Engineering a ribozyme cleavage-induced split fluorescent aptamer complementation assay.
Engineering a ribozyme cleavage-induced split fluorescent aptamer complementation assay.
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DOI：
10.1093/nar/gkw117
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发表时间：
2016-06-02
影响因子：
14.9
通讯作者：
Fussenegger M
中科院分区：
文献类型：
--
作者：
Ausländer S;Fuchs D;Hürlemann S;Ausländer D;Fussenegger M
锤头状核酶是能够调节活细胞中基因表达的自切割RNA分子。它们的切割性能受到分子内环-环相互作用的强烈影响，这是通过现代预测算法不易获得的特征。核酶工程和基于核酶的基因开关的有效实施需要个体自切割性能的详细知识。通过合理的设计，我们设计了荧光适配体-核酶RNA结构，允许实时测量核酶体外自切割活性。工程化的核酸分子实现分裂的Spinach适体序列，其在核酶自切割时可用于链置换，从而补充荧光Spinach适体。这种完全基于RNA的核酶性能测定将核酶切割活性与菠菜荧光相关联，以提供用于验证锤头状核酶中的环-环相互作用的快速且直接的技术。
Hammerhead ribozymes are self-cleaving RNA molecules capable of regulating gene expression in living cells. Their cleavage performance is strongly influenced by intra-molecular loop–loop interactions, a feature not readily accessible through modern prediction algorithms. Ribozyme engineering and efficient implementation of ribozyme-based genetic switches requires detailed knowledge of individual self-cleavage performances. By rational design, we devised fluorescent aptamer-ribozyme RNA architectures that allow for the real-time measurement of ribozyme self-cleavage activity in vitro. The engineered nucleic acid molecules implement a split Spinach aptamer sequence that is made accessible for strand displacement upon ribozyme self-cleavage, thereby complementing the fluorescent Spinach aptamer. This fully RNA-based ribozyme performance assay correlates ribozyme cleavage activity with Spinach fluorescence to provide a rapid and straightforward technology for the validation of loop–loop interactions in hammerhead ribozymes.