The overall aim of this research is to implement surface plasmon resonance (SPR) imaging methods in the study of protein-DNA interactions. The sequence-specific binding of proteins to DNA plays a pivotal role in the regulation and control of gene expression, replication and recombination. In addition, enzymes that recognize and modify specific sequences are critical components of biological DNA manipulation and repair systems. An enhanced understanding of how these proteins recognize certain oligonucleotide sequences would aid in the design of biomedical systems which could, for example, be used to regulate the expression of therapeutic proteins. For this reason, the study of protein-DNA interactions is a rapidly growing area of molecular biology, aided in part by recent advances in NMR and X-ray structural determination methods. At the same time, the explosive increase in the amount of available genomic sequence information obtained from large scale DNA sequencing efforts creates the need to survey this vast amount of new DNA sequence data for genes, operator sequences and other protein binding sites. In support of this effort, our goal is to use SPR imaging techniques as a rapid and efficient method for screening the sequence-specific binding of proteins to large arrays of double-stranded DNA molecules immobilized at chemically modified gold surfaces. Specific goals of this project will be to: (i) fabricate arrays of single- and double-stranded oligonucleotide sequences to chemically modified gold surfaces, (ii) demonstrate the sensitivity of a white light-based SPR imaging system to monitor in situ both DNA hybridization adsorption and protein adsorption, (iii) monitor the enzymatic manipulation of surface-bound oligonucleotides by T4 DNA ligase, Exonuclease I, and various restriction endonucleases, (iv) monitor the binding of the lac repressor protein of E. coli to arrays of double-stranded oligonucleotide sequences which model both the wild type and mutations of the two-fold symmetric lac operator site, and (v) investigate the sequence specificity of the binding of the protein MutS (which is involved in the methyl-directed DNA mismatch correction system of E. coli) to heteroduplexes with single base mismatches and 1 to 4 base insertions.
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