Directed evolution of ribozymes with novel catalytic functions

已经建立了用于选择能够催化苯甲醇还原的Ribox/Combox变体的替代系统。第一个目的是在最初的步骤中鉴定核酶的变体，能够与目标底物(苯甲醇)结合更高的亲和力。该系统基于Capture-SELEX，在第二步将使用基于荧光的分析来筛选与该系统识别的变体的催化活性。此外，我们还确定了可能具有更高反应活性的替代底物。这类化合物包括芳环上带有一个或多个硝基或羧基的苯甲醇和苯甲醛衍生物。第二个策略是基于前面提到的选择系统，该系统利用氧化NAD+对厌氧生长的需求(Selle Vida，Murray和Heap，2021)。Combox变异体文库以及野生型Combox已被克隆到用于选择系统的适当质粒中。此外，一个经典的蛋白质苯甲醇脱氢酶也被克隆到同一载体中。我们证明了转化苯甲醇脱氢酶蛋白的细胞可以在添加苯甲醇的培养基中厌氧生长，证明这对底物和酶活适合于选择系统。我们目前正在将该系统应用于基于RNA的苯甲醇脱氢酶。

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.