Transciption Factor Interactions Using Addressed Arrays

使用寻址数组的转录因子相互作用

• 批准号: 6737228
• 负责人: Bryce P Nelson
• 金额: $ 19.23万
• 依托单位: GENTEL BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-16 至 2005-08-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6737228
• 关键词: DNA binding protein; binding sites; gene expression; intermolecular interaction; microarray technology; protein binding; protein structure function; surface plasmon resonance; technology /technique development; transcription factor

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.

描述（由申请人提供）：该I期SBIR项目旨在证明系统的实用性，以有效筛选小分子对蛋白质-DNA结合的影响。DNA结合蛋白是基因表达的基本调节因子，通过识别和结合特定的DNA序列。使用治疗剂恢复突变蛋白质（例如，肿瘤抑制因子p53）一直是药物研究人员的长期目标，但已证明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先导物的有效方法。需要新的工具来帮助筛选可以调节特定基因和基因家族表达的潜在化学和生物方法。DNA阵列为快速筛选提供了一种特别有前途的方法，但传统的DNA阵列只能测试单个溶液条件或每个阵列的化合物。我们建议开发一种新型的DNA阵列，该阵列使用固定在平面表面上的许多微米大小的DNA探针线和交叉的微流体通道的组合，这些微流体通道可以在芯片上携带多达数百个离散的溶液。每个“交叉点”都是一个独特的反应位点，每个阵列可以同时进行10，000多个检测。对于检测，该技术使用无标记技术，该技术能够进行动力学分析，表面等离子体共振成像（SPRi），这是传统SPR测量技术的微阵列适合的适应。我们的目标是通过开发一个四元件DNA LineArray和一个四通道微流体系统来证明我们的策略的可行性，以证明合作蛋白质-DNA结合的调制。探针DNA将被固定到平面表面，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的测定的低纳摩尔范围灵敏度。我们预计，与我们的系统，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 LineArrays和微通道的密度以及研究细胞提取物的DNA结合活性来扩大第一阶段提案中描述的研究范围。