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Helicases are essential for nearly all nucleic acid processes across the tree of life, yet detailed understanding of how they couple ATP hydrolysis to translocation and unwinding remains incomplete because their small (∼300 picometer), fast (∼1 ms) steps are difficult to resolve. Here, we use Nanopore Tweezers to observe single Escherichia coli RecQ helicases as they translocate on and unwind DNA at ultrahigh spatiotemporal resolution. Nanopore Tweezers simultaneously resolve individual steps of RecQ along the DNA and conformational changes of the helicase associated with stepping. Our data reveal the mechanochemical coupling between physical domain motions and chemical reactions that together produce directed motion of the helicase along DNA. Nanopore Tweezers measurements are performed under either assisting or opposing force applied directly on RecQ, shedding light on how RecQ responds to such forces in vivo. Determining the rates of translocation and physical conformational changes under a wide range of assisting and opposing forces reveals the underlying dynamic energy landscape that drives RecQ motion. We show that RecQ has a highly asymmetric energy landscape that enables RecQ to maintain velocity when encountering molecular roadblocks such as bound proteins and DNA secondary structures. This energy landscape also provides a mechanistic basis making RecQ an ‘active helicase,’ capable of unwinding dsDNA as fast as it translocates on ssDNA. Such an energy landscape may be a general strategy for molecular motors to maintain consistent velocity despite opposing loads or roadblocks.

Techniques that can directly sequence RNAs and determine secondary structures are essential to establish how an RNA molecule folds and how folding affects its function. We describe the development of a direct RNA nanopore sequencing technique using an engineeredBombyx moriretroelement reverse transcriptase (RT) to thread RNA through theMycobacterium smegmatisporin A (MspA) nanopore in single-nucleotide steps. First, we establish the map that correlates RNA sequence and MspA ion current. We find that during sequencing the RT can sense the strength of RNA secondary structures 11-12 nt downstream of its front boundary, modifying its stepping dynamics accordingly. Using this feature, we achieve simultaneous RNA nanopore sequencing and structure detection, without the need of prior conversion to complementary DNA (cDNA) or RNA modifications. The sequence-dependent ion currents open the way to utilize the MspA nanopore to investigate the single-molecule activity of other processive RNA translocases such as the ribosome.

The genome of SARS-CoV-2 encodes for a helicase called nsp13 that is essential for viral replication and highly conserved across related viruses, making it an attractive antiviral target. Here we use nanopore tweezers, a high-resolution single-molecule technique, to gain detailed insight into how nsp13 turns ATP-hydrolysis into directed motion along nucleic acid strands. We measured nsp13 both as it translocates along single-stranded DNA or unwinds short DNA duplexes. Our data confirm that nsp13 uses the inchworm mechanism to move along the DNA in single-nucleotide steps, translocating at ~1000 nt/s or unwinding at ~100 bp/s. Nanopore tweezers’ high spatio-temporal resolution enables observation of the fundamental physical steps taken by nsp13 even as it translocates at speeds in excess of 1000 nucleotides per second enabling detailed kinetic analysis of nsp13 motion. As a proof-of-principle for inhibition studies, we observed nsp13’s motion in the presence of the ATPase inhibitor ATPγS. Our data reveals that ATPγS interferes with nsp13’s action by affecting several different kinetic processes. The dominant mechanism of inhibition differs depending on the application of assisting force. These advances demonstrate that nanopore tweezers are a powerful method for studying viral helicase mechanism and inhibition.

Single-molecule picometer resolution nanopore tweezers (SPRNT) is a technique for monitoring the motion of individual enzymes along a nucleic acid template at unprecedented spatiotemporal resolution. We review the development of SPRNT and the application of single-molecule kinetics theory to SPRNT data to develop a detailed model of helicase motion along a single-stranded DNA substrate. In this review, we present three examples of questions SPRNT can answer in the context of the Superfamily 2 helicase Hel308. With Hel308, SPRNT’s spatiotemporal resolution enables resolution of two distinct enzymatic substates, one which is dependent upon ATP concentration and one which is ATP independent. By analyzing dwell-time distributions and helicase back-stepping, we show, in detail, how SPRNT can be used to determine the nature of these observed steps. We use dwell-time distributions to discern between three different possible models of helicase backstepping. We conclude by using SPRNT’s ability to discern an enzyme’s nucleotide-specific location along a DNA strand to understand the nature of sequence-specific enzyme kinetics and show that the sequence within the helicase itself affects both step dwell-time and backstepping probability while translocating on single-stranded DNA.

