Nonenzymatic copying of RNA templates with activated nucleotides is a useful model for studying the emergence of heredity at the origin of life. Previous experiments with defined-sequence templates have pointed to the poor fidelity of primer extension as a major problem. Here we examine the origin of mismatches during primer extension on random templates in the simultaneous presence of all four 2-aminoimidazole-activated nucleotides. Using a deep sequencing approach that reports on millions of individual template-product pairs, we are able to examine correct and incorrect polymerization as a function of sequence context. We have previously shown that the predominant pathway for primer extension involves reaction with imidazolium-bridged dinucleotides, which form spontaneously by the reaction of two mononucleotides with each other. We now show that the sequences of correctly paired products reveal patterns that are expected from the bridged dinucleotide mechanism, whereas those associated with mismatches are consistent with direct reaction of the primer with activated mononucleotides. Increasing the ratio of bridged dinucleotides to activated mononucleotides, either by using purified components or by using isocyanide-based activation chemistry, reduces the error frequency. Our results point to testable strategies for the accurate nonenzymatic copying of arbitrary RNA sequences.

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桥接的二核苷酸和活化的单核苷酸之间的竞争决定了非酶RNA引物延伸的错误频率

具有活化核苷酸的RNA模板的非酶复制是研究生命起源遗传的有用模型。先前使用限定序列模板进行的实验已指出引物延伸的保真度差是一个主要问题。在这里，我们在所有四个2-氨基咪唑活化的核苷酸同时存在的情况下，检查了随机模板上引物延伸过程中错配的起源。使用深度测序方法报告数百万个单独的模板产品对，我们能够根据序列上下文检查正确和不正确的聚合。先前我们已经表明，引物延伸的主要途径涉及与咪唑桥连的二核苷酸的反应，其通过两个单核苷酸相互反应而自发形成。现在，我们显示正确配对的产物的序列揭示了预期的桥接二核苷酸机制的模式，而那些与错配有关的序列与引物与活化的单核苷酸的直接反应相一致。通过使用纯化的组分或通过使用基于异氰酸酯的活化化学方法来增加桥接的二核苷酸与活化的单核苷酸的比率，可以减少错误发生的频率。我们的结果指出了可检测的策略，用于任意RNA序列的准确非酶复制。通过使用纯化的组分或通过使用基于异氰酸酯的活化化学方法来增加桥接的二核苷酸与活化的单核苷酸的比率，可以减少错误发生的频率。我们的结果指出了可检测的策略，用于任意RNA序列的准确非酶复制。通过使用纯化的组分或通过使用基于异氰酸酯的活化化学方法来增加桥接的二核苷酸与活化的单核苷酸的比率，可以减少错误发生的频率。我们的结果指出了可检测的策略，用于任意RNA序列的准确非酶复制。