Sequence-controlled RNA self-processing: computational design, biochemical analysis, and visualization by AFM
Sequence-controlled RNA self-processing: computational design, biochemical analysis, and visualization by AFM
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DOI：
10.1261/rna.047670.114
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发表时间：
2015-07-01
期刊：
影响因子：
4.5
通讯作者：
Mueller, Sabine
中科院分区：
文献类型：
--
作者：
Petkovic, Sonja;Badelt, Stefan;Mueller, Sabine
可逆化学允许分子实体的组装和分解，对于生物自组织是重要的。因此，既支持磷酸二酯键的切割又支持磷酸二酯键的形成的核酶可能是早期生命中调节RNA功能多样性和复杂性增加的原因之一。我们之前已经设计了一种发夹状核酶的变体，它显示了核酶如何通过形成串联体来环化或延长自己的长度。使用维也纳RNA包，我们现在优化了这个发夹状核酶变体，并选择了四个不同的RNA序列，预计它们在转录时会更有效地循环或形成更长的连锁体。(2)双向PAGE分析证实：(I)所有四种选定的核酶都具有催化活性，(Ii)获得了高产率的环状物种。原子力显微镜成像结合RNA结构预测使我们能够计算单体和自连接的二聚体和三聚体的分布。我们的结果表明，经过计算优化的分子确实形成了合理数量的三聚体，这在原始系统中还没有观察到，我们证明了理论预测、生化和物理分析的结合是准确预测核酶行为和设计具有预定功能的核酶的一种有前途的方法。
Reversible chemistry allowing for assembly and disassembly of molecular entities is important for biological self-organization. Thus, ribozymes that support both cleavage and formation of phosphodiester bonds may have contributed to the emergence of functional diversity and increasing complexity of regulatory RNAs in early life. We have previously engineered a variant of the hairpin ribozyme that shows how ribozymes may have circularized or extended their own length by forming concatemers. Using the Vienna RNA package, we now optimized this hairpin ribozyme variant and selected four different RNA sequences that were expected to circularize more efficiently or form longer concatemers upon transcription. (Two-dimensional) PAGE analysis confirms that (i) all four selected ribozymes are catalytically active and (ii) high yields of cyclic species are obtained. AFM imaging in combination with RNA structure prediction enabled us to calculate the distributions of monomers and self-concatenated dimers and trimers. Our results show that computationally optimized molecules do form reasonable amounts of trimers, which has not been observed for the original system so far, and we demonstrate that the combination of theoretical prediction, biochemical and physical analysis is a promising approach toward accurate prediction of ribozyme behavior and design of ribozymes with predefined functions.