SYNTHESIS AND CHARACTERIZATION OF FLUORESCENT OLIGONUCLEOTIDES - EFFECT OF INTERNAL LABELING ON PROTEIN RECOGNITION

Fluorescently labelled 42 base pair DNA duplexes were synthesised to examine the interaction between the TyrR repressor protein of Escherichia coli and its DNA recognition sequence. An Fmoc-protected 5-(3-aminoprop-1-yn-1-yl)-2'-deoxyuridine phosphoramidite was synthesised and incorporated into oligonucleotides using standard beta-cyanoethyl phosphoramidite chemistry. Oligonucleotides containing the 3-aminopropynyl nucleotide at internal positions were reacted with fluorescein isothiocyanate to generate fluorescent DNA molecules useful for characterising interactions between DNA and proteins. Short DNA duplexes were investigated with respect to their melting temperatures and their ability to bind TyrR. Oligonucleotides containing a TyrR binding site were labelled in the central region of the recognition sequence or near the 5' edge of the recognition sequence. Fluorescein-labelled oligonucleotides could hybridise to form duplex DNA, and gel retardation experiments showed that the presence of the dye did not alter the binding affinity for the TyrR protein significantly. Fluorescence anisotropy measurements were used to examine the binding equilibrium in low and high salt buffers. A dissociation constant of 200-500 nM was obtained for the interaction of the TyrR dimer with a 42 bp duplex containing a centrally located 22 bp TyrR binding site.