Motor Enzymes Research Articles

Inherently disordered regions of axonemal dynein assembly factors.

The dynein-driven beating of cilia is required to move individual cells and to generate fluid flow across surfaces and within cavities. These motor enzymes are highly complex and can contain upwards of 20 different protein components with a total mass approaching 2 MDa. The dynein heavy chains are enormous proteins consisting of ~4500 residues and ribosomes take approximately 15 min to synthesize one. Studies in a broad array of organisms ranging from the green alga Chlamydomonas to humans has identified 19 cytosolic factors (DNAAFs) that are needed to specifically build axonemal dyneins; defects in many of these proteins lead to primary ciliary dyskinesia in mammals which can result in infertility, severe bronchial problems, and situs inversus. How all these factors cooperate in a spatially and temporally regulated manner to promote dynein assembly in cytoplasm remains very uncertain. These DNAAFs contain a variety of well-folded domains many of which provide protein interaction surfaces. However, many also exhibit large regions that are predicted to be inherently disordered. Here I discuss the nature of these unstructured segments, their predicted propensity for driving protein phase separation, and their potential for adopting more defined conformations during the dynein assembly process.

Anti-KIF20B autoantibodies in systemic autoimmune rheumatic diseases: Their high prevalence in systemic lupus erythematosus.

The kinesin superfamily protein 20B (KIF20B), also known as M-phase phosphoprotein-1, is a plus-end-directed motor enzyme for cytokinesis. Anti-KIF20B antibodies have been reported in idiopathic ataxia, but no previous studies have examined anti-KIF20B antibodies in systemic autoimmune rheumatic diseases (SARDs). We aimed to establish methods for detecting anti-KIF20B antibodies and to investigate the clinical significance of these antibodies in SARDs. Serum samples from 597 patients with various SARDs and 46 healthy controls (HCs) were included. Fifty-nine samples that had been examined by immunoprecipitation using the recombinant KIF20B protein produced by in vitro transcription/translation were used for establishing the ELISA cutoff with the same recombinant protein for measuring the anti-KIF20B antibodies. The ELISA performed well, showing close agreement with the immunoprecipitation results (Cohen's κ >0.8). The ELISA results for 643 samples showed the prevalence of anti-KIF20B to be higher in the systemic lupus erythematosus (SLE) patients than in the HCs (18/89 vs. 3/46, P = 0.045). Since no SARD other than SLE had higher frequencies of anti-KIF20B antibodies than those of the HCs, we investigated the clinical characteristics of anti-KIF20B antibody-positive cases in SLE. The score on the SLE Disease Activity Index-2000 (SLEDAI-2K) was significantly higher for the anti-KIF20B-positive SLE patients than for the anti-KIF20B-negative SLE patients (P = 0.013). In a multivariate regression analysis of the anti-single-stranded deoxyribonucleic acid, anti-double-stranded deoxyribonucleic acid, and anti-KIF20B antibodies, the presence of anti-KIF20B antibody was significantly associated with high SLEDAI-2K scores (P = 0.003). Anti-KIF20B antibodies were found in ~20% of patients with SLE and were associated with high SLEDAI-2K scores. Much larger cohort and longitudinal studies are needed to confirm the association between anti-KIF20B antibodies and SLE.

Assessing Readability of an 8-Letter Expanded Deoxyribonucleic Acid Alphabet with Nanopores.

Chemists have now synthesized new kinds of DNA that add nucleotides to the four standard nucleotides (guanine, adenine, cytosine, and thymine) found in standard Terran DNA. Such "artificially expanded genetic information systems" are today used in molecular diagnostics; to support directed evolution to create medically useful receptors, ligands, and catalysts; and to explore issues related to the early evolution of life. Further applications are limited by the inability to directly sequence DNA containing nonstandard nucleotides. Nanopore sequencing is well-suited for this purpose, as it does not require enzymatic synthesis, amplification, or nucleotide modification. Here, we take the first steps to realize nanopore sequencing of an 8-letter "hachimoji" expanded DNA alphabet by assessing its nanopore signal range using the MspA (Mycobacterium smegmatis porin A) nanopore. We find that hachimoji DNA exhibits a broader signal range in nanopore sequencing than standard DNA alone and that hachimoji single-base substitutions are distinguishable with high confidence. Because nanopore sequencing relies on a molecular motor to control the motion of DNA, we then assessed the compatibility of the Hel308 motor enzyme with nonstandard nucleotides by tracking the translocation of single Hel308 molecules along hachimoji DNA, monitoring the enzyme kinetics and premature enzyme dissociation from the DNA. We find that Hel308 is compatible with hachimoji DNA but dissociates more frequently when walking over C-glycoside nucleosides, compared to N-glycosides. C-glycocide nucleosides passing a particular site within Hel308 induce a higher likelihood of dissociation. This highlights the need to optimize nanopore sequencing motors to handle different glycosidic bonds. It may also inform designs of future alternative DNA systems that can be sequenced with existing motors and pores.

