Deoxyribozymes with 2′-5′ RNA ligase activity
Deoxyribozymes with 2′-5′ RNA ligase activity
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DOI：
10.1021/ja028774y
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发表时间：
2003-03-05
影响因子：
15
通讯作者：
Silverman, SK
中科院分区：
文献类型：
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作者：
Flynn-Charlebois, A;Wang, YM;Silverman, SK
体外选择用于鉴定连接两个RNA底物的脱氧核酶。在连接反应中，由2 '，3'-环磷酸和5 '-羟基产生2'-5 ' RNA磷酸二酯键。新的Mg 2+依赖性脱氧核酶在pH 7.5和37 ℃下过夜孵育时提供50-60%的连接RNA产率，并且在pH 9.0和37 ℃下1小时内提供40-50%的产率。各种RNA底物序列可以通过与两种RNA底物相互作用的DNA结合臂的简单沃森-克里克共变来连接。目前的脱氧核酶在紧邻连接点周围的核苷酸处具有一些RNA底物序列要求（UAUA下箭头GGAA或UAUN下箭头GGAA，其中箭头表示连接位点，N等于任何核苷酸）。一种新的脱氧核酶被用于通过连接制备四膜虫I组内含子RNA P4-P6结构域，一种代表性的结构RNA。非变性凝胶电泳显示P4-P6的核苷酸A233和G234之间的2 '-5'连接不破坏其Mg 2+依赖性折叠（Δ Δ G度' < 0.2 kcal/mol）。这表明2 '-5'键不一定干扰折叠RNA中的结构。因此，当研究结构/功能关系时，这些非天然连接在修饰的RNA中可能是可接受的。连接RNA的脱氧核酶对于制备用于研究RNA结构、折叠和催化的位点特异性修饰的RNA应该是特别有用的。
In vitro selection was used to identify deoxyribozymes that ligate two RNA substrates. In the ligation reaction, a 2'-5' RNA phosphodiester linkage is created from a 2',3'-cyclic phosphate and a 5'-hydroxyl group. The new Mg2+-dependent deoxyribozymes provide 50-60% yield of ligated RNA in overnight incubations at pH 7.5 and 37 degreesC, and they afford 40-50% yield in 1 h at pH 9.0 and 37 degreesC. Various RNA substrate sequences may be joined by simple Watson-Crick covaration of the DNA binding arms that interact with the two RNA substrates. The current deoxyribozymes have some RNA substrate sequence requirements at the nucleotides immediately surrounding the ligation junction (either UAUAdown arrowGGAA or UAUNdown arrowGGAA, where the arrow denotes the ligation site and N equals any nucleotide). One of the new deoxyribozymes was used to prepare by ligation the Tetrahymena group I intron RNA P4-P6 domain, a representative structured RNA. Nondenaturing gel electrophoresis revealed that a 2'-5' linkage between nucleotides A233 and G234 of P4-P6 does not disrupt its Mg2+-dependent folding (DeltaDeltaGdegrees' < 0.2 kcal/mol). This demonstrates that a 2'-5' linkage does not necessarily interfere with structure in a folded RNA. Therefore, these non-native linkages may be acceptable in modified RNAs when structure/function relationships are investigated. Deoxyribozymes that ligate RNA should be particularly useful for preparing site-specifically modified RNAs for studies of RNA structure, folding, and catalysis.