Direct Measurement of HIV-1 Reverse Transcriptase Binding Kinetics, One Complex at a Time

Abstract

HIV-1 reverse transcriptase (RT) is entirely responsible for the conversion of the viral RNA genome into double stranded DNA. Due to its essential role in virus replication, it is a primary target for drug development. In order to rationally modulate RT binding to its Template/Primer (T/P) substrate using novel inhibitory compounds, an accurate description of DNA-protein binding kinetics in the absence of ensemble averaging is crucial. We are therefore developing a robust single-molecule assay to directly measure the association/dissociation rates of protein-nucleic acid complexes at equilibrium. Utilizing this method in comparison with bulk methods, we describe in detail the effects of various known RT inhibitors (RTIs) as well as novel RTIs under development in our laboratory on the binding kinetics of individual RT-T/P complexes.