Continuous millisecond conformational cycle of a DEAH box helicase reveals control of domain motions by atomic-scale transitions

Helicases are motor enzymes found in every living organism and viruses, where they maintain the stability of the genome and control against false recombination. The DEAH-box helicase Prp43 plays a crucial role in pre-mRNA splicing in unicellular organisms by translocating single-stranded RNA. The molecular mechanisms and conformational transitions of helicases are not understood at the atomic level. We present a complete conformational cycle of RNA translocation by Prp43 in atomic detail based on molecular dynamics simulations. To enable the sampling of such complex transition on the millisecond timescale, we combined two enhanced sampling techniques, namely simulated tempering and adaptive sampling guided by crystallographic data. During RNA translocation, the center-of-mass motions of the RecA-like domains followed the established inchworm model, whereas the domains crawled along the RNA in a caterpillar-like movement, suggesting an inchworm/caterpillar model. However, this crawling required a complex sequence of atomic-scale transitions involving the release of an arginine finger from the ATP pocket, stepping of the hook-loop and hook-turn motifs along the RNA backbone, and several others. These findings highlight that large-scale domain dynamics may be controlled by complex sequences of atomic-scale transitions.

Fingerprinting full-length proteins through single-file translocations.

Biological nanopore is a revolutionary approach to single molecule detection of biological polymers including proteins. However, for full-length protein fingerprinting/sequencing, the nonuniform charged backbone, complex secondary structures, and intra-protein interactions, present multiple challenges in protein capture, threading, and transport with velocity control. Here we introduce an approach for realizing unidirectional transport of full-length proteins through nanopores. To achieve this, we combined a high chemical resistant biological nanopore platform with high concentration of denaturant, guanidinium chloride to unfold, capture and translocate full-length proteins through a biological nanopore by an electroosmotic effect. The uniform and slow (∼10 µs/amino acid) translocation of proteins, combined with supervised machine learning, allows us to use their electrical current blockade signatures to discern their threading orientation and identity. When combined with a method for tail-modification of native proteins, our study can pave the way to direct single-molecule protein fingerprinting without the requirement of a motor enzyme. (Yu et al., BioRxiv 2021, https://doi.org/10.1101/2021.09.28.4621550)

Molecular dynamics simulations of F1-ATPase's angular fluctuations and associated restoring force.

Molecular dynamics (MD) is a computational method that allows researchers to analyze the movement of atoms and molecules on the nanosecond timescale. The runtime for these simulations is limited to the hardware specifications of the computers. One frame at a time, fluctuations in the positions of organic molecules can be recorded with a image resolutions far beyond what can be seen by single-molecule imaging. In this poster, we examine a rotary motor enzyme, F1-ATPase. The rotor's angular displacement can be tracked and associated with an instantaneous velocity at each timestep. By analyzing such data, we found that a restoring force exists to regulate the rotation around a dwell angle. As fluctuations drive the angular position of the rotor to deviate from the center of the dwell angle, the restoring force motivates the rotor to return to the original position. We hope to combine our present data with measurements collected from single-molecule imaging to calculate mathematical model from the atomistic fluctuations.

A scalable framework for the discovery of functional helicase substrates and helicase-driven regulatory switches

Helicases are ubiquitous motor enzymes that remodel nucleic acids (NA) and NA-protein complexes in key cellular processes. To explore the functional repertoire and specificity landscape of helicases, we devised a screening scheme-Helicase-SELEX (Systematic Evolution of Ligands by EXponential enrichment)-that enzymatically probes substrate and cofactor requirements at global scale. Using the transcription termination Rho helicase of Escherichia coli as a prototype for Helicase-SELEX, we generated a genome-wide map of Rho utilization (Rut) sites. The map reveals many features, including promoter- and intrinsic terminator-associated Rut sites, bidirectional Rut tandems, and cofactor-dependent Rut sites with inverted G > C skewed compositions. We also implemented an H-SELEX variant where we used a model ligand, serotonin, to evolve synthetic Rut sites operating invitro and invivo in a ligand-dependent manner. Altogether, our data illustrate the power and flexibility of Helicase-SELEX to seek constitutive or conditional helicase substrates in natural or synthetic NA libraries for fundamental or synthetic biology discovery.

