Chemical Determinants of DNA Ligase Fidelity

DNA 连接酶保真度的化学决定因素

• 批准号: 8012837
• 负责人: Natasha Paul
• 金额: $ 36.64万
• 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 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8012837
• 关键词: Amino Acids; Amino Sugars; Base Pairing; Base Sequence; Biological; Biotechnology; Buffers; Charge; Chemicals; DNA; DNA Ligases; Detection; Diagnostic; Discrimination; Effectiveness; Enzymes; Escherichia coli; Evaluation; Foundations; Generations; Goals; Government; Hydroxyl Radical; Investigation; Lead; Ligase; Ligation; Location; Marketing; Measures; Modification; Molecular; Molecular Biology; Nucleic Acids; Nucleotides; Oligonucleotides; Performance; Persons; Phase; Point Mutation; Polymorphism Analysis; Positioning Attribute; Proteins; Reaction; Role; Running; Series; Side; Single Nucleotide Polymorphism; Source; Specificity; Structure-Activity Relationship; T4 DNA Ligase; Testing; Variant; Vertebral column; Work; adenylate; alpha-thioadenosine triphosphate; analog; base; cofactor; commercialization; improved; inorganic phosphate; methylphosphonate; next generation; novel; novel strategies; phase 1 study; phase 2 study; phosphodiester; phosphonate; phosphorothioate; public health relevance; research study; success; sugar; technology development; 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 $20.5 billion. 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连接酶更频繁地用作分子生物学应用中的工具，所述分子生物学应用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“下一代”连接测序。随着生物技术领域中对DNA连接酶的需求增加，也需要提高连接的保真度。尽管已经采用了许多提高连接保真度的方法，但大多数方法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和改进的反应条件。在此，我们提出了一种稍微不同的方法来提高连接的严格性，其采用了一组化学修饰的连接组件。在我们的三管齐下的方法中，我们提出了ATP辅因子，供体探针和受体探针的化学修饰变体的评估。这种方法的意义是巨大的，因为这三种组分中的每一种都与连接酶内的不同关键氨基酸接触。希望DNA连接酶的核酸组分的细微化学改变可以反过来诱导连接保真度的改善。 公共卫生相关性：分子诊断领域是一个不断增长的市场，目前估计价值为205亿美元。在这些努力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，我们提出了化学修饰组分的研究。