FLUORESCENT LABELING OF NUCLEIC ACIDS

Boston College, Chestnut Hill MA

Investigators

Linked publications & trials

Abstract

A group of DNA conjugates composed of a probe sequence and a linker which tethers a Hoechst 33258 analogue will be employed for complex formation with double-stranded DNA/RNA targets. We will further develop various aspects of these conjugates including the nature of the linker and the ligand, the structure of nucleic acid sequence that functions as the target, and we will examine the addition of peptides conjugated to the 3'- terminus of the sequences. The various conjugated complexes prepared will be characterized using spectroscopy and calorimetry. We will determine how the effects of single base-pair mismatches affect the stability of the conjugate complexes, and establish the ability of these conjugated probes to discriminate the correct from the incorrect sequence target. Both sequence targeting abilities and biological properties, particularly their ability to inhibit RNA polymerases, will be examined in more detail. With a view towards targeting RNA sequences, we will develop the tethered ligand approach with selected aminoglycoside antibiotics known to have affinity for RNA. We will examine the structure stabilizing abilities of Tobramycin, Neomycin B, and various analogues constructed on a Neamine core disaccharide. The final aspect of this proposal will develop the methodology for affinity labeling, particularly as it is applied to protein-DNA complexes.