Chemoproteomic profiling unveils binding and functional diversity of endogenous proteins that interact with endogenous triplex DNA

Triplex DNA structures, formed by a third DNA strand wrapped around the major groove of double helix, are key molecular regulators and genomic threats that are pharmacologically exploitable. However, the regulatory network governing triplex DNA dynamics are poorly understood. Here we performed chemoproteomic profiling of triplex DNA interactome in live cells to address this knowledge gap. We developed and validated a chemical probe that exhibits exceptional specificity for recognizing triplex DNA structures. By employing a co-binding-mediated proximity capture strategy, we enriched triplex DNA interactome for quantitative proteomics analysis. This enabled the identification of a comprehensive list of triplex DNA interacting proteins, characterized by diverse binding properties and regulatory mechanisms in native chromatin context. As a demonstration, we further validated DDX3X as the first ATP-independent helicase capable of resolving triplex DNA structures with 5' overhangs on the third DNA strand to prevent triplex-DNA-induced DNA damages. Overall, our triplex DNA interactome offers a valuable resource for investigating the biology of triplex DNA in both health and disease.