Protein-DNA interactions are essential for fundamental cellular processes such as transcription, DNA damage repair, and apoptosis. As such, small molecule disruptors of these interactions could be powerful tools for investigation of these biological processes, and such compounds would have great potential as therapeutics. Unfortunately, there are few methods available for the rapid identification of compounds that disrupt protein-DNA interactions. Here we show that photonic crystal (PC) technology can be utilized to detect protein-DNA interactions, and can be used in a high-throughput screening mode to identify compounds that prevent protein-DNA binding. The PC technology is used to detect binding between protein-DNA interactions that are DNA-sequencedependent (the bacterial toxin-antitoxin system MazEF) and those that are DNA-sequenceindependent (the human apoptosis inducing factor (AIF)). The PC technology was further utilized in a screen for inhibitors of the AIF-DNA interaction, and through this screen aurin tricarboxylic acid was identified as the first in vitro inhibitor of AIF. The generality and simplicity of the photonic crystal method should enable this technology to find broad utility for identification of compounds that inhibit protein-DNA binding.High-throughput screening (HTS) of compound collections is now a staple of modern drug discovery. In the most common incarnation, in vitro enzyme inhibition screens of large (>100,000 members) compound libraries are conducted using substrates that provide an easily quantified chromogenic/ fluorescent readout. Such screens have led to the discovery of many novel enzyme inhibitors and drug leads (1,2). Unfortunately, many potential drug targets are not enzymes, and thus for these systems high-throughput methods are needed that go beyond enzyme inhibition assays and directly report on small molecule-protein binding events.One area in which small molecule ligands for nonenzyme proteins would be useful is in the disruption of protein-macromolecule interactions. The identification of compounds that perturb protein-protein or protein-nucleic acid interactions is extremely challenging (3-6), and this is partly due to the paucity of good high-throughput screens. Successes in modulating protein-protein and protein-nucleic acid interactions with small molecules fall into a few classes: surface receptor-ligand interactions (integrins (7-9), IL-1/2 (10,11), TNFα (12)), (47). Another limitation is the potential for false positives due to fluorescent compounds, which is an inherent limitation of any fluorescence-based HTS method. Given the largely unexplored pharmacological realm that is protein-nucleic acid interactions, HTS assays independent of fluorescent tags would be extremely useful, especially in those cases where fluorescence anisotropy is not possible. In this report we describe the first use of photonic crystal technology for the development of an assay capable of detecting protein-DNA binding and further apply it in a high-throughput screening mode fo...

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