Systematic minimization of RNA ligase ribozyme through large-scale design-synthesis-sequence cycles
Systematic minimization of RNA ligase ribozyme through large-scale design-synthesis-sequence cycles
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DOI：
10.1093/nar/gkz729
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发表时间：
2019-09-26
影响因子：
14.9
通讯作者：
Yokobayashi, Yohei
中科院分区：
文献类型：
--
作者：
Nomura, Yoko;Yokobayashi, Yohei
由RNA酶（核酶）催化的模板指导的RNA连接是一种合理且重要的反应，可能参与了前生物进化过程中的遗传信息传递。实验室进化实验已经产生了几类连接酶核酶，但它们的最低序列要求仍然在很大程度上未被探索。由于选择实验强烈倾向于高活性序列，活性较低但较小的催化基序可能在这些实验中被忽略。我们使用了大规模的DNA合成和高通量核酶测定，通过深度测序系统地最大限度地减少了以前实验室进化的连接酶核酶。在设计和评估> 10000个序列后，我们鉴定了小至18个连续碱基的催化核心，其催化模板指导的区域特异性RNA连接。如此短的序列可以催化这一关键反应的事实表明，类似简单或甚至更简单的基序可能占据了RNA序列空间，而这些空间本来可以被益生元核酶所接近。
Template-directed RNA ligation catalyzed by an RNA enzyme (ribozyme) is a plausible and important reaction that could have been involved in transferring genetic information during prebiotic evolution. Laboratory evolution experiments have yielded several classes of ligase ribozymes, but their minimal sequence requirements remain largely unexplored. Because selection experiments strongly favor highly active sequences, less active but smaller catalytic motifs may have been overlooked in these experiments. We used large-scale DNA synthesis and high-throughput ribozyme assay enabled by deep sequencing to systematically minimize a previously laboratory-evolved ligase ribozyme. After designing and evaluating >10 000 sequences, we identified catalytic cores as small as 18 contiguous bases that catalyze template-directed regiospecific RNA ligation. The fact that such a short sequence can catalyze this critical reaction suggests that similarly simple or even simpler motifs may populate the RNA sequence space which could have been accessible to the prebiotic ribozymes.