Directed Evolution of DNA and RNA Polymerases

DNA 和 RNA 聚合酶的定向进化

• 批准号: 6738558
• 负责人: REBECCA C HOLMBERG
• 金额: $ 4.3万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6738558
• 关键词: DNA directed DNA polymerase; DNA directed RNA polymerase; chemical synthesis; directed evolution; genetic library; genetic transcription; mutant; nucleic acid biosynthesis; nucleic acid structure; nucleobase; nucleotides; phage display; polymerase chain reaction; technology /technique development

DESCRIPTION (provided by applicant): Our goal is to evolve enzymes that synthesize DNA and RNA containing unnatural nucleobases using phage display technology. Our objective is to expand the genetic alphabet and to create novel, stable, replicable biopolymers useful in biotechnical applications. Specific Aim I involves evolving DNA polymerases that incorporate unnatural nucleobases. First, a library of mutant polymerases for the Stoffel fragment DNA polymerase will be generated (Specific Aim, I. a). This library will then be displayed on phage and used to select for efficient incorporation, extension, and fidelity of unnatural deoxynucleobases with a template attached to the same phage particle (Specific Aim I.b). Concurrently, for Specific Aim II similar studies will be conducted for transcription with RNA polymerases. An assay will be developed to determine the kinetic capabilities of T7 RNA polymerase (Specific Aim II.a). The directed evolution selection process will then be employed for non-canonical nucleotides that do not show ease of incorporation and extension by the wild-type enzyme. First, T7 RNA polymerase will be displayed on phage (Specific Aim II.b). Once successful, a mutant library of T7 polymerase will be generated for display (Specific Aim II.c). Finally, a selection with the mutant library will be executed for incorporation of two types of unnatural ribonucleotides (Specific Aim II.d): One set comprised of sugar modified nucleotides, and another set composed of modified nucleobases.

描述（由申请人提供）：我们的目标是使用噬菌体展示技术进化合成含有非天然核碱基的DNA和RNA的酶。我们的目标是扩大遗传字母表，并创造新的，稳定的，可复制的生物聚合物在生物技术应用中有用。特定目标I涉及进化并入非天然核碱基的DNA聚合酶。首先，产生斯托费尔片段DNA聚合酶的突变聚合酶文库（Specific Aim，I. a）。然后将该文库展示在噬菌体上，并用于选择非天然脱氧核碱基与连接至相同噬菌体颗粒的模板的有效掺入、延伸和保真度（特异性目标I.B）。同时，对于特异性目的II，将对RNA聚合酶的转录进行类似的研究。将开发一种测定T7 RNA聚合酶动力学能力的试验（特异性目的II. a）。然后将定向进化选择过程用于不显示易于通过野生型酶掺入和延伸的非规范核苷酸。首先，将T7 RNA聚合酶展示在噬菌体上（特异性Aim II.B）。一旦成功，将生成T7聚合酶的突变体文库用于展示（特异性目的II. c）。最后，将使用突变体文库进行选择以掺入两种类型的非天然核糖核苷酸（特定目的II.d）：一组由糖修饰的核苷酸组成，另一组由修饰的核碱基组成。