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. ? ? ?
