Directed evolution of ribozymes with novel catalytic functions

具有新催化功能的核酶的定向进化

• 批准号: 21F20810
• 负责人: 横林 洋平
• 金额: $ 1.47万
• 依托单位: Okinawa Institute of Science and Technology Graduate University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for JSPS Fellows
• 财政年份: 2021
• 资助国家: 日本
• 起止时间: 2021-07-28 至 2023-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-21F20810/
• 关键词: Ribozyme; In vitro evolution; Directed evolution; SELEX; 進化工学; 核酸化学; RNA; リボザイム

Alternative systems for the selection of ribox/combox variants able to catalyze the reduction of benzyl alcohol have been set up. The first one aims to identify in an initial step variants of the ribozyme able to bind with higher affinity to the target substrate (benzyl alcohol). The system is based on capture-SELEX, and variants identified with the system will be screened for catalytic activity in a second step using a fluorescence-based assay. Additionally, we have also identified alternative substrates likely to have a higher reactivity. These include benzyl alcohol and benzaldehyde derivatives with one or more nitro or carboxyl substituents in the aromatic ring. Iterative rounds of capture-SELEX with the different substrates are currently being performed.The second strategy is based on the previously mentioned selection system which exploits the requirement of oxidized NAD+ for anaerobic growth (Selles Vidal, Murray and Heap, 2021). The library of combox variants, as well as wild-type combox, have been cloned into the appropriate plasmids for the selection system. Additionally, a classical protein benzyl alcohol dehydrogenase has been also cloned into the same vector. We have demonstrated that cells transformed with the protein benzyl alcohol dehydrogenase can grow anaerobically in medium supplemented with benzyl alcohol, proving that this pair of substrate and enzymatic activity are suitable for the selection system. We are currently applying the system to the RNA-based benzyl alcohol dehydrogenases.

已经建立了选择能够催化苯甲醇还原的 Ribox/combox 变体的替代系统。第一个目标是在初始步骤中识别能够以更高亲和力与目标底物（苯甲醇）结合的核酶变体。该系统基于 capture-SELEX，在第二步中将使用基于荧光的测定来筛选用该系统识别的变体的催化活性。此外，我们还发现了可能具有更高反应性的替代底物。这些包括在芳环中具有一个或多个硝基或羧基取代基的苯甲醇和苯甲醛衍生物。目前正在对不同底物进行多轮捕获-SELEX 迭代。第二种策略基于前面提到的选择系统，该系统利用氧化 NAD+ 进行厌氧生长的需求（Selles Vidal、Murray 和 Heap，2021）。组合盒变体文库以及野生型组合盒已被克隆到用于选择系统的适当质粒中。此外，经典的蛋白质苯甲醇脱氢酶也被克隆到同一载体中。我们已经证明用蛋白质苯甲醇脱氢酶转化的细胞可以在补充有苯甲醇的培养基中厌氧生长，证明这对底物和酶活性适合选择系统。我们目前正在将该系统应用于基于 RNA 的苯甲醇脱氢酶。