Mapping Protein-ssDNA Interactions at Single Nucleotide Resolution by Dual Nucleases-Assisted Next-Generation Sequencing.

Protein interactions with single-stranded DNA (ssDNA) are critical for DNA repair, recombination, and other biological processes. We present a dual-nuclease-assisted next-generation sequencing method for mapping protein-ssDNA interactions at single-nucleotide resolution. By combining enzymatic digestion with Supernuclease and Snake Venom Phosphodiesterase, protein-bound ssDNA fragments are selectively protected from cleavage and sequenced, enabling precise binding landscape identification. Applying this approach to Escherichia coli single-stranded DNA-binding protein (EcSSB) revealed that secondary stem-loop structures modulate its binding preferences and distribution patterns. Our platform establishes a rapid, cost-effective, and high-resolution framework for studying dynamic protein-ssDNA interactions, supporting the analysis of both well-characterized and unexplored ssDNA-binding proteins.