Koizumi M, Kerr J N, Soukup G A, Breaker R R
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.
Nucleic Acids Symp Ser. 1999(42):275-6. doi: 10.1093/nass/42.1.275.
We have engineered allosteric ribozymes by combining modular rational design with combinatorial strategies. This new procedure was used to create allosteric ribozymes that are activated by specific nucleoside 3',5'-cyclic monophosphates (cNMPs). A random-sequence domain was attached to stem II of hammerhead ribozymes via a communication module that serves as an interface between ribozyme and the effector binding site. Subjecting this initial random pool to in vitro selection methods produced populations that respond, or cleave, only in the presence of specific effector molecules. From generation 18, 20 and 23, cGMP, cCMP and cAMP-specific responsive ribozymes, respectively, were isolated and characterized. These methods show great promise for engineering allosteric ribozymes and for creating new ligand-specific aptamers.
我们通过将模块化的合理设计与组合策略相结合，构建了变构核酶。这一新方法被用于创建由特定的核苷3',5'-环一磷酸（cNMP）激活的变构核酶。一个随机序列结构域通过一个通信模块连接到锤头状核酶的茎II上，该通信模块作为核酶与效应物结合位点之间的接口。对这个初始随机文库进行体外筛选，产生了仅在特定效应分子存在时才会响应或切割的群体。分别从第18代、20代和23代中分离并鉴定出了对cGMP、cCMP和cAMP特异响应的核酶。这些方法在构建变构核酶和创建新的配体特异性适体方面显示出巨大的潜力。
