A guide to accelerated direct digital counting of single nucleic acid molecules by FRET-based intramolecular kinetic fingerprinting.

Cell-free nucleic acids (cfNAs) such as short non-coding microRNA (miRNA) and circulating tumor DNA (ctDNA) that reside in bodily fluids have emerged as potential cancer biomarkers. Methods for the rapid, highly specific, and sensitive monitoring of cfNAs in biofluids have, therefore, become increasingly attractive as clinical diagnosis tools. As a next generation technology, we provide a practical guide for an amplification-free, single molecule Förster resonance energy transfer (smFRET)-based kinetic fingerprinting approach termed intramolecular single molecule recognition through equilibrium Poisson sampling, or iSiMREPS, for the rapid detection and counting of miRNA and mutant ctDNA with virtually unlimited specificity and single molecule sensitivity. iSiMREPS utilizes a pair of fluorescent detection probes, wherein one probe immobilizes the target molecules on the surface, and the other probe transiently and reversibly binds to the target to generate characteristic time-resolved fingerprints as smFRET signal that are detected in a total internal reflection fluorescence microscope. Analysis of these kinetic fingerprints enables near-perfect discrimination between specific binding to target molecules and nonspecific background binding. By accelerating kinetic fingerprinting using the denaturant formamide and reducing background signals by removing target-less probes from the surface via toehold-mediated strand displacement, iSiMREPS has been demonstrated to count miR-141 and EGFR exon 19 deletion ctDNA molecules with a limit of detection (LOD) of ~1 and 3 fM, respectively, as well as mutant allele fractions as low as 0.0001%, during a standard acquisition time of only ~10 s per field of view. In this review, we provide a detailed roadmap for implementing iSiMREPS more broadly in research and clinical diagnostics, combining rapid analysis, high specificity, and high sensitivity.

Direct kinetic fingerprinting and digital counting of single protein molecules.

The sensitive and accurate quantification of protein biomarkers plays important roles in clinical diagnostics and biomedical research. Sandwich ELISA and its variants accomplish the capture and detection of a target protein via two antibodies that tightly bind at least two distinct epitopes of the same antigen and have been the gold standard for sensitive protein quantitation for decades. However, existing antibody-based assays cannot distinguish between signal arising from specific binding to the protein of interest and nonspecific binding to assay surfaces or matrix components, resulting in significant background signal even in the absence of the analyte. As a result, they generally do not achieve single-molecule sensitivity, and they require two high-affinity antibodies as well as stringent washing to maximize sensitivity and reproducibility. Here, we show that surface capture with a high-affinity antibody combined with kinetic fingerprinting using a dynamically binding, low-affinity fluorescent antibody fragment differentiates between specific and nonspecific binding at the single-molecule level, permitting the direct, digital counting of single protein molecules with femtomolar-to-attomolar limits of detection (LODs). We apply this approach to four exemplary antigens spiked into serum, demonstrating LODs 55- to 383-fold lower than commercially available ELISA. As a real-world application, we establish that endogenous interleukin-6 (IL-6) can be quantified in 2-µL serum samples from chimeric antigen receptor T cell (CAR-T cell) therapy patients without washing away excess serum or detection probes, as is required in ELISA-based approaches. This kinetic fingerprinting thus exhibits great potential for the ultrasensitive, rapid, and streamlined detection of many clinically relevant proteins.

Direct Kinetic Fingerprinting for High-Accuracy Single-Molecule Counting of Diverse Disease Biomarkers.

Methods for detecting and quantifying disease biomarkers in biofluids with high specificity and sensitivity play a pivotal role in enabling clinical diagnostics, including point-of-care tests. The most widely used molecular biomarkers include proteins, nucleic acids, hormones, metabolites, and other small molecules. While numerous methods have been developed for analyzing biomarkers, most techniques are challenging to implement for clinical use due to insufficient analytical performance, high cost, and/or other practical shortcomings. For instance, the detection of cell-free nucleic acid (cfNA) biomarkers by digital PCR and next-generation sequencing (NGS) requires time-consuming nucleic acid extraction steps, often introduces enzymatic amplification bias, and can be costly when high specificity is required. While several amplification-free methods for detecting cfNAs have been reported, these techniques generally suffer from low specificity and sensitivity. Meanwhile, the quantification of protein biomarkers is generally performed using immunoassays such as enzyme-linked immunosorbent assay (ELISA); the analytical performance of these methods is often limited by the availability of antibodies with high affinity and specificity as well as the significant nonspecific binding of antibodies to assay surfaces. To address the drawbacks of existing biomarker detection methods and establish a universal diagnostics platform capable of detecting different types of analytes, we have developed an amplification-free approach, named single-molecule recognition through equilibrium Poisson sampling (SiMREPS), for the detection of diverse biomarkers with arbitrarily high specificity and single-molecule sensitivity. SiMREPS utilizes the transient, reversible binding of fluorescent detection probes to immobilized target molecules to generate kinetic fingerprints that are detected by single-molecule fluorescence microscopy. The analysis of these kinetic fingerprints enables nearly perfect discrimination between specific binding to target molecules and any nonspecific binding. Early proof-of-concept studies demonstrated the in vitro detection of miRNAs with a limit of detection (LOD) of approximately 1 fM and >500-fold selectivity for single-nucleotide polymorphisms. The SiMREPS approach was subsequently expanded to the detection of rare mutant DNA alleles from biofluids at mutant allele fractions of as low as 1 in 1 million, corresponding to a specificity of >99.99999%. Recently, SiMREPS was generalized to protein quantification using dynamically binding antibody probes, permitting LODs in the low-femtomolar to attomolar range. Finally, SiMREPS has been demonstrated to be suitable for the in situ detection of miRNAs in cultured cells, the quantification of small-molecule toxins and drugs, and the monitoring of telomerase activity at the single-molecule level. In this Account, we discuss the principles of SiMREPS for the highly specific and sensitive detection of molecular analytes, including considerations for assay design. We discuss the generality of SiMREPS for the detection of very disparate analytes and provide an overview of data processing methods, including the expansion of the dynamic range using super-resolution analysis and the improvement of performance using deep learning algorithms. Finally, we describe current challenges, opportunities, and future directions for the SiMREPS approach.

