CAREER: Development of DNA polymerases capable of high fidelity modified DNA synthesis

众所周知，DNA是对所有生命形式的信息进行编码的材料。除了这个角色，DNA也是现代生物技术中最有价值的工具之一。基础技术，如聚合酶链式反应和DNA测序，以及新兴技术，如DNA编码药物发现，都依赖于DNA的独特功能。然而，基于DNA的工具的有用性受到生物环境中DNA易受破坏的限制。通过这一奖项，化学部生命过程化学计划资助克莱蒙特·麦肯纳学院、皮策学院和斯克里普斯学院W.M.Keck科学系的Aaron Leconte博士开发蛋白质，这种蛋白质可以合成在生物环境中比自然DNA更稳定的化学修饰形式的DNA。因此，修饰后的DNA有可能扩大基于DNA的工具的应用，例如高通量DNA测序和用于许多治疗和临床诊断的合成生物学。这样的研究可能会通过改善人类健康而造福社会。莱孔特教授结合本科生实验室课程的研究经验，为这些学生的STEM研究生学习和职业生涯做好准备。他还在开发视频，记录现任和曾经的本科生的研究经验，可以用来激励和吸引更多的学生参加STEM研究。这项研究项目旨在开发突变的DNA聚合酶，能够高保真地合成被称为M-DNA的修饰形式的DNA。这种DNA对核酸酶有抵抗力。M-DNA通常不会被天然DNA聚合酶扩增。突变的DNA聚合酶能够合成长的、修饰的DNA，直到最近才被发现。虽然这些突变的DNA聚合酶是向前迈出的令人兴奋的一步，但这些酶经常出错，限制了它们的应用。莱孔特实验室的研究重点是最近发现的突变DNA聚合酶SFP1的特性和工程。初步数据表明，这种酶能够合成M-DNA，而且它比以前鉴定的其他M-DNA聚合酶的错误要少得多。勒孔特团队开发了高通量测序方法，可以定量评估M-DNA合成。通过这些方法获得的高通量测序数据为工程工作提供了信息，以生产在M-DNA合成过程中具有较低错误频率的DNA聚合酶。新的聚合酶能够以更高的保真度执行修饰的DNA合成，具有在从临床诊断到合成生物学等领域的一系列应用的潜力。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

DNA is widely known as the material that encodes information in all life forms. Besides this role, DNA is also one of the most valuable tools in modern biotechnology. Foundational technologies, such as the polymerase chain reaction and DNA sequencing, as well as emerging technologies, such as DNA-encoded drug discovery, rely on the unique functions of DNA. However, the usefulness of DNA-based tools is limited by DNA's susceptibility to destruction in a biological environment. With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Aaron Leconte from the W. M. Keck Science Department of Claremont McKenna, Pitzer, and Scripps Colleges to develop proteins that can synthesize chemically-modified forms of DNA that are more stable than natural DNA in a biological environment. Consequently, the modified DNAs have the potential to expand the applications of DNA-based tools, such as high throughput DNA sequencing and synthetic biology used in a number of therapeutic and clinical diagnostics. Such research may benefit society by improving human health. Professor Leconte integrates research experiences in laboratory courses for undergraduate students to prepare these students for STEM graduate studies and careers. He is also developing videos that document the research experiences of current and former undergraduate students that can be used to inspire and attract more students to STEM studies. This research project seeks to develop mutant DNA polymerases capable of high fidelity synthesis of modified forms of DNA called M-DNA. This DNA is resistant to nucleases. M-DNAs are generally not amplified by native DNA polymerases. Mutant DNA polymerases capable of synthesizing long, modified DNAs have been identified only recently. While these mutant DNA polymerases represent an exciting step forward, these enzymes make frequent errors, limiting their application. The research in the Leconte laboratory focuses on the characterization and engineering of a recently discovered mutant DNA polymerase, SFP1. Preliminary data suggest that this enzyme is capable of M-DNA synthesis and that it makes far fewer errors than other previously characterized M-DNA polymerases. The Leconte group develops high throughput sequencing methods that can quantitatively evaluate M-DNA synthesis. The high-throughput sequencing data obtained by these methods inform engineering efforts to produce DNA polymerases that have a lower error frequency during M-DNA synthesis. The new polymerases that can perform modified DNA synthesis with higher fidelity have the potential to enable an array of applications in fields that range from clinical diagnostics to synthetic biology.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.