Electronic Measurements of Single-Molecule Processing by DNA Polymerase I (Klenow Fragment)

Abstract

Bioconjugating single molecules of the Klenow fragment of DNA polymerase I into electronic nanocircuits allowed electrical recordings of enzymatic function and dynamic variability with the resolution of individual nucleotide incorporation events. Continuous recordings of DNA polymerase processing multiple homopolymeric DNA templates extended over 600 s and through >10,000 bond forming events. An enzymatic processivity of 42 nucleotides for a template of the same length was directly observed. Statistical analysis determined key kinetic parameters for the enzyme’s open and closed conformations. Consistent with these nanocircuit-based observations, the enzyme closed complex forms a phosphodiester bond in a highly efficient process >99.8% of the time with a mean duration of only 0.3 ms for all four dNTPs. The rate-limiting step for catalysis occurs during the enzyme open state, but with a nearly two-fold longer duration for dATP or dTTP incorporation than for dCTP or dGTP into complementary, homopolymeric DNA templates. Taken together, the results provide a wealth of new information complementing prior work on the mechanism and dynamics of DNA polymerase I.

Processive Incorporation of Deoxynucleoside Triphosphate Analogs by Single-Molecule DNA Polymerase I (Klenow Fragment) Nanocircuits

“…33,34 Purification of KF to >95% ensured its homogeneity (Figure S1). A fluorescence-based assay confirmed activity of the bulk enzyme prior to attachment (Figure S2).…”

“…The drain electrode was biased at 100 mV, and the electrolyte, which served as a gate electrode, was held at or near 0 V. Incubation of the device with any template-primer and its complementary dNTPs transduced fluctuations, Δ I ( t ), whereas these fluctuations were absent with non-complementary dNTPs or in control measurements missing the template-primer or KF attachment. 33 I ( t ) fluctuations were amplified (Keithley 428), digitized at 100 kHz, and stored as uninterrupted, 600 s data sets for later analysis. Between measurements, the KF nanocircuits were rinsed twice with activity buffer, incubated in buffer for 5 min, then rinsed twice with buffer before introducing another nucleotide and template-primer.…”

“…32 KF conjugated to SWCNT-FETs uncovered significant kinetic and conformational differences between the enzyme-catalyzed formation of A•T/T•A and G•C/C•G sets of base pairs. 33 …”

Recent progress in dissecting molecular recognition by DNA polymerases with non-native substrates

“…Another DNA polymerase-monitoring technology relies on voltage-gating of single-walled carbon nanotube field-effect transistors (SWCNT-FETs). These devices, developed in our lab, provide a readout of the charged amino acids on the enzyme’s surface that move within 1 nm of the nanotube during catalysis [4]. …”

“…A new single-molecule electronic technique for examining DNA polymerase was initially developed in our lab to detect conformational transitions during catalysis by various enzymes, including KF [4]. Although the exact nature of the observed conformations for KF is unclear, distinct states have been observed for dNTP analogs [5].…”