直很困难。在这里，我们报告了一种在水缓冲液中通过原位激活来连接核糖核苷酸二聚体和三聚体的方法。我们测试了所有 16 种不同的二聚体和两种三聚体。通过 NMR 的结合研究表明，代表二聚体与发夹之间的复合物的毫摩尔解离常数低，这些发夹模拟了引物-模板双链体，这证实了弱模板效应不是这些短寡聚物连接性能差的原因。相反，环化被发现与连接竞争，在测定过程中，多达 90%的二聚体转化为环状形式。这种副反应强烈依赖于序列，并且对于二聚体比对三聚体更为明显。在优化的反应条件下，在 3'-末端具有强配对嘌呤的情况下，观察到高收率。这些结果表明，核糖核苷酸的短寡聚物是无酶复制中的合格反应物。
