Nanopore Tweezers Research Articles

Nanopore tweezers show fractional-nucleotide translocation in sequence-dependent pausing by RNA polymerase

RNA polymerases (RNAPs) carry out the first step in the central dogma of molecular biology by transcribing DNA into RNA. Despite their importance, much about how RNAPs work remains unclear, in part because the small (3.4 Angstrom) and fast (~40 ms/nt) steps during transcription were difficult to resolve. Here, we used high-resolution nanopore tweezers to observe the motion of single Escherichia coli RNAP molecules as it transcribes DNA ~1,000 times improved temporal resolution, resolving single-nucleotide and fractional-nucleotide steps of individual RNAPs at saturating nucleoside triphosphate concentrations. We analyzed RNAP during processive transcription elongation and sequence-dependent pausing at the yrbL elemental pause sequence. Each time RNAP encounters the yrbL elemental pause sequence, it rapidly interconverts between five translocational states, residing predominantly in a half-translocated state. The kinetics and force-dependence of this half-translocated state indicate it is a functional intermediate between pre- and post-translocated states. Using structural and kinetics data, we show that, in the half-translocated and post-translocated states, sequence-specific protein-DNA interaction occurs between RNAP and a guanine base at the downstream end of the transcription bubble (core recognition element). Kinetic data show that this interaction stabilizes the half-translocated and post-translocated states relative to the pre-translocated state. We develop a kinetic model for RNAP at the yrbL pause and discuss this in the context of key structural features.

Observing inhibition of the SARS-CoV-2 helicase at single-nucleotide resolution.

The genome of SARS-CoV-2 encodes for a helicase (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 double-stranded DNA. Our data reveal nsp13's single-nucleotide steps, translocating at ∼1000 nt/s or unwinding at ∼100 bp/s. Nanopore tweezers' high spatiotemporal resolution enables 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. We construct a detailed picture of inhibition in which ATPγS has multiple mechanisms of inhibition. The dominant mechanism of inhibition depends on the application of assisting force. This lays the groundwork for future single-moleculeinhibition studies with viral helicases.

Probing protein structural dynamics at the single-molecule level by nanopore tweezers.

Proteins are dynamic entities that sample multiple conformations over a range of time scales. Understanding how proteins work requires a full description of their conformational dynamics over time. Nuclear magnetic resonance (NMR) spectroscopy can measure protein dynamics at multiple time scales, however, application is laborious and limited to relatively small proteins that can be stably prepared at high concentrations. Conformational heterogeneity, such as multiple weakly populated conformational states, is challenging to resolve from ensemble methods such as NMR due to averaging effects. Integrative approaches and single-molecule techniques can be advantageous for revealing transient protein dynamics and functional heterogeneities. Nanopore tweezers have emerged as a powerful, single-molecule analytical tool to monitor the protein's conformational dynamics. Nanopore tweezers confine a single protein within the pore lumen and measure ionic current changes induced by the protein analyte's motion. Here we will present several studies of ClyA nanopore tweezers to showcase their utility in resolving ligand interaction modes, screening allosteric drugs and investigating intrinsically disordered protein interactions.

Nanopore tweezers measurements of RecQ conformational changes reveal the energy landscape of helicase motion.

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.

Sequence-dependent mechanochemical coupling of helicase translocation and unwinding at single-nucleotide resolution

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 invivo, 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.

Mapping the conformational energy landscape of Abl kinase using ClyA nanopore tweezers

Protein kinases play central roles in cellular regulation by catalyzing the phosphorylation of target proteins. Kinases have inherent structural flexibility allowing them to switch between active and inactive states. Quantitative characterization of kinase conformational dynamics is challenging. Here, we use nanopore tweezers to assess the conformational dynamics of Abl kinase domain, which is shown to interconvert between two major conformational states where one conformation comprises three sub-states. Analysis of kinase-substrate and kinase-inhibitor interactions uncovers the functional roles of relevant states and enables the elucidation of the mechanism underlying the catalytic deficiency of an inactive Abl mutant G321V. Furthermore, we obtain the energy landscape of Abl kinase by quantifying the population and transition rates of the conformational states. These results extend the view on the dynamic nature of Abl kinase and suggest nanopore tweezers can be used as an efficient tool for other members of the human kinome.

