Multiplex Single-Molecule Kinetics of Nanopore-Coupled Polymerases

DNA polymerases have revolutionized the biotechnology field due to their ability to precisely replicate stored genetic information. Screening variants of these enzymes for specific properties gives the opportunity to identify polymerases with different features. We have previously developed a single-molecule DNA sequencing platform by coupling a DNA polymerase to an α-hemolysin pore on a nanopore array. Here, we use this approach to demonstrate a single-molecule method that enables rapid screening of polymerase variants in a multiplex manner. In this approach, barcoded DNA strands are complexed with polymerase variants and serve as templates for nanopore sequencing. Nanopore sequencing of the barcoded DNA reveals both the barcode identity and kinetic properties of the polymerase variant associated with the cognate barcode, allowing for multiplexed investigation of many polymerase variants in parallel on a single nanopore array. Further, we develop a robust classification algorithm that discriminates kinetic characteristics of the different polymerase mutants. As a proof of concept, we demonstrate the utility of our approach by screening a library of ∼100 polymerases to identify variants for potential applications of biotechnological interest. We anticipate our screening method to be broadly useful for applications that require polymerases with altered physical properties.DNA聚合酶(DNA polymerase)凭借其精准复制存储遗传信息的能力，彻底变革了生物技术领域。针对特定特性筛选这类酶的变体，有助于发掘具备差异化功能特征的聚合酶。我们此前曾通过将DNA聚合酶与纳米孔阵列(nanopore array)上的α-溶血素孔(α-hemolysin pore)相结合，开发出一套单分子DNA测序平台(single-molecule DNA sequencing platform)。本文中，我们依托该方法展示了一种可实现聚合酶变体多重快速筛选的单分子技术。该技术中，带条形码的DNA链(barcoded DNA strands)会与聚合酶变体结合，并作为纳米孔测序的模板。对带条形码DNA进行纳米孔测序，可同时获取条形码的身份信息，以及与该同源条形码(cognate barcode)对应的聚合酶变体的动力学特性，从而实现在单张纳米孔阵列上并行开展大量聚合酶变体的多重研究。此外，我们还开发了一款可区分不同聚合酶突变体(polymerase mutant)动力学特征的稳健分类算法(robust classification algorithm)。作为概念验证(proof of concept)，我们通过筛选一个包含约100种聚合酶的文库来发掘具备生物技术应用潜力的变体，以此证明本方法的实用性。我们预计，该筛选方法可广泛应用于需要具备改性物理特性聚合酶的各类场景。