A simple, versatile and site-specific labeling technique permits the incorporation of fluorophores or other agents into nucleic acids. The backbone labeling procedures proposed in the previous application have been achieved during the last award period. In the present proposal, we will exploit these procedures in a number of areas. (i) The ability to easily tether a specific reporter group to a DNA sequence provides the foundation for the development of a series of DNA diagnostics. These materials will exploit complementary base pairing interactions for sequence recognition (and targeting) while delivering the diagnostic agent. In this proposal we will synthesize and study the properties of a nucleic acid hybridization probe. This conjugate will tether a minor groove binding fluorophore (Hoechst 33258). Upon location of the target DNA sequence, hybridization generates the minor groove structure in which the tethered fluorophore binds and the fluorescence signal is generated. (ii) Photoaffinity materials can be a powerful group of agents for understanding complex structures or processes. We will employ two types of backbone photoaffinity agents, the perfluoroazides and a simple benzophenone derivative, to study a protein-DNA complex (the trp repressor-operator) and to study a hammerhead ribozyme. (iii) Fluorescence resonance energy transfer (FRET) is an important biophysical technique that permits the measurement of distances from approximately 10 - 60 Angstroms. We will continue the FRET studies initiated during the previous application to study both the trp repressor- operator complex and the hammerhead ribozyme. Many of the DNA/RNA sequences used in (ii) and (iii) are similar but will vary in the agent (photoaffinity label or fluorophore) tethered to the sequence.
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