Enzyme-free ligation of dimers and trimers to RNA primers
Enzyme-free ligation of dimers and trimers to RNA primers
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DOI：
10.1093/nar/gkz160
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发表时间：
2019-05-07
影响因子：
14.9
通讯作者：
Richert, Clemens
中科院分区：
文献类型：
--
作者：
Sosson, Marilyne;Pfeffer, Daniel;Richert, Clemens
核酸分子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。对于用单核苷酸肽延伸引物已经满足了这一挑战，但是短寡核苷酸（二聚体或三聚体）的连接已经被证明是困难的。在这里，我们报告了一种方法连接二聚体和三聚体的核糖核苷酸使用原位激活在水性缓冲液中。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，低的毫摩尔解离常数之间的复合物的代表性二聚体和发夹模仿引物-模板双链体，确认弱模板效应是不是这些短的低聚物的连接性能差的原因。相反，发现环化与连接竞争，在测定过程中高达90%的二聚体转化为环状形式。该副反应是强烈的序列依赖性的，并且对于二聚体比对于三聚体更明显。在优化的反应条件下，观察到高产率与强配对的嘌呤在3 '-末端。这些结果表明，核糖核苷酸的短寡聚体在无酶复制中是胜任的反应物。
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.