Bhuyan Soubhagya K, He Weisi, Cui Jingyu, Tanner Julian A
School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, P.R. China.
Advanced Biomedical Instrumentation Centre, Hong Kong Science Park, Shatin, Hong Kong SAR, P.R. China.
Biol Methods Protoc. 2024 Dec 13;10(1):bpae088. doi: 10.1093/biomethods/bpae088. eCollection 2025.
Peroxidase DNAzymes are single-stranded, stable G-quadruplexes structures that exhibit catalytic activity with cofactor hemin. This class of DNAzymes offers several advantages over traditional protein and RNA catalysts, including thermal stability, resistance to hydrolysis, and easy of synthesis in the laboratory. However, their use in medicine, biology, and chemistry is limited due to their low catalytic rates. Selecting and evolving for higher catalytic rates has been challenging due to limitations in selection methodology which generally use affinity as the selection pressure instead of kinetics. We previously evolved a new peroxidase DNAzyme (mSBDZ-X-3) through a directed evolution method, which was subsequently used for proximity labelling in a proteomic experiment in cell culture. Herein, we present a detailed protocol for this function-based laboratory evolution method to evolve peroxidase DNAzymes for future laboratory implementation. This approach is based on capturing self-biotinylated DNA, which is catalyzed by intrinsic peroxidase activity to select for DNAzyme molecules. The selection method uses fluorescence-based real-time monitoring of the DNA pools, allowing for the enrichment of catalytic activity and capture of catalytic DNA across evolutionary selection rounds. The evolved mSBDZ-X-3 DNAzyme attributes parallel G-quadruplex structure and demonstrates better catalytic properties than DNAzyme variants evolved previously. The influence of critical reaction parameters is outlined. This protocol enables discovery of improved peroxidase DNAzyme/RNAzyme variants from natural or chemical-modified nucleotide libraries. The approach could be applicable for the selection of catalytic activities in a variety of directed molecular evolution contexts.
过氧化物酶DNA酶是单链、稳定的G-四链体结构，在辅因子血红素存在下表现出催化活性。这类DNA酶相对于传统的蛋白质和RNA催化剂具有多个优势，包括热稳定性、抗水解性以及易于在实验室合成。然而，由于其催化速率较低，它们在医学、生物学和化学领域的应用受到限制。由于选择方法的局限性，通常使用亲和力作为选择压力而非动力学，因此选择和进化出更高催化速率一直具有挑战性。我们之前通过定向进化方法进化出了一种新的过氧化物酶DNA酶（mSBDZ-X-3），随后将其用于细胞培养蛋白质组学实验中的邻近标记。在此，我们提供了一种基于功能的实验室进化方法的详细方案，用于进化过氧化物酶DNA酶以便未来在实验室实施。这种方法基于捕获自我生物素化的DNA，其由内在过氧化物酶活性催化以选择DNA酶分子。该选择方法使用基于荧光的DNA文库实时监测，从而在进化选择轮次中实现催化活性的富集和催化DNA的捕获。进化后的mSBDZ-X-3 DNA酶具有平行G-四链体结构，并且比之前进化的DNA酶变体表现出更好的催化特性。文中概述了关键反应参数的影响。该方案能够从天然或化学修饰的核苷酸文库中发现改进的过氧化物酶DNA酶/RNA酶变体。该方法可适用于各种定向分子进化背景下催化活性的选择。
