Deep Mutational Scan of a DNA Polymerase via Compartmentalized Self-Replication

We present a novel platform for high-resolution mapping of DNA polymerase activity and stability under the effects of harsh chemistries that are incompatible with most other mutational scanning methods. This approach pairs compartmentalized self-replication (CSR), a high-throughput method for polymerase directed evolution, with deep mutational scanning (DMS), a method of quantifying variant effect via next-generation sequencing of libraries that are subjected to a functional selection. We demonstrate the validity of this “CSR-DMS” platform by showing that it identifies loss-of-function variants at sites with known DNA binding or catalytic activity in the wild-type protein. We further explore the efficacy of this method by imposing denaturing selective pressures (heat or guanidinium thiocyanate) during screening and showing that variants with high positive enrichment under these selective pressures possess higher resistance to denaturation in activity assays. These variants may be useful for “direct” diagnostic workflows that detect biomarkers from crude sample matrices with little to no sample purification. Furthermore, the mechanisms of stabilizing mutations can be inferred from trends in the scores of similar mutations in sequence-to-function heatmaps and corroborated by the behavior of residues of interest in molecular dynamics simulations of the wild-type protein. These mechanisms, uncovered by CSR-DMS, inform future approaches to rational design of extremely stable DNA polymerases. Overall, we propose that CSR-DMS could be used both for the study of the biophysical mechanisms of selective pressures and for the engineering of polymerases with novel capabilities. Overall design: A barcoded single amino acid variant library of DNA polymerase spanning its entire sequence was expressed in E. coli. Cells were encapsulated in oil-aqueous emulsion droplets and subjected to PCR cycling either alone, in conjunction with excessive heat treatment, or in conjunction with added guanidinium. The PCR cycling forced variant polymerases to amplify barcode sequences from their own coding plasmids under a "compartmentalized self-replication" selection scheme. The emulsion was broken after 20 cycles of PCR and barcoded DNA fragments were isolated and sequenced with Illumina sequencing. Barcodes were associated back to corresponding DNA polymerase variants by comparison with a barcode-variant map created by sequencing the variant library plasmid using PacBio HiFi long-read sequencing.

本研究报道一种全新平台，可在多数其他突变扫描方法无法兼容的严苛化学条件下，实现DNA聚合酶（DNA polymerase）活性与稳定性的高分辨率图谱绘制。该方法将用于聚合酶定向进化的高通量技术分隔式自我复制（compartmentalized self-replication, CSR），与通过对经功能选择的文库进行下一代测序（next-generation sequencing）来量化变异体效应的深度突变扫描（deep mutational scanning, DMS）相结合。本研究通过验证该"CSR-DMS"平台可在野生型蛋白的已知DNA结合或催化活性位点处识别功能丧失型变异体，证实了该平台的有效性。本研究进一步在筛选过程中施加变性选择压力（高温或硫氰酸胍（guanidinium thiocyanate）），并证实经此类选择压力下富集度显著升高的变异体在活性测定中具有更强的抗变性能力，以此探究该方法的效能。此类变异体可应用于无需或仅需极少量样本纯化的"直接式"诊断流程，用于从粗制样本基质中检测生物标志物。此外，可通过序列-功能热图（sequence-to-function heatmaps）中相似突变的得分趋势推断稳定型突变的作用机制，并通过野生型蛋白分子动力学模拟（molecular dynamics simulations）中目标残基的行为验证该机制。本研究通过CSR-DMS揭示的这些机制，可为未来极度稳定型DNA聚合酶的理性设计提供指导。综上，本研究认为CSR-DMS既可用于研究选择压力的生物物理机制，也可用于工程化改造具备全新功能的聚合酶。

整体实验设计：构建覆盖DNA聚合酶全序列的带条形码单氨基酸变异体文库，并在大肠杆菌（E. coli）中表达该文库。将细胞封装于油-水乳液微滴中，分别在单独条件、伴随过度热处理条件或添加硫氰酸胍的条件下进行PCR循环扩增。PCR循环过程按照"分隔式自我复制"选择方案，迫使变异体聚合酶扩增自身编码质粒中的条形码序列。PCR循环20轮后破乳，分离带条形码的DNA片段并通过Illumina测序进行测序。通过比对利用PacBio HiFi长读长测序技术对变异体文库质粒进行测序得到的条形码-变异体映射表，可将条形码与对应的DNA聚合酶变异体一一关联。