Chemical Determinants of DNA Ligase Fidelity

DNA 连接酶保真度的化学决定因素

• 批准号: 7537086
• 负责人: Natasha Paul
• 金额: $ 8.9万
• 依托单位: TRILINK BIOTECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-07 至 2010-01-06
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7537086
• 关键词: Amino Acids; Base Sequence; Binding Sites; Biological; Biological Assay; Biotechnology; Chemicals; Class; Condition; DNA; DNA Ligases; Deoxyribose; Detection; Diagnostic; Diphosphates; Discrimination; Engineering; Enzymes; Evaluation; Goals; Government; Hydroxyl Radical; Investigation; Left; Ligase; Ligation; Marketing; Measures; Minor Groove; Modification; Molecular; Molecular Biology; Nucleic Acids; Numbers; Object Attachment; Oligonucleotides; Performance; Persons; Play; Point Mutation; Polymerase Chain Reaction; Polymorphism Analysis; Proteins; Public Health; Purpose; Reaction; Relative (related person); Reporter; Role; Series; Single Nucleotide Polymorphism; Source; Specificity; Sulfur; System; Time; Variant; Vertebral column; Work; base; cofactor; improved; methylphosphonate; next generation; novel strategies; phosphodiester; phosphorothioate; recombinational repair; research study; sugar; tool

DESCRIPTION (provided by applicant): DNA ligases are more frequently being used as a tool in molecular biology applications that include nucleotide sequence detection, single nucleotide polymorphism (SNP) detection, protein detection, and "next generation" sequencing by ligation. With the increased demand for DNA ligases in the field of biotechnology, so is the need for improved fidelity of ligation. Although many approaches to improving ligation fidelity have been employed, most involve use of ligases from different biological sources, point mutations of key amino acid residues, and modified reaction conditions. Herein, we propose a slightly different approach to improving the stringency of ligation, which employs a set of chemically modified ligation components. In our three-pronged approach, we propose the evaluation of chemically modified variants of the ATP cofactor, the donor probe, and the acceptor probe. The significance of this approach is great because each of these three components makes contacts with different key amino acid contacts within the ligase. It is hoped that subtle chemical alterations to the nucleic acid component of DNA ligase may in turn induce an improvement in the fidelity of ligation. PUBLIC HEALTH RELEVANCE: The field of molecular diagnostics is a growing market with a current estimated value of $800 million. One key class of enzymes that are used in these efforts is the DNA dependent DNA ligases. To further improve the accuracy of the DNA joining reaction catalyzed by DNA ligases, we propose the investigation of chemically modified components.

描述（由申请人提供）：DNA 连接酶更常被用作分子生物学应用中的工具，包括核苷酸序列检测、单核苷酸多态性 (SNP) 检测、蛋白质检测和通过连接进行的“下一代”测序。随着生物技术领域对 DNA 连接酶的需求不断增加，对提高连接保真度的需求也在增加。尽管已经采用了许多提高连接保真度的方法，但大多数涉及使用来自不同生物来源的连接酶、关键氨基酸残基的点突变以及修改的反应条件。在此，我们提出了一种略有不同的方法来提高连接的严格性，该方法采用一组化学修饰的连接组件。在我们的三管齐下的方法中，我们建议评估 ATP 辅因子、供体探针和受体探针的化学修饰变体。这种方法的意义重大，因为这三个组件中的每一个都与连接酶内不同的关键氨基酸接触点接触。人们希望，DNA 连接酶核酸成分的细微化学改变可能会反过来导致连接保真度的提高。 公共健康相关性：分子诊断领域是一个不断增长的市场，目前估计价值为 8 亿美元。在这些工作中使用的一类关键酶是 DNA 依赖性 DNA 连接酶。为了进一步提高DNA连接酶催化的DNA连接反应的准确性，我们建议对化学修饰成分进行研究。