Docking and Activity of DNA Polymerase on Solid-State Nanopores.

Integration of motor enzymes with biological nanopores has enabled commercial DNA sequencing technology; yet studies of the similar principle applying to solid-state nanopores are limited. Here, we demonstrate the real-life monitoring of phi29 DNA polymerase (DNAP) docking onto truncated-pyramidal nanopore (TPP) arrays through both electrical and optical readout. To achieve effective docking, atomic layer deposition of hafnium oxide is employed to reduce the narrowest pore opening size of original silicon (Si) TPPs to sub-10 nm. On a single TPP with pore opening size comparable to DNAP, ionic current measurements show that a polymerase–DNA complex can temporally dock onto the TPP with a certain docking orientation, while the majority become translocation events. On 5-by-5 TPP arrays, a label-free optical detection method using Ca2+ sensitive dye, are employed to detect the docking dynamics of DNAP. The results show that this label-free detection strategy is capable of accessing the docking events of DNAP on TPP arrays. Finally, we examine the activity of docked DNAP by performing on-site rolling circle amplification to synthesize single-stranded DNA (ssDNA), which serves as a proof-of-concept demonstration of utilizing this docking scheme for emerging nanopore sensing applications.

A chemical thermodynamic model of motor enzymes unifies chemical-Fx and powerstroke models

Molecular motors play a central role in many biological processes, ranging from pumping blood and breathing to growth and wound healing. Through motor-catalyzed chemical reactions, these nanomachines convert the chemical free energy from ATP hydrolysis into two different forms of mechanical work. Motor enzymes perform reversible work, wrev, through an intermediate step in their catalyzed reaction cycle referred to as a working step, and they perform Fx work when they move a distance, x, against a force, F. In a powerstroke model, wrev is performed when the working step stretches a spring within a given motor enzyme. In a chemical-Fx model, wrev is performed in generating a conserved Fx potential defined external to the motor enzyme. It is difficult to find any common ground between these models even though both have been shown to account for mechanochemical measurements of motor enzymes with reasonable accuracy. Here, I show that, by changing one simple assumption in each model, the powerstroke and chemical-Fx model can be reconciled through a chemical thermodynamic model. The formal and experimental justifications for changing these assumptions are presented. The result is a unifying model for mechanochemical coupling in motor enzymes first presented by A.V. Hill in 1938 that is consistent with single-molecule structural and mechanical data.

Control of subunit stoichiometry in single-chain MspA nanopores

Transmembrane protein channels enable fast and highly sensitive detection of single molecules. Nanopore sequencing of DNA was achieved using an engineered Mycobacterium smegmatis porin A (MspA) in combination with a motor enzyme. Due to its favorable channel geometry, the octameric MspA pore exhibits the highest current level compared with other pore proteins. To date, MspA is the only protein nanopore with a published record of DNA sequencing. While widely used in commercial devices, nanopore sequencing of DNA suffers from significant base-calling errors due to stochastic events of the complex DNA-motor-pore combination and the contribution of up to five nucleotides to the signal at each position. Different mutations in specific subunits of a pore protein offer an enormous potential to improve nucleotide resolution and sequencing accuracy. However, individual subunits of MspA and other oligomeric protein pores are randomly assembled in vivo and in vitro, preventing the efficient production of designed pores with different subunit mutations. In this study, we converted octameric MspA into a single-chain pore by connecting eight subunits using peptide linkers. Lipid bilayer experiments demonstrated that single-chain MspA formed membrane-spanning channels and discriminated all four nucleotides identical to MspA produced from monomers in DNA hairpin experiments. Single-chain constructs comprising three, five, six, and seven connected subunits assembled to functional channels, demonstrating a remarkable plasticity of MspA to different subunit stoichiometries. Thus, single-chain MspA constitutes a new milestone in the optimization of MspA as a biosensor for DNA sequencing and many other applications by enabling the production of pores with distinct subunit mutations and pore diameters.

