Single molecule kinetics of reverse transcriptase

We use single molecule methods to directly observe the enzymatic behavior of individual reverse transcriptase (RT) molecules on single-stranded DNA template strands in vitro. A flow-stretched DNA assay is used, allowing for characterization of enzymatic rate, processivity, and pausing of RT during DNA synthesis as a function of template base pair content, secondary structure, and applied template tension. We study the kinetics of plus-strand DNA synthesis catalyzed by RT derived from the Moloney murine leukemia virus (M-MLV) with inhibited RNase H activity, an enzyme commonly used for RT-PCR. Initial observations show an average enzymatic rate of ∼5-10 bp/sec. Understanding potentially diverse kinetic mechanisms in molecular subpopulations may allow for development of more effective treatments for retroviral infections leading to cancer.