This Phase I SBIR project is intended to demonstrate the utility of a system to efficiently screen small molecules for their effect on protein-DNA binding. DNA binding proteins are the fundamental regulators of gene expression through their recognition and binding to specific DNA sequences. Using therapeutics to restore normal DNA binding activity of a mutated protein (e.g., the tumor suppressor p53) has been a long-standing goal of pharmaceutical researchers, but has proven to be a challenging technical task. One reason is the lack of effective methods for rapidly and comprehensively profiling relevant proteins and small molecule leads. New tools are needed to help screen for potential chemical and biological methods that can modulate the expression of specific genes and gene families. DNA arrays offer a particularly promising approach to rapid screening, but conventional DNA arrays can test only a single solution condition or compound per array. We propose to develop a novel type of DNA array that uses the combination of many micrometer-size lines of DNA probes affixed to a planar surface and intersecting microfluidic channels that can carry as many as several hundred discrete solutions across the chip. Each """"""""intersection"""""""" is a unique reaction site, enabling more than 10,000 simultaneous assays per array. For detection, this technology uses a label-free technique that enables kinetic analysis, surface plasmon resonance imaging (SPRi), which is a microarray-suitable adaptation of conventional SPR measurement techniques. The objective will be to demonstrate the feasibility of our strategy by developing a four-element DNA LineArray and a four-channel microfluidics system to demonstrate modulation of cooperative protein-DNA binding. The probe DNA will be affixed to the planar surface and the microchannels will transport the potential binding proteins. SPRi will determine whether cooperative binding has occurred. Our preliminary results indicate low nanomolar-range sensitivity for this SPRi -based assay. We anticipate that with our system, researchers can study protein-DNA binding for many recombinant and cellular DNA binding proteins. In Phase II, we plan to expand the scope of the research described in this Phase I proposal by increasing the density of the DNA LineArrays and microchannels and by studying DNA binding activities of cellular extracts. ? ?