Fast state detection in F1-ATPase rotation enhanced by theory of mixed states and external torque

During brief 120° transitions between long catalytic dwells, single F1-ATPase molecules exhibit angular jumps that vary with rotation angles. Using the angular jump profile enables the detection of fast states in the mechano-chemical scheme of the enzyme, states that are difficult to capture from single-molecule trajectories due to the fluctuations of the imaging nanoprobe. In a previous work, a short-lived, three occupancy state was postulated from a multi-state, probabilistic theory to explain the mean angular jump profile. An assumption in the theory was that the ‘mixing’ of chemical states is negligible during jumps. In a mixing event, two subsequent angular positions recorded by the imaging apparatus belong to two different chemical states of the motor enzyme due to fast reactions within a recording frame. In this paper, we provide an enhanced method for the detection of fast states. On one hand, we show using Langevin simulations that state mixing leads to faster mean angular jump, shifting up the profile. Consequently, the improved method provides a correction to the angular position and lifetime of the postulated three-occupancy metastable state. On the other hand, we show that when F1-ATPase is subject to torques opposing rotation in hydrolysis direction, the torques shift down the dwell angles without affecting the angle-dependent reaction rates. The torques improve detection capability for the fast state by increasing dwell times which is made evident by the flattening of the mean angular jump profile within 40°–60° from the catalytic dwell. In the three-occupancy state release of ADP occurs in concert with the binding of ATP to a different site in the F1-ATPase. Similarly, in the full ATP synthase when torques are created by the proton gradient in the FO region, the release of the product ATP is presumably accelerated by the binding of ADP to a different site in the F1 domain.

Force spectroscopy with electromagnetic tweezers.

Force spectroscopy using magnetic tweezers (MTs) is a powerful method to probe the physical characteristics of single polymers. Typically, molecules are functionalized for specific attachment to a glass surface at one end and a micrometer-scale paramagnetic bead at the other end. By applying an external magnetic field, multiple molecules can be stretched and twisted simultaneously without exposure to potentially damaging radiation. The majority of MTs utilize mobile, permanent magnets to produce forces on the beads (and the molecule under test). However, translating and rotating the permanent magnets may require expensive precision actuators, limit the rate at which force can be changed, and may induce vibrations that disturb tether dynamics and bead tracking. Alternatively, the magnetic field can be produced with an electromagnet, which allows fast force modulation and eliminates motor-associated vibration. Here, we describe a low-cost quadrapolar electromagnetic tweezer design capable of manipulating DNA-tethered MyOne paramagnetic beads with forces as high as 15 pN. The solid-state nature of the generated B-field modulated along two axes is convenient for accessing the range of forces and torques relevant for studying the activity of DNA motor enzymes like polymerases and helicases. Our design specifically leverages technology available at an increasing number of university maker spaces and student-run machine shops. Thus, it is an accessible tool for undergraduate education that is applicable to a wide range of biophysical research questions.

Recent insights into the relative timing of myosin's powerstroke and release of phosphate.

Myosin is a motor enzyme that converts the chemical energy in ATP into mechanical work to drive a myriad of intracellular processes, from muscle contraction to vesicular transport. Key steps in the transduction of energy are the force-generating powerstroke, and the release of phosphate (Pi ) from the nucleotide-binding site. Both events occur rapidly after binding to actin, making it difficult to determine which event occurs first. Early efforts suggested that these events occur simultaneously; however, recent findings indicate that they are separate and distinct events that occur at different rates. High-resolution crystal structures of myosin captured in intermediate states of the ATPase cycle suggest that when Pi is in the active site it prevents the powerstroke from occurring, leading to the hypothesis that Pi -release precedes the powerstroke. However, advances in functional assays, enabling sub-millisecond temporal and nanometer spatial resolution, are challenging this hypothesis. For example, Föster Resonance Energy Transfer (FRET) based assays, as well as single molecule laser trap assays, suggest the opposite; that the powerstroke occurs prior to the release of Pi from myosin's active site. This review provides some historical context and then highlights recent reports that reveal exciting new insight into this fundamental mechanism of energy transduction by this prototypical motor enzyme.

Fast ATP-Dependent Subunit Rotation in Reconstituted FoF1-ATP Synthase Trapped in Solution.

