The objective of this proposal is the further development and application of a spin assay for nucleic acid binding proteins. The assay provides a rapid, sensitive, and safe (no radiolabeling) means for detecting complexes between nucleic acids and nucleic acid binding proteins by electron spin resonance (ESR) without potential interference by a matrix such as filters or gels. The complexes are detected with lst and 2nd generation spin labeled nucleic acids through nitroxide radicals as reporter groups which are incorporated at random to a small extent (nitroxide/nucleotide varies between 0.002 to 0.02 into the nucleic acids). 1st generation spin labeled nucleic acids are labeled non-site specifically by chemical means, whereas 2nd generation spin labeled nucleic acids (labeling specificity with respect to nucleotide) are prepared enzymatically. It is proposed to also include 3rd generation spin labeled nucleic acids (labeling specificity with respect to nucleotide and sequence) in the spin assay. The spin assay employing these spin labeled nucleic acids will be used either to detect binding of a particular protein to a given nucleic acid under a variety of conditions (protein modification, salt concentration, etc.) or to determine the relative affinity of a variety of nucleic acids for a particular protein through competition experiments with spin labeled and unlabeled nucleic acids. Recently developed computer programs will be used to determine the fraction F of nucleic acids in the complexed state directly from the ESR spectrum. The assay will be applied to follow the binding of gene 32 and gene 5 proteins to nucleic acids, the binding of nucleic acid inhibitors and their analogs to AMV reverse transcriptase, and to further define the role of gene 32 protein and SSB protein of E. coli during the replicative process.
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