The hybridization-stabilization assay: a solution-based isothermal method for rapid screening and determination of sequence preference of ligands that bind to duplexed nucleic acids

The gene-to-drug quest will be most directly served by the discovery and de velopment of small molecules that bind to nucleic acids and modulate gene e xpression at the level of transcription and/or inhibit replication of infec tious agents. Full realization of this potential will require implementatio n of a complete suite of modern drug discovery technologies. Towards this e nd, here we describe our initial results with a new assay for identificatio n and characterization of novel nucleic acid binding ligands. It is based o n the well recognized property of stabilization of hybridization of complem entary oligonucleotides by groove and/or intercalation binding ligands. Unl ike traditional thermal melt methodologies, this assay is isothermal and, u nlike gel-based footprinting techniques, the assay also is performed in sol ution and detection can be by any number of highly sensitive, non-radioisot opic modalities, such as fluorescence resonance energy transfer, described herein. Thus, the assay is simple to perform, versatile in design and amena ble to miniaturization and high throughput automation. Assay validation was performed using various permutations of direct and competitive binding for mats and previously well studied ligands, including pyrrole polyamide and i ntercalator natural products, designed hairpin pyrrole-imidazole polyamides and furan-based non-polyamide dications. DNA specific ligands were identif ied and their DNA binding site size and sequence preference profiles were d etermined. A systematic approach to studying the relationship of binding se quence specificity with variation in ligand structure was demonstrated, and preferred binding sites in longer DNA sequences were found by pseudo-footp rinting, with results that are in accord with established findings. This as say methodology should promote a more rapid discovery of novel nucleic acid ligands and potential drug candidates.