FoF1-ATP synthases are ubiquitous membrane-bound, rotary motor enzymes that can catalyze ATP synthesis and hydrolysis. Their enzyme kinetics are controlled by internal subunit rotation, by substrate and product concentrations, and by mechanical inhibitory mechanisms but also by the electrochemical potential of protons across the membrane. Single-molecule Förster resonance energy transfer (smFRET) has been used to detect subunit rotation within FoF1-ATP synthases embedded in freely diffusing liposomes. We now report that kinetic monitoring of functional rotation can be prolonged from milliseconds to seconds by utilizing an anti-Brownian electrokinetic trap (ABEL trap). These extended observation times allowed us to observe fluctuating rates of functional rotation for individual FoF1-liposomes in solution. Broad distributions of ATP-dependent catalytic rates were revealed. The buildup of an electrochemical potential of protons was confirmed to limit the maximum rate of ATP hydrolysis. In the presence of ionophores or uncouplers, the fastest subunit rotation speeds measured in single reconstituted FoF1-ATP synthases were 180 full rounds per second. This was much faster than measured by biochemical ensemble averaging, but not as fast as the maximum rotational speed reported previously for isolated single F1 complexes uncoupled from the membrane-embedded Fo complex. Further application of ABEL trap measurements should help resolve the mechanistic causes of such fluctuating rates of subunit rotation.

Evaluation of P. denitrificans F1-ATPase Rotary Motion using Automated Methods to Detect Hidden States

F-ATPase is a rotary motor enzyme with the biological function of synthesizing ATP, the “fuel molecule” of cells. A subsystem of F-ATPase, F1-ATPase, functions in reverse: it hydrolyses ATP into ADP and inorganic phosphate. During this process, the rotation of F1-ATPase's shaft-like rotor is driven by chemical free energy. Samples of paracoccus denitrificans F1-ATPase are studied in single-molecule imaging experiments while the enzyme undergoes ATP synthesis. This single-molecule experiment is performed through the use of a 40 nanometer gold nano-probe used to record rotation angles at a microsecond temporal resolution.

Chloroplast ATP synthase is reduced by both f-type and m-type thioredoxins

The activity of the molecular motor enzyme, chloroplast ATP synthase, is regulated in a redox-dependent manner. The γ subunit, CF1-γ, is the central shaft of this enzyme complex and possesses the redox-active cysteine pair, which is reduced by thioredoxin (Trx). In light conditions, Trx transfers the reducing equivalent obtained from the photosynthetic electron transfer system to the CF1-γ. Previous studies showed that the light-dependent reduction of CF1-γ is more rapid than those of other Trx target proteins in the stroma. Although there are multiple Trx isoforms in chloroplasts, it is not well understood as to which chloroplast Trx isoform primarily contributes to the reduction of CF1-γ, especially under physiological conditions. We therefore performed direct assessment of the CF1-γ reduction capacity of each of the Trx isoforms. The kinetic analysis of the reduction process showed no significant difference in the reduction efficiency between two major chloroplast Trxs, namely Trx-f and Trx-m. Based on the thorough analyses of the CF1-γ redox dynamics in Arabidopsis thaliana Trx mutant plants, we found that lack of Trx-f or Trx-m had no significant impact on the in vivo light-dependent reduction of CF1-γ. The results showed that CF1-γ can accept the reducing power from both Trx-f and Trx-m in chloroplasts.

F-ATP-ase of Escherichia coli membranes: The ubiquitous MgADP-inhibited state and the inhibited state induced by the ε–subunit's C-terminal domain are mutually exclusive

ATP synthases are important energy-coupling, rotary motor enzymes in all kingdoms of life. In all F-type ATP synthases, the central rotor of the catalytic F1 complex is composed of the γ subunit and the N-terminal domain (NTD) of the ε subunit. In the enzymes of diverse bacteria, the C-terminal domain of ε (εCTD) can undergo a dramatic conformational change to trap the enzyme in a transiently inactive state. This inhibitory mechanism is absent in the mitochondrial enzyme, so the εCTD could provide a means to selectively target ATP synthases of pathogenic bacteria for antibiotic development. For Escherichia coli and other bacterial model systems, it has been difficult to dissect the relationship between ε inhibition and a MgADP-inhibited state that is ubiquitous for FOF1 from bacteria and eukaryotes. A prior study with the isolated catalytic complex from E. coli, EcF1, showed that these two modes of inhibition are mutually exclusive, but it has long been known that interactions of F1 with the membrane-embedded FO complex modulate inhibition by the εCTD. Here, we study membranes containing EcFOF1 with wild-type ε, ε lacking the full εCTD, or ε with a small deletion at the C-terminus. By using compounds with distinct activating effects on F-ATP-ase activity, we confirm that εCTD inhibition and ubiquitous MgADP inhibition are mutually exclusive for membrane-bound E. coli F-ATP-ase. We determine that most of the enzyme complexes in wild-type membranes are in the ε-inhibited state (>50%) or in the MgADP-inhibited state (30%).

