Investigating the Determinants of Polymerase Specificity by Droplet Microfluidics

我们星球上的每一个生物体都需要DNA聚合酶在细胞分裂过程中忠实而有效地复制其遗传信息（DNA）。在聚合酶链式反应等应用中利用DNA合成的能力已经彻底改变了医学诊断，法医学和分子生物学。 未来的技术进步将需要定制DNA聚合酶以用于特定性质和特定应用。 该项目的目标是确定DNA聚合酶如何识别它们的DNA靶标，并使用这些信息定制用于识别和复制新靶标的聚合酶。该项目还包含一个重要的教育组成部分，旨在吸引和保持学生对生物科学的兴趣。工程DNA聚合酶，以合成任何类型的非天然核酸聚合物与其天然对应物具有相同的催化效率，将需要更好地了解聚合酶序列如何与其生化功能相关。在这项提案中，我们将通过应用融合科学方法来解决这个问题，该方法将高通量微流体筛选与下一代测序相结合，以评估复制型DNA聚合酶催化结构域中发现的所有可能的单点突变的功能活性。结果的大规模测序数据将被组织成蛋白质适应度图，揭示每个突变（有益的，中性的或负面的）对一组定义的合成类似物的功能后果。促进强合成活性的突变将被重组并通过严格筛选以产生具有上级催化活性的新聚合酶。最好的酶将使用动力学，生物化学和结构方法来表征，这些方法定义了合成的速率，保真度和机制。这项研究的成功完成将提供关于聚合酶工程极限的新的基础知识，该工程使用尖端微流体技术，允许在油包水微滴内大规模并行筛选新型聚合酶活性。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Every living organism on our planet requires a DNA polymerase to faithfully and efficiently copy its genetic information (DNA) during cell division. The ability to harness DNA synthesis in applications such as the polymerase chain reaction has revolutionized medical diagnostics, forensics and molecular biology. Future technological advances will require the customization of DNA polymerases for particular properties and particular applications. The goal of this project is to determine how DNA polymerases recognize their DNA targets and to use this information to customize a polymerase for recognition and copying of novel targets. This project also contains a significant educational component that is designed to attract and maintain student interest in the biological sciences.Engineering DNA polymerases to synthesize any type of unnatural nucleic acid polymer with the same catalytic efficiency as their natural counterparts will require a better understanding of how a polymerase sequence relates to its biochemical function. In this proposal, we will address this problem by applying a convergent science approach that combines high-throughput microfluidic screening with next-generation sequencing to assess the functional activity of all possible single-point mutations found in the catalytic domain of a replicative DNA polymerase. The large-scale sequencing data that results will be organized into protein fitness maps that reveal the functional consequences of each mutation (beneficial, neutral, or negative) against a defined set of synthetic analogs. Mutations that promote strong synthesis activity will be recombined and taken through stringent screens to create new polymerases with superior catalytic activity. The best enzymes will be characterized using kinetic, biochemical, and structural methods that define the rate, fidelity, and mechanism of synthesis. Successful completion of this study will provide new fundamental knowledge about the limits of polymerase engineering using cutting-edge microfluidic techniques that allow for massively parallel screening of novel polymerase activities inside water-in-oil microdroplets.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.