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

人类DNA易受来自内源性和环境的化学和物理因子的影响， 来源，产生各种DNA修饰。研究已经记录了大量的DNA修饰， 包括超过50种内源性核碱基修饰和许多衍生自 环境化学品。某些DNA修饰在基因调控中起作用，而其他病变则具有 致突变和致病作用。最近的测序数据显示，DNA的分布 基因组中的修饰是不均匀的。在全基因组范围内绘制DNA修饰图对于 阐明它们在遗传调节、发育和发病机制中的作用。不幸的是，目前的方法 测序DNA修饰在灵敏度、特异性、分辨率 和吞吐量。该提案通过开发一种新的DNA测序方法来解决这些限制。 Illumina测序仪可以同时绘制10种以上的DNA修饰。成功完成本 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。这项研究利用了DNA修复的化学过程， 用于以单核苷酸分辨率对多个DNA修饰进行测序的特异性化学探针。这些新颖 化学物质捕获并富集脱碱基（AP）位点，这是DNA修复的中心中间体。此外，两种化学 在扩增过程中，探针作为唯一的定位码，允许测序读数。同时映射 不同的DNA损伤将通过偶联损伤特异性DNA修复酶与多重 测序该提案基于我们令人信服的数据，证明了两种合成药物的可行性 探针标记和富集AP DNA具有高特异性和灵敏度。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修饰目的二是合成另一种新的胞嘧啶测序化合物 修改和错配。预期的结果是，拟议的方法将解决一个主要的未满足的需求 在Illumina测序仪上对多个DNA修饰进行测序。从长远来看，先进的技术将 有助于产生高分辨率的各种DNA修饰的单核苷酸分辨率基因组图谱， 产量和成本效益的方式。这项研究意义重大，因为与其他Illumina相比， 基于方法，该技术将允许比现有技术更大的一个数量级改进， 方法在测序的修改数量，补充了最近的进展与PacBio和 纳米孔技术。该项目的创新在于开发新型化学探针， 便于富集，创造性地使用多种修复酶，以确保定位的准确性， 定位探针以允许扩增和测序读出。创新的方法将共同实现 前所未有的特异性和灵敏度，这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.