Chemical Determinants of DNA Ligase Fidelity

描述(申请人提供)：DNA连接酶越来越多地被用作分子生物学应用的工具，包括核苷酸序列检测、单核苷酸多态(SNP)检测、蛋白质检测和连接测序。随着生物技术领域对DNA连接酶需求的增加，对连接保真度的要求也越来越高。虽然已经采用了许多方法来提高连接的保真度，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以及改进的反应条件。在此，我们提出了一种略微不同的方法来提高结扎的严密性，该方法使用了一组化学修饰的结扎组件。在我们的三管齐下的方法中，我们提出了对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，因为这三种成分中的每一种都与连接酶中不同的关键氨基酸接触。人们希望，对DNA连接酶的核酸成分进行细微的化学改变，可能会反过来提高连接的保真度。 与公共卫生相关：分子诊断领域是一个不断增长的市场，目前估计价值205亿美元。在这些努力中使用的一类关键酶是DNA依赖的DNA连接酶。为了进一步提高DNA连接酶催化DNA连接反应的准确性，我们提出了化学修饰成分的研究。

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.