Discovery of the widespread site-specific single-stranded nuclease family Ssn

Martin Chenal, Alex Rivera-Millot, Luke B. Harrison, Ahmed S. Khairalla, Cecilia Nieves, Ève Bernet, Mansoore Esmaili, Manel Belkhir, Jonathan Perreault & Frédéric J. Veyrier

Nature Communications volume 16, Article number: 2388 (2025) 

Published: 10 March 2025

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.

See https://www.nature.com/articles/s41467-025-57514-1

Figure 1: Hairpin repeats surround key genes in Neisseria and other bacteria.

a Logos and predicted secondary structures of repeated sequences from N. meningitidis (dRS3, NTS) and N. wadsworthii (NTS_var). For the secondary structure, the different colors represent the base-pair probability. b Core-genome phylogeny of Neisseria species with their associated repeated elements. The four color gradients represent the relative abundance of the different enumerated elements. c Circular map of the N. meningitidis 8013 genome with its DUS (green) and NTS (red) repeats depicted. Major clusters of NTS are highlighted with black arrows pointing to the corresponding genomic landscape. Those major clusters are emphasized with schematic representations of the loci of interest (not drawn to the scale). d Logos and predicted structures and (e) whole-genome distribution of SRM repeats in R. wittichi and W. pipientis.