PROJECT SUMMARY Our research program seeks to develop innovative technologies to investigate the role of ADP-ribosylation, an under-studied DNA modification implicated in critical biological processes, including DNA repair, genome replication, and pathogen defense. Though prevalent across all kingdoms of life, from bacteria to humans, the precise function and extent of DNA ADP-ribosylation remain elusive. Current knowledge largely stems from studies on the bacterial DarT-DarG toxin-antitoxin system, and more recently, the discoveries of DNA modification by the anticancer drug target PARP1 in human cells. One significant challenge in the field is the absence of technology capable of identifying DNA ADP-ribosylation sites across the whole genome. Past efforts have involved meticulous mutagenesis using radioactive labeling on predefined oligo sequences or breaking down the genome to individual bases for modification assessment. However, these methods are limited in their ability to provide the broader genomic context of DNA ADP-ribosylation. Our goal is to bridge this gap through the development of two complementary techniques, using the well- characterized DarT-mediated ADP-ribosylation as our model. The first repurposes tools we have developed to study protein ADP-ribosylation and applies them to DNA, using high-throughput Illumina-based sequencing to pinpoint the modification site. The second approach involves nanopore sequencing, using a modified transmembrane protein as both a channel for DNA passage and a biosensor for base identification. By monitoring changes in electric current, we can distinguish specific bases and detect DNA modifications. To optimize this method, we will collaborate with leading nanopore epigenetics expert Dr. Winston Timp at Hopkins and adapt established machine learning approaches for electric current data analyses. We will test and refine our methods using DarT-modified oligos with defined sequences and genomic DNA standards. In summary, this program aims to unravel the complexities of DNA ADP-ribosylation using cutting-edge sequencing methods. These strategies will enable the investigation of the sequence context of ADP- ribosylation sites and, in due course (beyond the current scope), the study of motifs from diverse pathogen toxins and other modifying enzymes such as human PARPs. By developing tools to reveal the mechanisms of DNA repair, gene regulation, and pathogen defense, we anticipate that our work may catalyze significant advancements in healthcare, potentially leading to the development of new treatment and detection tools for a range of diseases, from infections to cancers.

PROJECT NARRATIVE This research program aims to advance our understanding of ADP-ribosylation—a recently discovered DNA modification implicated in critical biological processes, including DNA repair, gene regulation, and pathogen defense. By developing new tools to study this modification, we can deepen our understanding of how our bodies protect against diseases, including infections and cancer. Ultimately, our work could pave the way for novel diagnostic tools and therapeutic strategies, potentially enhancing public health outcomes.