Single-Molecule Activity of φ29 DNA Polymerase Monitored by Nanoscale Transistors

Single-molecule techniques are enabling the rapid advancement of next-generation DNA sequencing. Recently, electronic nanocircuits functionalized with DNA polymerases have arisen as a new, potentially high-density and high-throughput platform for single-molecule DNA sequencing and enzyme kinetic studies [1]. Here, we describe single-molecule electronic measurements of φ29 DNA polymerase, an enzyme having extremely high fidelity and processivity. Using φ29 linked to carbon nanotube transistors, base-by-base activity of individual polymerase molecules was continuously monitored for up to 30 min as the enzymes replicated single-stranded templates. Base incorporation rates averaged 20 s^-1 but varied widely. Rates up to 200 and 400 s^-1 occurred when φ29 encountered homopolymeric sequences of poly(dT) and poly(dC), respectively. On the other hand, the processing of poly(dA) and poly(dG) sequences was dominated by pauses lasting 50 to 300 s. Such observations of sequence-dependent activity demonstrate how single-molecule methods can excel over traditional, ensemble-based techniques like PCR sequencing and gel chromatography assays.

[1] T. J. Olsen et. al., JACS 135, 7855 (2013); O. T. Gul et. al., Biosensors 6, 29 (2016)