PROJECT SUMMARY This proposal aims to develop the first potent and specific antagonists of the pro-mutagenic effects of APOBEC DNA deaminase enzymes in cells. Access to whole genome sequences has helped reveal common mutational signatures across various cancers. One such prominent mutational signature, termed SBS2, includes hypermutated clusters containing a high density of C to T/G substitutions on the same strand, a phenomenon known as kataegis. These features, in addition to the enrichment of the mutations in 5’-TC motifs, point to a causative role for APOBEC3 (A3) family enzymes. While these enzymes normally mutate and restrict retroviruses or retroelements, studies have confirmed that two family members, APOBEC3A (A3A) and APOBEC3B (A3B), have a prominent role in pathological mutagenesis targeting the host genome. The relative contributions of each enzyme remain a matter of vigorous debate, as genetic approaches specifically targeting A3A or A3B are limited by their high homology to one another and juxtaposition on the genome. Furthermore, no molecular tools currently exist that can disrupt A3 function. There is therefore a pressing need for molecular probes that can either inhibit or deplete A3 enzymes from cells. This proposal builds on the hypothesis that insights into the mechanism and substrate selectivity of A3 enzymes can be leveraged to design potent and specific antagonists. Specifically, we have demonstrated that mechanism-based inhibitor moieties can be presented in preferred secondary structures and engineered into exonuclease-resistant DNA molecules to yield potent nanomolar inhibitors of A3A. These molecules present the opportunity for facile functionalization, which can be utilized to convert classical inhibitors into molecules capable of inducing the catalytic degradation of the target APOBEC enzymes in cells, via an unprecedented combination of nucleic acid inhibitors and proteasome targeting (PROTAC) technology. Taken together, this proposal aims to fill a critical gap in the field by introducing tools to perturb APOBEC function in cells in order to reveal their underlying biology and offer a roadmap for potential therapeutics.