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.

1. A. Chien, D. B. Edgar, J. M. Trela, J. Bacteriology 127 1550 (1976).
2. Y. Choi, et. al., Science 335 319 (2012); T. J. Olsen et. al., JACS 135 7855 (2013).