C-Glycoside DNA bases from an artificially expanded genetic information system reduce processivity in a SF2 helicase as revealed by nanopore tweezers

Artificially expanded genetic information systems (AEGISs) incorporating nucleobases beyond the standard adenine, thymine, cytosine, and guanine have been developed and used for applications such as diagnostics, information storage, and understanding the origin of life. However, these applications are limited by the availability of methods to efficiently sequence AEGISs due to the high cost of sample preparation and limited compatibility between sequencing-critical enzymes and non-standard bases.

Modelling single-molecule kinetics of helicase translocation using high-resolution nanopore tweezers (SPRNT).

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.

SV40 Large T Antigen Helicase Unwinds Double-Stranded DNA in Single-Nucleotide Steps as Revealed by Nanopore Tweezers

SV40 Large T Antigen Helicase Unwinds Double-Stranded DNA in Single-Nucleotide Steps as Revealed by Nanopore Tweezers

Tracking Single RNAP Enzyme Steps and State Transitions During Elongation and Pausing with Nanopore Tweezers

Tracking Single RNAP Enzyme Steps and State Transitions During Elongation and Pausing with Nanopore Tweezers

Single-Molecule Analysis and Engineering of DNA Motors.

Molecular motors are diverse enzymes that transduce chemical energy into mechanical work and, in doing so, perform critical cellular functions such as DNA replication and transcription, DNA supercoiling, intracellular transport, and ATP synthesis. Single-molecule techniques have been extensively used to identify structural intermediates in the reaction cycles of molecular motors and to understand how substeps in energy consumption drive transitions between the intermediates. Here, we review a broad spectrum of single-molecule tools and techniques such as optical and magnetic tweezers, atomic force microscopy (AFM), single-molecule fluorescence resonance energy transfer (smFRET), nanopore tweezers, and hybrid techniques that increase the number of observables. These methods enable the manipulation of individual biomolecules via the application of forces and torques and the observation of dynamic conformational changes in single motor complexes. We also review how these techniques have been applied to study various motors such as helicases, DNA and RNA polymerases, topoisomerases, nucleosome remodelers, and motors involved in the condensation, segregation, and digestion of DNA. In-depth analysis of mechanochemical coupling in molecular motors has made the development of artificially engineered motors possible. We review techniques such as mutagenesis, chemical modifications, and optogenetics that have been used to re-engineer existing molecular motors to have, for instance, altered speed, processivity, or functionality. We also discuss how single-molecule analysis of engineered motors allows us to challenge our fundamental understanding of how molecular motors transduce energy.

Fractional‐nucleotide translocation in sequence‐dependent pausing by RNA polymerase: Single‐molecule picometer‐resolution nanopore tweezers (SPRNT)

Single‐molecule picometer‐resolution nanopore tweezers (SPRNT) enables monitoring of translocation of a nucleic‐acid motor protein on a nucleic‐acid track with sequence registration, sub‐nucleotide spatial resolution, sub‐millisecond temporal resolution, and the ability to apply assisting and opposing forces.We have demonstrated the ability of SPRNT to monitor translocation of RNA polymerase (RNAP) relative to the DNA template strand, the DNA non‐template strand, and the RNA product strand in transcription, and we have applied SPRNT to analyze RNAP translocation in transcription elongation and sequence‐dependent transcriptional pausing.The results show that sequence‐dependent transcriptional pausing involves a “half‐translocated” state in which RNAP translocates relative to the DNA template strand by 0.5 nucleotides. The “half‐translocated” state is unable to bind the next NTP substrate, presumably because RNAP translocation by 0.5 nt does not align the next template‐strand nucleotide with the RNAP active‐center NTP addition site. The half‐translocated state interconverts rapidly (~0.1–1 s) with the “pre‐translocated” state and converts slowly (~1 s) to the NTP‐binding‐competent “post‐translocated state.” In the presence of saturating NTPs, conversion to the post‐translocated state is followed by pause escape. In the presence of limiting NTPs, conversion to the post‐translocated state is followed by return to interconverting half‐translocated and pre‐translocated states and continued pausing.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Taking a Closer Look at RecQ Helicase with Nanopore Tweezers

Single-molecule picometer resolution nanopore tweezers (SPRNT) is a single-molecule tool for studying enzymes that move on nucleic acids. SPRNT measures enzyme position with sub-Angstrom spatial resolution on millisecond timescales while also providing the DNA sequence passing through the enzyme. We use SPRNT to monitor the kinetics of the superfamily 2 helicase RecQ as it translocates on single-stranded DNA and as it unwinds double-stranded DNA. We observe two kinetic states, which have not previously been distinguished (or identified), for each nucleotide translocated by RecQ in both translocase and helicase modes. We discuss implications of these kinetic states for the ATPase cycle of RecQ and discuss our results in the context of complementary magnetic tweezers assays of RecQ.

