Novel Chemical Probes for Sequencing Multiple DNA Modifications at Single-Nucleotide Resolution

用于以单核苷酸分辨率对多个 DNA 修饰进行测序的新型化学探针

• 批准号: 10675459
• 负责人: Linlin Zhao
• 金额: $ 30.08万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-02 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10675459
• 关键词: Address; Base Pairing; Chemicals; Chemistry; Code; Complement; Coupling; Creativeness; Cytosine; DNA; DNA Damage; DNA Modification Process; DNA Repair; DNA Repair Enzymes; DNA glycosylase; DNA lesion; DNA mapping; DNA sequencing; Data; Deamination; Detection; Development; Disease; Endogenous Factors; Engineering; Ensure; Epigenetic Process; Etiology; Frequencies; Gene Expression Regulation; Generations; Genetic; Genome; Genomic DNA; Genomics; Goals; Hela Cells; Intervention; Label; Lesion; Location; Malignant Neoplasms; Maps; Methods; Mismatch Repair Deficiency; Mitochondrial DNA; Modeling; Modification; Mutagenesis; Nucleotides; Outcome; Pathogenesis; Pathogenicity; Pathology; Pattern; Play; Polymerase Chain Reaction; Positioning Attribute; Predisposition; Preparation; Property; Regulation; Research; Resolution; Role; Sampling; Sensitivity and Specificity; Signal Transduction; Site; Source; Specificity; Spottings; Technology; Testing; Time; adduct; base; cost; cost effective; design; environmental chemical; genome-wide; human DNA; improved; innovation; insight; nanopore; novel; nucleobase; public health relevance; reference genome; repair enzyme; repaired; sequencing platform; therapeutically effective

Human DNA is susceptible to chemical and physical agents from endogenous and environmental sources, producing various DNA modifications. Research has documented a plethora of DNA modifications, including more than 50 endogenous nucleobase modifications and many covalent adducts derived from environmental chemicals. Certain DNA modifications function in gene regulation, whereas other lesions have mutagenic and pathogenic effects. Recent sequencing data have revealed that the distribution of DNA modifications in the genome is not uniform. Mapping DNA modifications on a genome-wide scale is critical for clarifying their roles in genetic regulation, development, and pathogenesis. Unfortunately, current methods for sequencing DNA modifications suffer from one or more drawbacks in terms of sensitivity, specificity, resolution, and throughput. This proposal addresses these limitations by developing a novel DNA sequencing method on Illumina sequencers to map more than 10 DNA modifications simultaneously. The successful completion of this proposal will facilitate the PI’s long-term goal of deciphering the functional importance of DNA modifications in mutagenesis and gene regulation. The research exploits the chemistry of DNA repair and develops highly specific chemical probes for sequencing multiple DNA modifications at single-nucleotide resolution. These novel chemicals capture and enrich abasic (AP) sites, a central intermediate in DNA repair. In addition, two chemical probes serve as unique locator codes during amplification, allowing sequencing readout. Simultaneous mapping of different DNA lesions will be achieved through coupling lesion-specific DNA repair enzymes with multiplex sequencing. The proposal is grounded on our compelling data demonstrating the feasibility of two synthetic probes to label and enrich AP DNA with high specificity and sensitivity. The proposed sequencing platform will be further developed and optimized via two aims. Aim 1 is to optimize the workflow for simultaneous sequencing multiple alkylated DNA modifications. Aim 2 is to synthesize another novel compound for sequencing cytosine modifications and mispairs. The expected outcome is that the proposed method will address a major unmet need in sequencing multiple DNA modifications on Illumina sequencers. In the long run, the developed technology will aid the generation of single-nucleotide resolution genomic maps for various DNA modifications in a high- throughput and cost-effective manner. The proposed research is significant because, compared to other Illumina- based methods, the technology will allow greater than one order of magnitude improvement over existing methods in the number of modifications sequenced, complementing the recent progress with PacBio and Nanopore technologies. The innovation of the project lies in the development of novel chemical probes to facilitate enrichment, creative use of multiple repair enzymes to ensure mapping accuracy, and the two unique locator probes to allow amplification and sequencing readout. Together, the innovative method will achieve unprecedented specificity and sensitivity, which reduce sequencing depth and costs.

人DNA容易受到内源和环境的化学和物理剂的影响 来源，产生各种DNA修饰。研究记录了大量DNA修饰， 包括超过50多个内源性核酶修饰和许多共价加合物 环境化学品。基因调控中某些DNA修饰功能，而其他病变具有 诱变和致病作用。最近的测序数据表明DNA的分布 基因组的修饰不是均匀的。在全基因组范围内映射DNA修饰对于 阐明它们在遗传调节，发育和发病机理中的作用。不幸的是，当前的方法 在敏感性，特异性，分辨率方面，测序DNA修饰具有一个或多个缺点 和吞吐量。该建议通过开发一种新的DNA测序方法来解决这些局限性 Illumina测序仪简单地绘制了10个以上的DNA修饰。成功完成 提案将促进PI的长期目标，即破译DNA修改的功能重要性 诱变和基因调节。该研究利用了DNA修复的化学反应，并高度发展 在单核苷酸分辨率下测序多种DNA修饰的特定化学问题。这些小说 化学物质捕获和富集脓肿位点，这是DNA修复中的中间中间。另外，两个化学物质 问题在放大过程中用作独特的定位器代码，从而允许测序读数。同时映射 通过将病变特异性的DNA修复酶与多路复用耦合，将实现不同的DNA病变 测序。该提案基于我们引人注目的数据，证明了两个合成的可行性 具有高特异性和灵敏度标记和富集AP DNA的问题。拟议的测序平台将 通过两个目标进一步开发和优化。 AIM 1是优化简单测序的工作流程 多种烷基化的DNA修饰。 AIM 2是合成另一种新型化合物以测序胞嘧啶 修改和错误。预期的结果是，所提出的方法将满足不满足的主要需求 在测序Illumina测序仪上多次DNA修饰时。从长远来看，开发的技术将 有助于产生单核苷酸分辨率基因组图，以在高位中进行各种DNA修饰 吞吐量和成本效益的方式。拟议的研究很重要，因为与其他Illumina-相比 基于方法，该技术将允许比现有的一个数量级以上 修改次数的方法是测序的，完成了PACBIO和PACBIO和 纳米技术。该项目的创新在于开发新的化学问题 促进富集，创造性地使用多种维修酶以确保映射准确性以及两个独特的 定位器问题可以放大和测序读数。共同的创新方法将实现 空前的特异性和敏感性，从而降低了测序深度和成本。