The conserved RNA helicase UPF1 coordinates nonsense-mediated mRNA decay (NMD) by engaging with mRNAs, RNA decay machinery and the terminating ribosome. UPF1 ATPase activity is implicated in mRNA target discrimination and completion of decay, but the mechanisms through which UPF1 enzymatic activities such as helicase, translocase, RNP remodeling, and ATPase-stimulated dissociation influence NMD remain poorly defined. Using high-throughput biochemical assays to quantify UPF1 enzymatic activities, we show that UPF1 is only moderately processive (

We used single-molecule picometer-resolution nanopore tweezers (SPRNT) to resolve the millisecond single-nucleotide steps of superfamily 1 helicase PcrA as it translocates on, or unwinds, several kilobase-long DNA molecules. We recorded more than two million enzyme steps under various assisting and opposing forces in diverse adenosine tri- and diphosphate conditions to comprehensively explore the mechanochemistry of PcrA motion. Forces applied in SPRNT mimic forces and physical barriers PcrA experiences in vivo , such as when the helicase encounters bound proteins or duplex DNA. We show how PcrA’s kinetics change with such stimuli. SPRNT allows for direct association of the underlying DNA sequence with observed enzyme kinetics. Our data reveal that the underlying DNA sequence passing through the helicase strongly influences the kinetics during translocation and unwinding. Surprisingly, unwinding kinetics are not solely dominated by the base pairs being unwound. Instead, the sequence of the single-stranded DNA on which the PcrA walks determines much of the kinetics of unwinding.

Helicases are essential for nearly all nucleic acid processes across the tree of life. Using Nanopore Tweezers we observed the small, fast steps taken by single RecQ helicases as they step along and unwind DNA at ultrahigh spatiotemporal resolution. By directly measuring conformational substates of RecQ we determine the coupling between helicase domain motions and chemical reactions that together produce forward motion along the DNA. Application of assisting and opposing forces shows that RecQ has a highly asymmetric energy landscape that reduces its sensitivity to opposing mechanical forces that could be encountered in vivo by molecular roadblocks such as DNA bound proteins. This energy landscape enables RecQ to maintain speed against an opposing load.

Unnatural base pairs (UBPs) have been developed and used for a variety of in vitro applications, as well as for the engineering of semi-synthetic organisms (SSOs) that store and retrieve increased information. However, these applications are limited by the availability of methods to rapidly and accurately determine the sequence of unnatural DNA. Here, we report the development and application of the MspA nanopore to sequence DNA containing the dTPT3-dNaM UBP. Analysis of two sequence contexts reveals that DNA containing the UBP is replicated with an efficiency and fidelity similar to that of natural DNA and sufficient for use as the basis of an SSO that produces proteins with non-canonical amino acids.

Motor enzymes that process nucleic-acid substrates play vital roles in all aspects of genome replication, expression, and repair. The DNA and RNA nucleobases are known to affect the kinetics of these systems in biologically meaningful ways. Recently, it was shown that DNA bases control the translocation speed of helicases on single-stranded DNA, however the cause of these effects remains unclear. We use single-molecule picometer-resolution nanopore tweezers (SPRNT) to measure the kinetics of translocation along single-stranded DNA by the helicase Hel308 from Thermococcus gammatolerans. SPRNT can measure enzyme steps with subangstrom resolution on millisecond timescales while simultaneously measuring the absolute position of the enzyme along the DNA substrate. Previous experiments with SPRNT revealed the presence of two distinct substates within the Hel308 ATP hydrolysis cycle, one [ATP]-dependent and the other [ATP]-independent. Here, we analyze in-depth the apparent sequence dependent behavior of the [ATP]-independent step. We find that DNA bases at two sites within Hel308 control sequence-specific kinetics of the [ATP]-independent step. We suggest mechanisms for the observed sequence-specific translocation kinetics. Similar SPRNT measurements and methods can be applied to other nucleic-acid-processing motor enzymes.

Significance DNA helicases are enzymes that use energy from ATP hydrolysis to move along nucleic acid tracks and unwind double-stranded DNA. Helicases are involved in every aspect of DNA metabolism and are therefore vital to maintaining genomic integrity. Using the single-molecule technique single-molecule picometer-resolution nanopore tweezers (SPRNT), which measures the position of DNA through the biological membrane protein MspA as an enzyme moves along the DNA, we monitored the kinetics of the helicase Hel308 at 1,000 times better temporal resolution than was previously possible. We derived a detailed mechanism for how ATP hydrolysis coordinates the motion of Hel308 along single-stranded DNA that can likely be applied to other structurally similar helicases and showed that the DNA sequence in Hel308 affects its kinetics.