Determining Dynamics of RNA Polymerase Elongation and Pausing using Nanopore Tweezers

Determining Dynamics of RNA Polymerase Elongation and Pausing using Nanopore Tweezers

Determining the effects of DNA sequence on Hel308 helicase translocation along single-stranded DNA using nanopore tweezers.

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.

High-Resolution Single-Molecule Analysis of UvrD Helicase using Nanopore Tweezers

Single-molecule methods reveal enzyme dynamics that cannot be observed with traditional bulk assays. We analyze UvrD helicase translocation on ssDNA using a new technique called Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT), which has unprecedented spatiotemporal resolution. In SPRNT, a membrane porin MspA in a phospholipid bilayer forms an electrical connection between two salt solutions. A voltage applied across the pore causes ion current to flow through and draws negatively charged DNA bound to UvrD into the pore. UvrD is too large to fit through MspA, and comes to rest on the rim, arresting the DNA's motion. Different DNA nucleotides in the pore modulate the ion current. As UvrD walks along the DNA, it draws the DNA out of the pore and changes the ion current. Therefore, the durations of ion-current states are kinetic measurements of UvrD. Using SPRNT, we show that UvrD moves in single nucleotide steps along DNA and characterize its ATPase activity.

Mechanism of Sequence Dependent Translocation of a Superfamily 2 Helicase on ssDNA

Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT) is a single-molecule method for monitoring the motion of nucleic acids controlled by a molecular motor enzyme through a nanopore at unprecedented spatiotemporal resolution with simultaneous readout of the nucleic acid sequence in the enzyme. We use this resolution to show that the translocation kinetics of the helicase Hel308 along single-stranded DNA depend on base-specific interactions between the DNA sequence and amino acids in Hel308. Combining our single-molecule data with structural data of Hel308, we propose a mechanism for sequence-specific translocation of Hel308 on ssDNA.

Analysis of Force Dependence of Translocation and Unwinding of Helicase PCRA using SPRNT

Using Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT), we analyzed the kinetics of a mutant PcrA ‘superhelicase’ under varying assisting and opposing forces as it tranlsocated along ssDNA and unwound dsDNA. The superhelicase, dubbed ‘PcrA-X,’ was created by constraining the flexible 2B domain into the ‘closed’ conformation by chemical crosslinking. With SPRNT we are able to resolve the helicase's stepping dynamics on millisecond timescales at sub-nucleotide spatial resolution. We find that the kinetics of both PcrA-X translocation on ssDNA and unwinding of dsDNA are force-dependent. By comparing ssDNA translocation to dsDNA unwinding, we examine how DNA unwinding relates to the helicase's stepping motion along its DNA track.

Revealing dynamics of helicase translocation on single-stranded DNA using high-resolution nanopore tweezers

Enzymes that operate on DNA or RNA perform the core functions of replication and expression in all of biology. To gain high-resolution access to the detailed mechanistic behavior of these enzymes, we developed single-molecule picometer-resolution nanopore tweezers (SPRNT), a single-molecule technique in which the motion of polynucleotides through an enzyme is measured by a nanopore. SPRNT reveals two mechanical substates of the ATP hydrolysis cycle of the superfamily 2 helicase Hel308 during translocation on single-stranded DNA (ssDNA). By analyzing these substates at millisecond resolution, we derive a detailed kinetic model for Hel308 translocation along ssDNA that sheds light on how superfamily 1 and 2 helicases turn ATP hydrolysis into motion along DNA. Surprisingly, we find that the DNA sequence within Hel308 affects the kinetics of helicase translocation.

Direct Single Molecule Measurement of ATP Hydrolysis Substates in Hel308 DNA Helicase using Nanopore Tweezers

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.