Microarrays for DNA binding proteins

描述（由申请人提供）：生物分子及其活性的高度多重检测一直是现代分子诊断、药物开发和基因组学和蛋白质组学研究的基本要素。存在完善的DNA阵列方法，其允许转录物的高度多重检测。用于检测或测量蛋白质活性的类似方法尚未得到很好的开发。我们建议在这个应用程序中开发的阵列能够高度多重检测序列特异性DNA结合蛋白。DNA结合蛋白是一类重要的生物分子，由于其在转录调节中的作用，对于所有细胞过程的调节至关重要。第一阶段提案的目标是证明这种方法的可行性，并建立制备这种阵列的一般方法。这一目标将通过实现以下两个目标来实现：目标#1。确定DNA结合蛋白微阵列是否可以达到所需的检测灵敏度。我们将使用纯化的模型DNA结合蛋白进行实验，建立最大化检测灵敏度的程序。我们将比较几种常用的寡核苷酸微阵列中的荧光标记在DNA结合蛋白的微阵列中的性能。我们将测试2种信号放大方案进一步提高检测灵敏度的能力。最后，我们将确定优化的微阵列是否能够以所需的（pM）灵敏度实现模型蛋白的可重复检测。目标2。建立DNA结合蛋白质微阵列是否可以分析复杂混合物中蛋白质的DNA结合活性。为了实现这一目标，我们将首先确定，如果相对水平的4个DNA结合蛋白可以忠实地报告的微阵列在样品中含有不同的和已知的相对量的这些蛋白质。其次，我们将测试检测SP1蛋白（组成型表达的转录因子）在其天然环境中的HeLa细胞提取物中的正常表达水平。该项目的长期目标是开发用于高度多重检测所有主要细胞通路中涉及的转录因子的阵列，并将这些阵列开发为检测异常细胞状态的诊断工具。本项目开发的DNA结合蛋白微阵列将成为研究和诊断人类疾病的有用工具。在研究中，它们将有助于识别与疾病相关的转录因子活动缺陷。在诊断方面，它们将提供一个机会，开发新的疾病检测工具，通过特定疾病特有的转录因子活动模式来识别疾病。

DESCRIPTION (provided by applicant): Highly multiplexed detection of biological molecules and their activities has been an essential element of modern molecular diagnosis, drug development and research in genomics and proteomics. Well established DNA array methodologies exist which permit highly multiplexed detection of transcripts. Similar methodologies for detecting or measuring activities of proteins are not well developed. We propose in this application development of arrays capable of highly multiplexed detection of sequence-specific DNA binding proteins. DNA binding proteins are an important class of biological molecule which, due to their role in transcription regulation, is of paramount importance for regulation of all cellular processes. The goal of this Phase I proposal will be to demonstrate the feasibility of the approach and to establish the general methodology for preparation of such arrays. This goal will be achieved by accomplishing the following 2 aims: Aim #1. To establish if microarrays for DNA binding proteins can achieve desired sensitivity of detection. We will use purified model DNA binding protein to perform experiments which will establish procedures to maximize detection sensitivity. We will compare several fluorescence labels commonly used in oligonucleotide microarrays for their performance in the microarray for DNA binding proteins. We will test 2 signal amplification schemes for their ability to further enhance detection sensitivity. Finally, we will determine if optimized microarrays will be capable of achieving reproducible detection of the model protein with a desired (pM) sensitivity. Aim #2. To establish if microarrays for DNA binding proteins can analyze DNA binding activities of proteins in complex mixtures. To achieve this goal we will first determine if relative levels of 4 DNA binding proteins could be faithfully reported by the microarray in samples containing varying and known relative amounts of these proteins. Secondly, we will test the detection of SP1 protein (a constitutively expressed transcription factor) at its normal level of expression in its native environment in HeLa cellular extracts. The long term goals of this project will be to develop arrays for highly multiplexed detection of transcription factors involved in all major cellular pathways will be developed and to develop these arrays as diagnostic tools for detecting abnormal cellular states. Microarrays for DNA binding proteins to be developed in this project will be a useful tool for research and diagnosis of human disease. In research, they will facilitate identification of defects in transcription factor activities linked to a disease. In diagnosis, they will provide an opportunity to develop new disease detection tools which will identify disease through a specific pattern of transcription factor activities characteristic for a particular disease.