INCORPORATION OF A FLUORESCENT GUANOSINE ANALOG INTO OLIGONUCLEOTIDESAND ITS APPLICATION TO A REAL-TIME ASSAY FOR THE HIV-1 INTEGRASE 3'-PROCESSING REACTION

We have synthesized a highly fluorescent (quantum yield 0.88) guanosin e analog, (3-methyl-8-(2-deoxy-beta-D-ribofuranosyl) isoxanthopterin ( 3-MI) in a dimethoxytrityl, phosphoramidite protected form, which can be site-specifically inserted into oligonucleotides through a 3',5'-ph osphodiester linkage using an automated DNA synthesizer Fluorescence i s partially quenched within an oligonucleotide and the degree of quenc h is a function of the fluorophore's proximity to purines and its posi tion in the oligonucleotide. As an example of the potential utility of this class of fluorophores, we developed a continuous assay for HIV-1 integrase 3'-processing reaction by incorporating 3-MI at the cleavag e site in a double-stranded oligonucleotide identical to the U5 termin al sequence of the HIV genome, Integrase cleaves the 3'-terminal dinuc leotide containing the fluorophore, resulting in an increase in fluore scence which can be monitored on a spectrofluorometer. Substitution of the fluorophore for guanosine at the cleavage site does not inhibit i ntegrase activity. This assay is specific for the 3'-processing reacti on. The change in fluorescence intensity is linear over time and propo rtional to the rate of the reaction. This assay demonstrates the poten tial utility of this new class of fluorophore for continuous monitorin g of protein/DNA interactions.