Selection of an improved RNA polymerase ribozyme with superior extension and fidelity
Selection of an improved RNA polymerase ribozyme with superior extension and fidelity
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DOI：
10.1261/rna.548807
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发表时间：
2007-07-01
期刊：
影响因子：
4.5
通讯作者：
Unrau, Peter J.
中科院分区：
文献类型：
--
作者：
Zaher, Hani S.;Unrau, Peter J.
我们目前对生物学的理解表明，早期生命主要依赖于RNA进行催化和复制。在这里，我们报告的RNA聚合酶核酶称为B6.61，表现出上级的延伸和保真度相对于它的祖先，轮-18聚合酶的分离。B6.61聚合酶通过使用新型大规模体外区室化系统从含有类似于9 × 10（14）个序列变体的诱变池中选择。B6.61比Round-18变体更快地聚合所有测试的引物-模板（PT）复合物。对于一个PT，B6.61表现出显着更快的延伸超过一个完整的螺旋旋转，并纳入至少20个核苷酸的序列，为RNA聚合酶核酶设置了新的延伸记录。B6.61结构效率的提高与保真度的改善有关，新变体比其亲本包含更少的不正确摆动碱基对。这种新的聚合酶证明了在可预见的未来发展人工RNA复制酶核酶的可行性。
Our current understanding of biology suggests that early life relied predominantly on RNA for catalysis and replication. Here, we report the isolation of an RNA polymerase ribozyme called B6.61 that exhibits superior extension and fidelity relative to its progenitor, the Round-18 polymerase. The B6.61 polymerase was selected from a mutagenized pool containing; similar to 9*10(14) sequence variants through the use of a novel large- scale in vitro compartmentalization system. B6.61 polymerized all tested primer-template ( PT) complexes faster than the Round-18 variant. For one PT, B6.61 exhibited dramatically faster elongation past one full helical turn and incorporated at least 20 nucleotides of sequence, setting a new extension record for an RNA polymerase ribozyme. The increased efficiency of the B6.61 construct was related to improvements in fidelity, with the new variant incorporating less incorrect wobble base pairs than its parent. This new polymerase demonstrates the feasibility of evolving an artificial RNA replicase ribozyme in the foreseeable future.