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

职业：开发能够进行高保真修饰 DNA 合成的 DNA 聚合酶

• 批准号: 1752924
• 负责人: Aaron Leconte
• 金额: $ 40万
• 依托单位: Claremont McKenna College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1752924&HistoricalAwards=false
• 关键词: CAREER; Development; DNA; polymerases; capable

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.

DNA被广泛认为是所有生命形式中编码信息的材料。 除此之外，DNA也是现代生物技术中最有价值的工具之一。 基础技术，如聚合酶链反应和DNA测序，以及新兴技术，如DNA编码的药物发现，都依赖于DNA的独特功能。 然而，基于DNA的工具的有用性受到DNA在生物环境中对破坏的敏感性的限制。 有了这个奖项，化学部的生命过程化学项目资助了W。M.凯克科学系的克莱蒙麦肯纳，皮策和斯克里普斯学院开发蛋白质，可以合成化学修饰形式的DNA，比自然DNA在生物环境中更稳定。 因此，修饰的DNA具有扩展基于DNA的工具的应用的潜力，例如用于许多治疗和临床诊断的高通量DNA测序和合成生物学。 这样的研究可以通过改善人类健康来造福社会。教授勒孔特整合了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，记录当前和以前的本科生的研究经验，可用于激励和吸引更多的学生到STEM研究。该研究项目旨在开发能够高保真合成修饰形式DNA（称为M-DNA）的突变DNA聚合酶。 这种DNA对核酸酶具有抗性。 M-DNA通常不被天然DNA聚合酶扩增。能够合成长的、修饰的DNA的突变DNA聚合酶最近才被鉴定。 虽然这些突变DNA聚合酶代表了令人兴奋的进步，但这些酶经常出错，限制了它们的应用。Leconte实验室的研究重点是最近发现的突变DNA聚合酶SFP1的表征和工程。初步数据表明，这种酶能够合成M-DNA，并且它产生的错误比其他先前表征的M-DNA聚合酶少得多。Leconte小组开发了可以定量评估M-DNA合成的高通量测序方法。 通过这些方法获得的高通量测序数据为生产在M-DNA合成期间具有较低错误频率的DNA聚合酶的工程努力提供了信息。 新的聚合酶能够以更高的保真度进行修饰的DNA合成，有可能在从临床诊断到合成生物学的各个领域实现一系列应用。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。