| 摘要 |
A simple, nondestructive, and high throughput method for establishing DNA binding affinity and sequence selectivity is based on the loss of fluorescence derived from the displacement of ethidium bromide or thiazole orange from the DNA of interest or, in selected instances, the change in intrinsic fluorescence of a DNA binding agent itself. The method is applicable for assessing relative or absolute DNA binding affinities. Enlisting a library of hairpin deoxyoligonucleotides containing all 5 base pair (512 hairpins) or 4 base pair (136 hairpins) sequences displayed in a 96-well format, a compound's rank order binding to all possible sequences is generated resulting in a high resolution definition of its sequence selectivity using this fluorescent intercalator displacement (FID) assay. As such, the technique complements the use of footprinting or affinity cleavage for the establishment of DNA binding selectivity and provides the information at a higher resolution. The merged bar graphs generated by this rank order binding provide a qualitative way to compare, or profile, DNA binding affinity and selectivity. |
