Distinguishing Catalytic and Noncatalytic Motions of Individual Taq Polymerase Molecules

Single-molecule field-effect transistors (smFETs) provide a powerful way to probe biochemical activity with a level of detail that is elusive to ensemble techniques. For example, smFETs have been used to record bond-by-bond catalysis by enzymes acting upon a variety of substrates [1], including the duplication of DNA by DNA polymerases [2]. With the temperature raised to 72 °C, the smFET method has now been applied to study Taq polymerase and key catalytic steps in the polymerase chain reaction (PCR). During PCR, Taq incorporates nucleotides into complementary Watson-Crick base pairs while rejecting noncomplementary mismatches. smFETs resolved both processes, even though the rejection events had durations averaging only 20 µs. This presentation will summarize the Taq immobilization orientations and analysis methods that best recorded and differentiated between catalytic and noncatalytic motions, producing detailed single-molecule kinetics and energy landscapes for the two processes.

[1] Y. Choi, et. al., Science 335 319 (2012).
[2] T. J. Olsen et. al., JACS 135, 7855 (2013); O. T. Gul et. al., Biosensors 6, 29 (2016)