Single-Molecule Kinetics of Taq Polymerase up to 94 °C

Single-molecule enzymology is a useful tool for understanding protein kinetics, function, and inhibition, but common techniques work in a limited temperature range. Here, we extend single-molecule science up to 94 °C with MHz resolution using solid-state, carbon nanotube biosensors. Their operation, which is essentially independent of temperature, provides unprecedented access to commercially important enzymes such as Taq, the DNA polymerase I from Thermus aquaticus. Taq is the linchpin protein for the polymerase chain reaction (PCR) amplification of DNA [1]. Our electronic biosensor approach provides direct access to single-nucleotide incorporation activity [2] from room temperature up to 94 °C. For example, the incorporation rate of a single Taq molecule was monitored from 1 bp/s at 22 °C to greater than 100 bp/s at 72 °C, the typical PCR operating temperature. In addition, the biosensor transduces the distinct motions of Taq subdomains, such as brief, 20-µsec flutters as the fingers domain tests nucleotides for complementarity.

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2. Y. Choi, et. al., Science 335 319 (2012); T. J. Olsen et. al., JACS 135 7855 (2013).