Nanopore sequencing of DNA is a single-molecule technique that may achieve long reads, low cost and high speed with minimal sample preparation and instrumentation. Here, we build on recent progress with respect to nanopore resolution and DNA control to interpret the procession of ion current levels observed during the translocation of DNA through the pore MspA. As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers). This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome. Furthermore, we show nanopore sequencing reads of phi X 174 up to 4,500 bases in length that can be unambiguously aligned to the phi X 174 reference genome, and demonstrate proof-of-concept utility with respect to hybrid genome assembly and polymorphism detection. This work provides the foundation for nanopore sequencing of long, complex, natural DNA strands.

Nanopore DNA sequencing is a promising single-molecule analysis technology. This technique relies on a DNA motor enzyme to control movement of DNA precisely through a nanopore. Specific experimental buffer conditions are required based on the preferred operating conditions of the DNA motor enzyme. While many DNA motor enzymes typically operate in salt concentrations under 100 mM, salt concentration simultaneously affects signal and noise magnitude as well as DNA capture rate in nanopore sequencing, limiting standard experimental conditions to salt concentrations greater than ~100 mM in order to maintain adequate resolution and experimental throughput. We evaluated the signal contribution from ions on both sides of the membrane (cis and trans) by varying cis and trans [KCl] independently during phi29 DNA Polymerase-controlled translocation of DNA through the biological porin MspA. Our studies reveal that during DNA translocation, the negatively charged DNA increases cation selectivity through MspA with the majority of current produced by the flow of K+ ions from trans to cis. Varying trans [K+] has dramatic effects on the signal magnitude, whereas changing cis [Cl-] produces only small effects. Good signal-to-noise can be maintained with cis [Cl-] as small as 20 mM, if the concentration of KCl on the trans side is kept high. These results demonstrate the potential of using salt-sensitive motor enzymes (helicases, polymerases, recombinases) in nanopore systems and offer a guide for selecting buffer conditions in future experiments to simultaneously optimize signal, throughput, and enzyme activity.

MspA is a biological porin that has demonstrated use in nanopore DNA sequencing. In this technique, an electric field is applied across a lipid bilayer containing MspA, and the flow of ions through the pore produces a measurable current. Negatively charged single stranded DNA molecules are driven through MspA according to the electric field. A polymerase or helicase bound to the DNA molecule controls the polymers’ translocation. The different DNA nucleotides uniquely modulate the flow of ions as the DNA translocates, allowing a sequence to be interpreted from the changes in current. As the observed current is due to the flow of positive and negative ions within MspA, varying the buffer salt concentration can have dramatic effects on the signal. With salt concentrations below 100 mM on both sides of the membrane, signal is reduced making sequencing challenging. In this study, we varied the concentrations on either side of the membrane (cis and trans) independently, creating asymmetric salt profiles. We observed the effects of these asymmetries on open pore current, DNA interaction rate, and DNA blockage currents during controlled translocation by phi29 DNA polymerase. MspA is highly cation selective during DNA translocation with the majority of current produced by the flow of K+ ions from trans to cis. Varying trans [K+] has dramatic effects on the signal magnitude, whereas changing cis [Cl−] produces only small effects. This means that useful signals can be gained with negligible cis [KCl], if the concentration of [KCl] on the trans side is kept high. Many DNA motor enzymes, whose properties may be ideal for sequencing, loose function at appreciable salt concentrations, and these results reveal that such enzymes could still be used in nanopore DNA sequencing with MspA.

Techniques for measuring the motion of single motor proteins, such as FRET and optical tweezers, are limited to a resolution of ∼300 pm. We use ion current modulation through the protein nanopore MspA to observe translocation of helicase Hel308 on DNA with up to ∼40 pm sensitivity. This approach should be applicable to any protein that translocates on DNA or RNA, including helicases, polymerases, recombinases and DNA repair enzymes.

The protein pore Mycobacterium smegmatis porin A (MspA) is enabling single-molecule nanopore DNA sequencing. In nanopore sequencing an electric field causes an ion current to flow through a single nano-meter sized pore embedded in a membrane. The electric field also draws single-stranded DNA through the pore. DNA bases inside the pore specifically modulate the measured ion current. We use molecular motors to regulate DNA's translocation speed through MspA. Here, we use this system to examine these motors by measuring the movement of DNA. With MspA, we can detect DNA motion as low as 35 picometers on sub millisecond timescales. To demonstrate the sensitivity of MspA we studied the detailed motions of DNA moved by a Super Family II helicase.