The Rotary Motor of Life: Single-Molecule Imaging and Molecular Dynamics Simulation of F1-ATPase

F-ATPase is a rotary motor enzyme and its biological function is synthesis of ATP, the “fuel molecule” of cells. A subsystem of F-ATPase, the F1-ATPase, functions in reverse: it hydrolyses ATP into ADP and inorganic phosphate and during this process a rotation of its shaft-like rotor is driven by the chemical free energy which powers the organic nano-motor. Single-molecule imaging experiments revealed that during this rotation F1-ATPase proceeds through steps, i.e, the rotor fluctuates around specific dwell angles then jumps to the next dwell angle. There is a concerted behavior during this stepping rotation in which binding of ATP molecules in one subunit induce the release of ADP (product) molecules from another. Due to the size of the particle, as well as the minute time the process occurs over, it is extremely difficult to obtain accurate information about this sequence of states in the rotation. We examined it using two methods: by simulating molecular dynamics, and the analysis of single-molecule imaging experimental data. In the former case we examined a simulated molecule with triple-occupancy over a finite time increment with a Phosphate and ADP molecule in a binding pocket, and analyzed the simulation trajectory data. Preliminary results suggest a new finding that the Phosphate when doubly protonated can “escape” the pocket via a side door at the interface between a and b subunits. For the latter method, we examined a single molecule undergoing ATP synthesis using a 40 nm gold nano-probe to record measurements. Preliminary results suggest that the ATP binding dwell can be resolved and it is possible to focus the analysis on a hidden, short-lived intermediate step during the binding of ATP and release of ADP.

A nucleobase-binding pocket in a viral RNA-dependent RNA polymerase contributes to elongation complex stability.

The enterovirus 71 (EV71) 3Dpol is an RNA-dependent RNA polymerase (RdRP) that plays the central role in the viral genome replication, and is an important target in antiviral studies. Here, we report a crystal structure of EV71 3Dpol elongation complex (EC) at 1.8 Å resolution. The structure reveals that the 5′-end guanosine of the downstream RNA template interacts with a fingers domain pocket, with the base sandwiched by H44 and R277 side chains through hydrophobic stacking interactions, and these interactions are still maintained after one in-crystal translocation event induced by nucleotide incorporation, implying that the pocket could regulate the functional properties of the polymerase by interacting with RNA. When mutated, residue R277 showed an impact on virus proliferation in virological studies with residue H44 having a synergistic effect. In vitro biochemical data further suggest that mutations at these two sites affect RNA binding, EC stability, but not polymerase catalytic rate (kcat) and apparent NTP affinity (KM,NTP). We propose that, although rarely captured by crystallography, similar surface pocket interaction with nucleobase may commonly exist in nucleic acid motor enzymes to facilitate their processivity. Potential applications in antiviral drug and vaccine development are also discussed.

Increasing the accuracy of nanopore DNA sequencing using a time-varying cross membrane voltage.

Nanopore DNA sequencing is limited by low base calling accuracy. Improved base-calling accuracy has so far relied on specialized base-calling algorithms, different nanopores and motor enzymes, or biochemical methods to re-read DNA molecules. Two primary error modes hamper sequencing accuracy: enzyme mis-steps and sequences with indistinguishable signals. We vary the driving voltage across an MspA nanopore between 100 to 200 mV with a frequency of 200 Hz, changing how the DNA strand moves through the nanopore. As a DNA helicase moves the DNA through the nanopore in discrete steps, the variable voltage positions the DNA continuously between these steps. The resulting electronic signal can be analysed to overcome the primary error modes in base-calling. Single-passage de novo base-calling accuracy in our device increases from 62.7 ± 0.5% with a constant driving voltage to 79.3 ± 0.3% with a variable driving voltage. Our variable-voltage sequencing mode is complementary to other advances in nanopore sequencing and is amenable to incorporation into other enzyme-actuated nanopore sequencing devices.