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

人类DNA容易受到来自内源性和环境的化学和物理因素的影响 来源，产生各种DNA修饰。研究记录了过多的DNA修饰， 包括50多种内源性碱基修饰和许多源自 环境化学品。某些DNA修饰在基因调节中起作用，而其他损伤则有 致突变和致病作用。最近的测序数据显示，DNA的分布 基因组中的修饰并不是统一的。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测序修饰在敏感性、特异性、分辨率、 和吞吐量。这项建议通过开发一种新的DNA测序方法来解决这些限制 Illumina测序仪可以同时映射10个以上的DNA修饰。成功地完成这项工作 该提案将促进PI破译DNA修饰在功能上的重要性的长期目标 诱变和基因调控。这项研究利用了DNA修复的化学原理，并取得了很大进展 用于在单核苷酸分辨率下对多个DNA修饰进行测序的特定化学探针。这些小说 化学物质捕获和丰富碱基(AP)位点，这是DNA修复的中心中间体。此外，还有两种化学物质 探针在扩增过程中充当唯一的定位码，允许测序读出。同步映射 不同的DNA损伤将通过将损伤特异性DNA修复酶与多重偶联来实现 测序。这项建议是基于我们令人信服的数据，证明了两种人工合成技术的可行性 标记和富集AP DNA的探针具有高度的特异性和敏感性。拟议的测序平台将 通过两个目标进一步发展和优化。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化合物 修饰和错配。预期的结果是，拟议的方法将解决一个主要的未得到满足的需求 在对Illumina测序仪上的多个DNA修饰进行测序时。从长远来看，开发的技术将 帮助生成用于各种DNA修饰的单核苷酸分辨率基因组图 吞吐量和成本效益高的方式。这项拟议的研究意义重大，因为与其他发光材料相比- 基于方法，该技术将允许比现有的改进一个数量级以上 方法在修改的数量测序，补充最近的进展与PacBio和 纳米孔技术。该项目的创新之处在于开发了新的化学探针，以 方便丰富，创造性地使用多个修复酶，以确保映射的准确性，并在两个独特的 定位探头，允许放大和测序读出。共同努力，创新的方法将实现 前所未有的特异性和敏感性，从而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.