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

人类 DNA 容易受到内源性和环境性化学和物理因素的影响 源，产生各种 DNA 修饰。研究记录了大量的 DNA 修饰， 包括超过 50 个内源核碱基修饰和许多源自 环境化学品。某些 DNA 修饰在基因调控中发挥作用，而其他损伤则具有 致突变和致病作用。最近的测序数据显示DNA的分布 基因组的修饰并不统一。在全基因组范围内绘制 DNA 修饰图对于 阐明它们在遗传调控、发育和发病机制中的作用。不幸的是，目前的方法 DNA修饰测序在灵敏度、特异性、分辨率、 和吞吐量。该提案通过开发一种新颖的 DNA 测序方法来解决这些限制 Illumina 测序仪可同时绘制 10 多个 DNA 修饰图。本次活动的顺利完成 该提案将促进 PI 的长期目标，即破译 DNA 修饰的功能重要性 诱变和基因调控。该研究利用了 DNA 修复的化学原理，并取得了高度进展 用于以单核苷酸分辨率对多个 DNA 修饰进行测序的特定化学探针。这些小说 化学物质捕获并富集脱碱基 (AP) 位点，这是 DNA 修复的核心中间体。此外，还有两种化学 探针在扩增过程中充当独特的定位代码，允许测序读数。同步测绘 通过将损伤特异性 DNA 修复酶与多重酶相结合，可以实现不同 DNA 损伤的修复。 测序。该提案基于我们令人信服的数据，证明了两种合成的可行性 探针以高特异性和灵敏度标记和富集 AP DNA。拟议的测序平台将 通过两个目标进一步开发和优化。目标 1 是优化同步测序的工作流程 多重烷基化 DNA 修饰。目标 2 是合成另一种用于胞嘧啶测序的新型化合物 修改和错误。预期结果是所提出的方法将解决主要的未满足需求 在 Illumina 测序仪上对多个 DNA 修饰进行测序。从长远来看，发达的技术将 有助于以高通量生成各种 DNA 修饰的单核苷酸分辨率基因组图谱 吞吐量和成本效益的方式。拟议的研究意义重大，因为与其他 Illumina- 基于方法，该技术将比现有技术实现一个数量级以上的改进 测序修饰数量的方法，补充了 PacBio 和 PacBio 的最新进展 纳米孔技术。该项目的创新点在于开发新型化学探针 方便富集，创造性地利用多种修复酶保证图谱准确度，两种独特的 定位探针允许扩增和测序读出。共同努力，创新方法将实现 前所未有的特异性和灵敏度，降低了测序深度和成本。

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.