Sequence-specific cleavage of the RNA strand in DNA-RNA hybrids by the fusion of ribonuclease H with a zinc finger.
Sequence-specific cleavage of the RNA strand in DNA-RNA hybrids by the fusion of ribonuclease H with a zinc finger.
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DOI：
10.1093/nar/gks885
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发表时间：
2012-12
影响因子：
14.9
通讯作者：
Bujnicki JM
中科院分区：
文献类型：
--
作者：
Sulej AA;Tuszynska I;Skowronek KJ;Nowotny M;Bujnicki JM
核糖核酸酶（Ribonucleases，RNases）是研究RNA序列、结构和功能的重要工具。它们的底物特异性仅限于识别底物中的单个碱基或不同的二级结构。目前，没有可用于RNA的纯序列依赖性片段化的RNA酶。在这里，我们报告了一种新的酶的发展，切割的RNA链的DNA-RNA杂交5核苷酸从一个nonanucleotide识别序列。该酶通过融合两个功能独立的结构域构建，一个是RNase HI，其以进行性和序列独立的方式水解DNA-RNA杂交体中的RNA，另一个是锌指，其识别DNA-RNA杂交体中的序列。融合酶的特异性的优化是由蛋白质-底物复合物的结构模型指导的，并且涉及许多步骤，包括RNA酶部分的定点诱变和结构域间接头长度的优化。用新的序列特异性工程化锌指结构域的方法是容易获得的，使得获得识别和切割多种序列的RNA酶文库是可行的，非常像市售的各种限制性酶。潜在地，除了体外应用之外，锌指-RNA酶HI融合物可以在体内用于靶向RNA降解。
Ribonucleases (RNases) are valuable tools applied in the analysis of RNA sequence, structure and function. Their substrate specificity is limited to recognition of single bases or distinct secondary structures in the substrate. Currently, there are no RNases available for purely sequence-dependent fragmentation of RNA. Here, we report the development of a new enzyme that cleaves the RNA strand in DNA–RNA hybrids 5 nt from a nonanucleotide recognition sequence. The enzyme was constructed by fusing two functionally independent domains, a RNase HI, that hydrolyzes RNA in DNA–RNA hybrids in processive and sequence-independent manner, and a zinc finger that recognizes a sequence in DNA–RNA hybrids. The optimization of the fusion enzyme’s specificity was guided by a structural model of the protein-substrate complex and involved a number of steps, including site-directed mutagenesis of the RNase moiety and optimization of the interdomain linker length. Methods for engineering zinc finger domains with new sequence specificities are readily available, making it feasible to acquire a library of RNases that recognize and cleave a variety of sequences, much like the commercially available assortment of restriction enzymes. Potentially, zinc finger-RNase HI fusions may, in addition to in vitro applications, be used in vivo for targeted RNA degradation.
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影响因子：
11.4
作者：
Nowotny, Marcin;Cerritelli, Susana M.;Yang, Wei
通讯作者：
Yang, Wei
DOI：
10.1038/nsb1196-940
发表时间：
1996-11-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
Kim, CA;Berg, JM
通讯作者：
Berg, JM
DOI：
10.1073/pnas.0609502103
发表时间：
2006-12-26
影响因子：
11.1
作者：
Minczuk, Michal;Papworth, Monika A.;Klug, Aaron
通讯作者：
Klug, Aaron
影响因子：
3.4
作者：
Jantz, Derek;Berg, Jeremy M.
通讯作者：
Berg, Jeremy M.
影响因子：
14.9
作者：
BRANLANT, C;KROL, A;JACOB, M
通讯作者：
JACOB, M