Abstract
Site-specific endonucleases that exclusively cut single-stranded DNA have hitherto never been described and constitute a barrier to the development of ssDNA-based technologies. We identify and characterize one such family, from the GIY-YIG superfamily, of widely distributed site-specific single-stranded nucleases (Ssn) exhibiting unique ssDNA cleavage properties. By first comprehensively studying the Ssn homolog from Neisseria meningitidis, we demonstrate that it interacts specifically with a sequence (called NTS) present in hundreds of copies and surrounding important genes in pathogenic Neisseria. In this species, NTS/Ssn interactions modulate natural transformation and thus constitute an additional mechanism shaping genome dynamics. We further identify thousands of Ssn homologs and demonstrate, in vitro, a range of Ssn nuclease specificities for their corresponding sequence. We demonstrate proofs of concept for applications including ssDNA detection and digestion of ssDNA from RCA. This discovery and its applications set the stage for the development of innovative ssDNA-based molecular tools and technologies.