Identification of Three-Way DNA Junction Ligands through Screening of Chemical Libraries and Validation by Complementary in Vitro Assays

The
human genome is replete with repetitive DNA sequences that
can fold into thermodynamically stable secondary structures such as
hairpins and quadruplexes. Cellular enzymes exist to cope with these
structures whose stable accumulation would result in DNA damage through
interference with DNA transactions such as transcription and replication.
Therefore, the chemical stabilization of secondary DNA structures
offers an attractive way to foster DNA transaction-associated damages
to trigger cell death in proliferating cancer cells. While much emphasis
has been recently given to DNA quadruplexes, we focused here on three-way
DNA junctions (TWJ) and report on a strategy to identify TWJ-targeting
agents through a combination of in vitro techniques (TWJ-screen, polyacrylamide
gel electrophoresis, fluorescence resonance energy transfer-melting,
electrospray ionization mass spectrometry, dialysis equilibrium, and
sulforhodamine B assays). We designed a complete workflow and screened
1200 compounds to identify promising TWJ ligands selected on stringent
criteria in terms of TWJ-folding ability, affinity, and selectivity.