Increasing the Utility of Polymerases by Directed Evolution

描述（由申请人提供）：DNA和RNA聚合酶及其技术使生物医学研究发生了革命性变化。然而，天然聚合酶的精细特异性限制了它们的潜在应用，涉及完全天然的生物聚合物，这是不适合许多诊断，治疗和DNA测序应用。前两个应用与人类健康有着明显的相关性，有望彻底改变疾病检测和治疗，后者有望迎来前所未有的个性化医疗时代。为了解决这些限制，我们开发了一种基于活性的选择系统，以发展识别修饰底物的聚合酶。该系统基于聚合酶文库和噬菌体颗粒上的底物的共展示，并允许它们以模仿自然界中达尔文进化的方式多样化和选择非天然活性。虽然我们已经确定了该系统的几个方面仍然需要优化，但我们已经使用它来进化几种“第一代”非天然DNA聚合酶，这些酶具有增强的合成由针对不同应用修饰的核苷酸组成的聚合物的能力。例如，SFM 19能够有效地合成由C2 '-OMe修饰的核苷酸组成的短链聚合物，其具有作为用于诊断和治疗应用的生物稳定聚合物的潜在应用。sf 197能够更有效地标记修饰的核苷酸，用于标记和下一代测序应用。虽然这两种进化的聚合酶代表了迈向实用酶的重要第一步，但它们仍需要进一步优化：SFM 19用于合成更长的修饰聚合物，Sf 197用于提高效率。 我们的第一个目标是进一步优化我们的选择系统，并使其适应RNA聚合酶的进化。我们的第二个目标是进化出具有真实的实用性的聚合酶。作为我们第二个目标的一部分，SFM 19和Sf 197将进一步多样化，并进行优化活性的选择。我们还将发展一种RNA聚合酶，以有效地识别C2 '-OMe核苷酸和一种DNA聚合酶，使甲基化胞嘧啶的直接测序成为可能，甲基化胞嘧啶是中心表观遗传标记，其在基因组中的分布具有重要的健康意义，但目前尚难以表征。 实现这些目标将提供一个强大的系统，用于开发具有专门定制的活性的聚合酶，以及四种具有直接和重要的健康相关应用的聚合酶。也许最重要的是，拟议的研究应该说明聚合酶进化的潜力，并将其减少到一个更实用和用户友好的系统，其目标是为更广泛的研究界提供一种普遍可用的方法，以定制聚合酶用于尽可能多的不同活动，因为有潜在的应用。

DESCRIPTION (provided by applicant): DNA and RNA polymerases and the technologies they enable have revolutionized biomedical research. However, the exquisite specificity of natural polymerases limits their potential applications to those involving the fully natural biopolymers, which are unsuitable for many diagnostic, therapeutic, and DNA sequencing applications. The first two applications are of obvious relevance to human health, promising to revolutionize disease detection and treatment, and the latter promising to usher in an unprecedented era of personalized medicine. To address these limitations, we developed an activity-based selection system to evolve polymerases that recognize modified substrates. The system is based on co-display of polymerase libraries and substrates on bacteriophage particles and which allows for their diversification and selection for unnatural activities in a manner that imitates Darwinian evolution in nature. While we have identified several aspects of the system that still require optimization, we have already used it to evolve several "first generation" unnatural DNA polymerases that possess increased abilities to synthesize polymers comprised of nucleotides modified for different applications. For example, SFM19 is able to efficiently synthesize short stretches of polymers comprised of C2'-OMe modified nucleotides, which have potential applications as biostable polymers for diagnostic and therapeutic applications. Sf197 is able to more efficiently polymerize nucleotides modified for labeling and next-generation sequencing applications. While both evolved polymerase represent important first steps toward practically useful enzymes, they both still require further optimization: SFM19 for the synthesis of longer modified polymers, and Sf197 for increased efficiency. Our first objective is to further optimize our selection system and to adapt it for the evolution of RNA polymerases. Our second objective is to evolve polymerases with real, practical utility. As part of our second objective, SFM19 and Sf197 will each be further diversified and subjected to selections for optimized activity. We will also evolve an RNA polymerase to efficiently recognize C2'-OMe nucleotides and a DNA polymerase that enables the direct sequencing of methylated cytosines, which are central epigenetic markers whose distribution through the genome has critical health implications, but which is currently challenging to characterize. Achieving these objectives will deliver a robust system for evolving polymerases with specifically tailored activities, and four evolved polymerases that have immediate and important health related applications. Perhaps most importantly, the proposed research should illustrate the potential of polymerase evolution and reduce it to a more practical and user friendly system, with the goal of providing to the broader research community a generally accessible method to tailor polymerases for as many different activities as there are potential applications.