Single-molecule picometer resolution nanopore tweezers (SPRNT) is a single molecule tool for studying enzymes that move on nucleic acids (NA). SPRNT measures NA position relative to the enzyme at sub-Angstrom spatial resolution on millisecond timescales, while simultaneously providing the NA sequence near the enzyme. We use this method to resolve two substates of the ATP hydrolysis cycle of a Hel308 DNA Helicase. By examining the dwell times of each state, we derive a kinetic model of Hel308's translocation along single-stranded DNA, and find that the model's rate constants depend on the DNA sequence within the enzyme. This is, to our knowledge, the first evidence of sequence-dependent translocase behavior in a helicase system.

Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT) is a new technique for observing the motion of single motor enzymes on nucleic acid tracks with more than an order of magnitude improvement in spatiotemporal resolution over existing single-molecule technologies such as optical tweezers. Because SPRNT is derived from nanopore DNA sequencing, it also provides a readout of the DNA sequence moving through the enzyme, allowing us to query how nucleic acid sequence regulates the motion of motor enzymes. We discuss how SPRNT works in the context of helicase and RNA polymerase systems to which we have applied SPRNT, and present results including a full kinetic model relating ATP-hydrolysis steps to sub-nucleotide helicase motion and observation of RNA polymerase pausing and termination at unprecedented resolution.

Both simulations and observations indicate that stars form in filamentary, hierarchically clustered associations, most of which disperse into their galactic field once feedback destroys their parent clouds. However, during their early evolution in these substructured environments, stars can undergo close encounters with one another that might have significant impacts on their protoplanetary disks or young planetary systems. We perform N-body simulations of the early evolution of dissolving, substructured clusters with a wide range of properties, with the aim of quantifying the expected number and orbital element distributions of encounters as a function of cluster properties. We show that the presence of substructure both boosts the encounter rate and modifies the distribution of encounter velocities compared to what would be expected for a dynamically relaxed cluster. However, the boost only lasts for a dynamical time, and as a result the overall number of encounters expected remains low enough that gravitational stripping is unlikely to be a significant effect for the vast majority of star-forming environments in the Galaxy. We briefly discuss the implications of this result for models of the origin of the Solar System, and of free-floating planets. We also provide tabulated encounter rates and orbital element distributions suitable for inclusion in population synthesis models of planet formation in a clustered environment., Comment: 17 Pages 19 Figures Accepted by Astrophysical Journal

We have developed a high-resolution nanopore sensor to observe enzyme activity with high positional sensitivity. In this method, single stranded DNA coupled to an enzyme is drawn through the engineered protein pore MspA. Nucleotides along DNA pass through the pore and modulate an ion current that flows through the pore. Analysis of this ion current indicates the precise position of the DNA and thereby provides a real-time record of the enzyme's activity. The position of DNA within MspA can be measured on millisecond time scales with a position resolution as small as ∼40 picometers. We demonstrate this sensitivity on a Hel308 helicase, where we observe two distinct sub-states for each nucleotide processed. One of these about half-nucleotide long steps is ATP-dependent and the other is ATP-independent. The spatial and temporal resolution of this low-cost single molecule technique allows exploration of hitherto unseen enzyme dynamics in real-time.

Recently, it was shown for the first time that Escherichia coli can efficiently propagate a genetic alphabet expanded from the four canonical DNA nucleotides to include the synthetic “alien” bases d5SICS and dNaM. Traditional sequencing platforms cannot detect these alien bases. We tested whether nanopore sequencing with the protein pore Mycobacterium smegmatis porin A (MspA) is sensitive to d5SICS and dNaM. In nanopore sequencing, a nanometer scale pore provides the only electrical connection between two electrolyte solutions. An applied voltage causes a current to flow through the pore. Negatively-charged single stranded DNA is drawn through the pore, causing a nucleotide specific reduction in the measured current. Thus-far, MspA is the only nanopore shown to demonstrate single-nucleotide sensitivity to standard nucleotides as well as methyl cytosine and hydroxymethyl cytosine. We use the φ29 DNA polymerase to regulate DNA motion to single-nucleotide steps. Here, we demonstrate the direct detection d5SICS and dNaM with MspA-enabled nanopore sequencing.