Single-molecule techniques based on optical, electronic, and mechanical mechanisms have all proven useful for studying enzymes like DNA polymerases. Electronic devices, in particular, provide exciting new opportunities for understanding polymerase activity, especially with regard to improving DNA sequencing technologies. Recently, single molecule kinetics of the Klenow fragment (KF) of polymerase I have been studied by bioconjugating KF to single-walled carbon nanotube field-effect transistors. Continuous recordings of KF processing various DNA templates have extended over 600 seconds and through >10,000 bond-forming events, providing new insights into KF's processivity, kinetic variability, and tolerance of various dNTP analogs (1,2). For example, we show that the average duration of each incorporation event appears invariant across all analog and native dNTPs, indicating that analog structure has no impact on the timing of this step of the catalytic cycle. Despite similar timings, however, polymerization with some analogs have revealed alternate conformational states that do not occur with native dNTPs. The electronic technique's particular sensitivity to allosteric motions implicates a special role for KF's O-helix as it tests the stability of nascent base pairs with these analogs. The results help explain polymerases’ tolerance for dNTP analogs, despite having highly evolved mechanisms for specific recognition of and discrimination among dNTPs.1. T.J. Olsen, et. al., “Electronic Measurements of Single-Molecule Processing by DNA polymerase I (Klenow fragment),” JACS 135, 7855 (2013).2. K.M. Pugliese, et. al., “Processive Incorporation of Deoxynucleoside Triphosphate Analogs by Single-Molecule DNA Polymerase I (Klenow Fragment) Nanocircuits.” JACS 137, 9587 (2015).
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