Directed Evolution of DNA and RNA Polymerases

DNA 和 RNA 聚合酶的定向进化

• 批准号: 6850684
• 负责人: REBECCA C HOLMBERG
• 金额: $ 1.03万
• 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 2005-03-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6850684
• 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; 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)：我们的目标是利用噬菌体展示技术进化出合成含有非天然核苷酸碱基的DNA和RNA的酶。我们的目标是扩大遗传字母表，创造可用于生物技术应用的新型、稳定、可复制的生物聚合物。具体目标I涉及进化DNA聚合酶，该聚合酶结合了非天然的核苷酸碱基。首先，将建立Stoffel片段DNA聚合酶的突变聚合酶文库(特定目标，I.a)。该文库随后将被展示在噬菌体上，并用于通过连接到相同噬菌体颗粒上的模板来选择非天然脱氧核苷酸的有效结合、延伸和保真度(特定目的I.B)。同时，针对特定目的II，将进行类似的RNA聚合酶转录研究。将开发一种测试来确定T7RNA聚合酶的动力学能力(特定目标二.a)。然后，定向进化选择过程将被用于不显示出容易被野生型酶结合和延伸的非规范核苷酸。首先，T7RNA聚合酶将展示在噬菌体上(特定目的II b)。一旦成功，T7聚合酶的突变文库将被生成用于展示(特定目的II c)。最后，将与突变体文库一起进行选择，以纳入两种类型的非天然核糖核苷酸(特定目标二.d)：一组由糖修饰的核苷酸组成，另一组由修饰的核苷酸组成。