Chemical Determinants of DNA Ligase Fidelity

描述（由申请人提供）：DNA连接酶更频繁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修改反应条件。在这里，我们提出了一种稍微不同的方法来提高结扎的严密性，它采用了一组化学修饰的结扎成分。在我们的三管齐下的方法中，我们提出了ATP辅助因子、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内不同的关键氨基酸接触。希望对DNA连接酶的核酸成分的细微化学改变可能反过来诱导连接保真度的提高。

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.