RESUMEN
Netropsin is one of the first ligands to be discovered that selectively binds to the minor groove of DNA and is actively used as a scaffold for developing potential anticancer and antibiotic agents. The mechanism by which netropsin binds to hairpin DNA remains controversial with two competing mechanisms having been proposed. In one mechanism, netropsin binding induces a hairpin-to-duplex DNA transition. Alternatively, netropsin binds in two thermodynamically different modes at a single duplexed AATT site. Here, results from native mass spectrometry (MS) with nanoscale ion emitters indicate that netropsin can simultaneously and sequentially bind to both hairpin and duplex DNA. Duplex DNA was not detected using conventional MS with larger emitters because nanoscale emitters significantly reduce the extent of salt adduction to ligand-DNA complex ions, including in the presence of relatively high concentrations of nonvolatile salts. Based on native MS and polyacrylamide gel electrophoresis results, the abundances of hairpin and duplex DNA are unaffected by the addition of netropsin. By native MS, the binding affinities for five ligand-DNA and DNA-DNA interactions can be rapidly obtained simultaneously. This research indicates a "simultaneous binding mechanism" for the interactions of netropsin with DNA.