REAL-TIME MEASUREMENTS OF ELONGATION BY A REVERSE-TRANSCRIPTASE USINGSURFACE-PLASMON RESONANCE

A rapid direct assay for polymerase-induced elongation along a given t emplate is an obligate requirement for understanding the processivity of polymerization and the mode of action of drugs and inhibitors on th is process. Surface plasmon resonance can be used to follow the associ ation and the dissociation rates of a given reverse transcriptase on D NA . RNA and DNA . DNA hybrids immobilized on a biotin-streptavidin su rface. The addition of nucleotides complementary to the template stran d produces an increase in the local mass, as deduced from an increase in the measured signal, due to elongation of the primer strand that al lows an estimation of both the extent and rate of the polymerization p rocess. The terminator drug 3'-deoxy-3'-azidothymidine triphosphate co mpletely abolishes the increase in signal as would be expected from an inhibition of elongation, This technique provides a sensitive assay f or the affinities of different polymerases for specific templates and for the effects of terminators of the elongation process.