DNA stretching and optimization of nucleobase recognition in enzymatic nanopore sequencing

Abstract

In nanopore sequencing, where single DNA strands are electrophoretically translocated through a nanopore and the resulting ionic signal is used to identify the four DNA bases, an enzyme has been used to ratchet the nucleic acid stepwise through the pore at a controlled speed. In this work, we investigated the ability of αHL nanopores to distinguish the four DNA bases under conditions that are compatible with the activity of DNA-handling enzymes. Our findings suggest that in immobilised strands, the applied potential exerts a force on DNA (~ 10 pN at +160 mV) that increases the distance between nucleobases by about 2.2 Å/V. The four nucleobases can be resolved over wide ranges of applied potentials (from +60 mV to +220 mV in 1 m KCl) and ionic strengths (from 200 mM KCl to 1 M KCl at +160 mV) and nucleobase recognition can be improved when the ionic strength on the side of the DNA-handling enzyme is low, while the ionic strength on the opposite side is high.

Thermal motion of DNA in an MspA pore

“…The parameter values extracted in this manner are σ eff = 0.54 ± 0.03 e / base , and D = D 0 / (5 ± 1), where D 0 = 2.94 ×10 -10 m 2 /s is the diffusion constant of a 1.5nm diameter bead free in solution, 1.5nm being the approximate Kuhn length of ssDNA. We account for the uncertainty in the length per base by using the value 0.5 ±0.02nm(23), which leads to a 4% relative uncertainty in the effective charge density.…”

“…We expect that as we update the reflecting boundary to a repulsive force F barrier ( x ) the position probability distribution should spread slightly beyond x = 0. This repulsive force would have a finite magnitude over a certain region due to effects like DNA stretching(23).…”

Charge, Diffusion, and Current Fluctuations of Single-Stranded DNA Trapped in an MspA Nanopore

“…The increased tension straightens out the thermal entropic fluctuating geometry of the molecule in this region and decreases the number of bases there. Previously, ssDNA elongation by stretching in a nanopore was reported by Stoddart et al (29) in the a-hemolysin nanopore.…”

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

“…Advances in the nanopore system itself, including the optimization of applied potential and salt concentration, increase the system’s sensitivity for identifying differences in nucleotide structure [48]. In one strategy, nanopores with internally bound proteins [49] could evaluate a single enzyme over long time scales by measuring small changes in the protein’s conformation.…”