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

Fluorescently labelled 42 base pair DNA duplexes were synthesised to e xamine the interaction between the TyrR repressor protein of Escherich ia coli and its DNA recognition sequence. An Fmoc-protected 5-(3-amino prop-1-yn-1-yl)-2'-deoxyuridine phosphoramidite was synthesised and in corporated into oligonucleotides using standard beta-cyanoethyl phosph oramidite chemistry. Oligonucleotides containing the 3-aminopropynyl n ucleotide at internal positions were reacted with fluorescein isothioc yanate to generate fluorescent DNA molecules useful for characterising interactions between DNA and proteins. Short DNA duplexes were invest igated with respect to their melting temperatures and their ability to bind TyrR. Oligonucleotides containing a TyrR binding site were label led in the central region of the recognition sequence or near the 5' e dge of the recognition sequence. Fluorescein-labelled oligonucleotides could hybridise to form duplex DNA, and gel retardation experiments s howed that the presence of the dye did not alter the binding affinity for the TyrR protein significantly. Fluorescence anisotropy measuremen ts were used to examine the binding equilibrium in low and high salt b uffers. A dissociation constant of 200-500 nM was obtained for the int eraction of the TyrR dimer with a 42 bp duplex containing a centrally located 22 bp TyrR binding site.