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Single-Molecule, Variable-Temperature Electronic Measurements of Taq DNA Polymerase Using Carbon Nanotube Transistors

  • Author(s): Turvey, Mackenzie
  • Advisor(s): Collins, Philip G
  • et al.
No data is associated with this publication.
Abstract

Single-molecule enzymology has proven to be a powerful technique for understanding the driving forces behind protein kinetics and function. Carbon nanotube field-effect transistors (CNTFETs) have been implemented as a novel platform to study the behavior of enzymes. This solid-state technique has contributed to the single-molecule field by providing both microsecond resolution and long-duration recordings. Bandwidths extending to 1 MHz have identified enzymatic conformations as short as 5 µs. Ten-minute recordings have shown enzymatic rates fluctuating over two orders of magnitude from minute to minute.

This dissertation documents multiple advances expanding the scope of this CNTFET technique. Progress was made on three fronts: incorporating variable temperature control, combining this solid-state electronic technique with TIRF to acquire simultaneous electronic and fluorescent recordings from a polymerase, and expanding experiments to new molecular catalysts.

Taq DNA polymerase was used as a model system to demonstrate variable temperature single-molecule enzymology. No other single-molecule technique can access high enough temperatures to study Taq polymerase, the industry standard for the billion dollar polymerase chain reaction (PCR) industry. Successful single-molecule measurements of Taq polymerase in four different orientations up to 85 °C are reported. Each attachment site transduced unique signals. One attachment site generated events with rates that increased with temperature and match ensemble catalytic rates. Taq’s catalytic activity ranged from 4 s-1 at 22 °C to up to 96 s-1 at 85 °C.

Other results from Taq polymerase focused on the importance of the attachment site and molecular orientation. Another attachment site transduced both catalytic turnovers, as well as another noncatalytic fluttering motion. In this signal, two distinct populations of events were detected: one population with 20 µs event durations and another with 125 µs event durations. The longer 125 µs events had a similar duration and rate as the catalytic events recorded by the first orientation. The rate of the 20 µs events depended upon nucleotide complementarity, allowing energetics of this enzymatic motion to be extracted. The two other attachment sites generated complex signals that were difficult to interpret. One orientation only transduced signal < 5% of the total measurement duration, remaining quiet for the vast majority of data sets. The last orientation produced signals that changed in shape and rate from minute to minute.

Chapter 4 describes experimental attempts to simultaneously measure electrical and fluorescent signals from Taq polymerase. Although ultimately unsuccessful due to apparent fluorophore quenching by the CNTFET, this dissertation documents the work for future experiments.

Finally, CNTFET measurements of two other catalytic systems are documented in Chapter 5. The enzyme dihydrofolate reductase (DHFR) and the Ruthenium-based Grubbs catalyst were both measured. DHFR produced signals that were not catalytic in nature, but were quenched upon introduction of an inhibitor. This process was reversible. This measurement of DHFR complements the Taq polymerase measurements in demonstrating the importance in proper choice of attachment sites. The Grubbs catalyst demonstrated too low of a catalytic rate to accumulate compelling statistics. The low catalytic rate was determined to be due to the linker ligand designed to conjugate the catalytic to the CNTFET.

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This item is under embargo until June 15, 2021.