Increasing the Utility of Polymerases by Directed Evolution

描述(由申请人提供)：DNA和RNA聚合酶及其实现的技术已经使生物医学研究发生了革命性的变化。然而，天然聚合酶的精致专一性限制了它们的潜在应用，仅限于涉及全天然生物聚合物的那些，这些生物聚合物不适合于许多诊断、治疗和DNA测序应用。前两个应用程序与人类健康有着明显的相关性，有望给疾病检测和治疗带来革命性的变化，而后者有望开启一个前所未有的个性化医学时代。为了解决这些限制，我们开发了一个基于活动的选择系统来进化识别修饰底物的聚合酶。该系统基于噬菌体颗粒上聚合酶文库和底物的共展示，并允许它们以模仿自然界达尔文进化的方式进行多样化和非自然活动的选择。虽然我们已经确定了该系统仍需优化的几个方面，但我们已经使用它来进化出几种“第一代”非天然DNA聚合酶，这些聚合酶具有更强的合成由针对不同应用而修饰的核苷酸组成的聚合物的能力。例如，SFM19能够有效地合成由C2‘-OMe修饰核苷酸组成的短延伸聚合物，这些聚合物具有潜在的生物稳定聚合物的诊断和治疗应用。Sf197能够更有效地聚合为标记和下一代测序应用而修改的核苷酸。虽然这两种进化的聚合酶都是迈向实用酶的重要第一步，但它们都还需要进一步优化：SFM19用于合成更长的改性聚合物，Sf197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真正实用的聚合酶。作为我们第二个目标的一部分，SFM19和Sf197将各自进一步多样化，并接受优化活动的选择。我们还将进化一种RNA聚合酶来有效识别C2‘-OME核苷酸，以及一种DNA聚合酶，它能够对甲基化的胞嘧啶进行直接测序，这些都是核心的表观遗传标记，其在基因组中的分布具有关键的健康影响，但目前很难表征它的特征。实现这些目标将提供一个强大的聚合酶进化系统，具有特别定制的活动，以及四个具有直接和重要的健康相关应用的进化聚合酶。也许最重要的是，拟议的研究应该说明聚合酶进化的潜力，并将其简化为更实用和用户友好的系统，目标是向更广泛的研究界提供一种普遍可用的方法，以适应尽可能多的不同活动和潜在的应用。 与公共卫生相关：虽然DNA聚合酶的可获得性使各种技术成为医学科学的革命性技术，但其精致的底物专一性限制了这些技术的应用。我们已经开发了一种能够进化聚合酶来识别修饰底物的选择系统，并已经使用它进化出了几种具有理想活性的第一代聚合酶。我们现在建议进一步优化该系统(目标是使其足够健壮，以供其他人普遍使用)，并进化出几种聚合酶，这些聚合酶将使重要的应用成为可能，例如作为诊断和治疗的修饰寡核苷酸的体外进化、通用DNA标记、下一代测序，甚至“表观测序”。

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. PUBLIC HEALTH RELEVANCE: While the availability of DNA polymerases has enabled a variety of technologies that have revolutionized the medical sciences, their exquisite substrate specificity limits the application of these technologies. We have developed a selection system that is capable of evolving polymerases to recognize modified substrates and already used it to evolve several "first generation" polymerases with desirable activities. We now propose to further optimize the system (with the goal of making it sufficiently robust for general use by others) and to evolve several polymerases that will enable important applications, such as the in vitro evolution of modified oligonucleotides as diagnostics and therapeutics, general DNA labeling, next generation sequencing, and even "epigenetic sequencing."