Transciption Factor Interactions Using Addressed Arrays

描述(由申请人提供)：这个第一阶段的SBIR项目旨在展示一个系统的效用，以有效地筛选小分子对蛋白质-DNA结合的影响。DNA结合蛋白通过识别和结合特定的DNA序列，是基因表达的基本调节因子。使用治疗药物来恢复突变蛋白(例如肿瘤抑制基因p53)的正常DNA结合活性一直是制药研究人员的长期目标，但事实证明这是一项具有挑战性的技术任务。一个原因是缺乏快速和全面分析相关蛋白质和小分子铅的有效方法。需要新的工具来帮助筛选潜在的化学和生物方法，这些方法可以调节特定基因和基因家族的表达。DNA阵列提供了一种特别有希望的快速筛选方法，但传统的DNA阵列只能测试每个阵列的单一溶液条件或化合物。我们建议开发一种新型的DNA阵列，它使用固定在平面上的许多微米大小的DNA探针直线和相交的微流控通道的组合，可以在芯片上携带多达数百个离散的解决方案。每个“交叉点”都是一个独特的反应部位，每个阵列可以同时进行10,000多次分析。对于检测，这项技术使用了一种无标记技术，实现了动力学分析，表面等离子体共振成像(SPRI)，这是一种适合于微阵列的传统SPR测量技术的改编。我们的目标是通过开发一个四元DNA线阵和一个四通道微流控系统来演示协同蛋白质-DNA结合的调节，从而证明我们策略的可行性。探针DNA将被固定在平面上，微通道将运输潜在的结合蛋白。SPRI将确定是否发生了协作绑定。我们的初步结果表明，这种基于SPRI的检测方法具有较低的纳摩尔范围灵敏度。我们预计，使用我们的系统，研究人员可以研究许多重组和细胞DNA结合蛋白的蛋白质-DNA结合。在第二阶段，我们计划通过增加DNA线阵列和微通道的密度以及通过研究细胞提取物的DNA结合活性来扩大第一阶段提案中描述的研究范围。

DESCRIPTION (provided by applicant): 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.