In vitro evolution of an RNA-cleaving DNA enzyme into an RNA ligase switches the selectivity from 3′-5′ to 2′-5′
In vitro evolution of an RNA-cleaving DNA enzyme into an RNA ligase switches the selectivity from 3′-5′ to 2′-5′
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DOI：
10.1021/ja0340331
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发表时间：
2003-05-07
影响因子：
15
通讯作者：
Silverman, SK
中科院分区：
文献类型：
--
作者：
Flynn-Charlebois, A;Prior, TK;Silverman, SK
连接 RNA 的脱氧核酶扩大了核酸催化的范围，并允许制备位点特异性修饰的 RNA。此前，通过从随机 DNA 池中进行体外选择来鉴定连接 5'-羟基和 2',3'-环状磷酸盐的脱氧核酶。在这里，体外进化的替代策略被用来将切割 RNA 的 8-17 脱氧核酶转化为连接 RNA 的 DNA 酶家族。亲本 8-17 DNA 酶可裂解天然 3'-5' 磷酸二酯键，但不裂解 2'-5' 键。令人惊讶的是，从 8-17 进化而来的新脱氧核酶仅产生 2'-5' 连接。因此，逆转 DNA 介导过程从连接到裂解的方向也会改变形成新磷酸二酯键的选择性。在 10-23 RNA 切割脱氧核酶进化为 RNA 连接酶时，观察到选择性的相同变化。先前从随机池中分离的 DNA 酶也产生 2'-5' 连接。因此，脱氧核酶介导的由 5'-羟基和 2',3'-环状磷酸酯形成非天然 2'-5' 磷酸二酯键在许多不同的情况下受到强烈青睐。
Deoxyribozymes that ligate RNA expand the scope of nucleic acid catalysis and allow preparation of site-specifically modified RNAs. Previously, deoxyribozymes that join a 5'-hydroxyl and a 2',3'-cyclic phosphate were identified by in vitro selection from random DNA pools. Here, the alternative strategy of in vitro evolution was used to transform the 8-17 deoxyribozyme that cleaves RNA into a family of DNA enzymes that ligate RNA. The parent 8-17 DNA enzyme cleaves native 3'-5' phosphodiester linkages but not 2'-5' bonds. Surprisingly, the new deoxyribozymes evolved from 8-17 create only 2'-5' linkages. Thus, reversing the direction of the DNA-mediated process from ligation to cleavage also switches the selectivity in forming the new phosphodiester bond. The same change in selectivity was observed upon evolution of the 10-23 RNA-cleaving deoxyribozyme into an RNA ligase. The DNA enzymes previously isolated from random pools also create 2'-5' linkages. Therefore, deoxyribozyme-mediated formation of a non-native 2'-5' phosphodiester linkage from a 5'-hydroxyl and a 2',3'-cyclic phosphate is strongly favored in many different contexts.