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International audienceThe quest for chemicals able to operate at selected genomic loci in a spatiotemporally controlled manner is desirable to create manageable DNA damages. Mounting evidence now shows that alternative DNA structures, including G-quadruplexes and branched DNA (or DNA junctions), might hamper proper progression of replication fork, thus triggering DNA damages and genomic instability. Therefore, small molecules that stabilize these DNA structures are currently scrutinized as a promising way to create genomic defects that cannot be dealt with properly by cancer cells. While much emphasis has been recently given to G-quadruplexes and related ligands, we report herein on three-way DNA junctions (TWJ) and related ligands. We first highlight the biological implications of TWJ and their strategic relevance as triggers for replicative stress. Then, we describe a new in vitro high-throughput screening assay, TWJ-Screen, which allows for identifying TWJ ligands with both high affinity and selectivity for TWJ over other DNA structures (duplexes and quadruplexes), in a convenient and unbiased manner as demonstrated by the screening of a library of 25 compounds from different chemical families. TWJ-Screen thus represents a reliable mean to uncover molecular tools able to foster replicative stress through an innovative approach, thus providing new strategic opportunities to combat cancers

TWJ-Screen: an isothermal screening assay to assess ligand/DNA junction interactions in vitro