DNA-Templated Timer Probes for Multiplexed Sensing.

Simultaneous analysis based on encoded fluorophores suffers from potential crosstalk between fluorophores and the limited number of colors that can be practically resolved. Inspired by nontrivial temporal patterns in living organisms, we developed a DNA-templated probe by utilizing DNA polymerase (DNAP) for multiplexed detection of nucleic acids. These probes use differential delay times of signaling by a DNAP-mediated extension to distinguish different targets, which serve as the primers. Taking advantage of the high processivity and the controllable kinetics of DNAP, we find that multiplexed detection can be achieved in homogeneous solution using a single fluorophore. As a proof of concept, we developed assays for genomic DNA from four different bacteria. In addition, we designed and implemented probes to undergo a single oscillation in signal as an alternative way for multiplexing. We anticipate this approach will find broad applications not only in sensing but also in synthetic DNA nanosystems.

Ultraspecific analyte detection by direct kinetic fingerprinting of single molecules.

The detection and quantification of biomarkers have numerous applications in biological research and medicine. The most widely used methods to detect nucleic acids require amplification via the polymerase chain reaction (PCR). However, errors arising from the imperfect copying fidelity of DNA polymerases, limited specificity of primers, and heat-induced damage reduce the specificity of PCR-based methods, particularly for single-nucleotide variants. Furthermore, not all analytes can be amplified efficiently. While amplification-free methods avoid these pitfalls, the specificity of most such methods is strictly constrained by probe binding thermodynamics, which for example hampers detection of rare somatic mutations. In contrast, single-molecule recognition through equilibrium Poisson sampling (SiMREPS) provides ultraspecific detection with single-molecule and single-nucleotide sensitivity by monitoring the repetitive interactions of a fluorescent probe with surface-immobilized targets. In this review, we discuss SiMREPS in comparison with other analytical approaches, and describe its utility in quantifying a range of nucleic acids and other analytes.

A guide to nucleic acid detection by single-molecule kinetic fingerprinting.

Conventional methods for detecting small quantities of nucleic acids require amplification by the polymerase chain reaction (PCR), which necessitates prior purification and introduces copying errors. While amplification-free methods do not have these shortcomings, they are generally orders of magnitude less sensitive and specific than PCR-based methods. In this review, we provide a practical guide to a novel amplification-free method, single-molecule recognition through equilibrium Poisson sampling (SiMREPS), that provides both single-molecule sensitivity and single-base selectivity by monitoring the repetitive interactions of fluorescent probes to immobilized targets. We demonstrate how this kinetic fingerprinting filters out background arising from the inevitable nonspecific binding of probes, yielding virtually zero background signal. As practical applications of this digital detection methodology, we present the quantification of microRNA miR-16 and the detection of the mutation EGFR L858R with an apparent single-base discrimination factor of over 3 million.

Ultraspecific and Amplification-Free Quantification of Mutant DNA by Single-Molecule Kinetic Fingerprinting.

Conventional techniques for detecting rare DNA sequences require many cycles of PCR amplification for high sensitivity and specificity, potentially introducing significant biases and errors. While amplification-free methods exist, they rarely achieve the ability to detect single molecules, and their ability to discriminate between single-nucleotide variants is often dictated by the specificity limits of hybridization thermodynamics. Here we show that a direct detection approach using single-molecule kinetic fingerprinting can surpass the thermodynamic discrimination limit by 3 orders of magnitude, with a dynamic range of up to 5 orders of magnitude with optional super-resolution analysis. This approach detects mutations as subtle as the drug-resistance-conferring cancer mutation EGFR T790M (a single C → T substitution) with an estimated specificity of 99.99999%, surpassing even the leading PCR-based methods and enabling detection of 1 mutant molecule in a background of at least 1 million wild-type molecules. This level of specificity revealed rare, heat-induced cytosine deamination events that introduce false positives in PCR-based detection, but which can be overcome in our approach through milder thermal denaturation and enzymatic removal of damaged nucleobases.

Single-Molecule Clocks Controlled by Serial Chemical Reactions.

Chemical clocks usually achieve well-defined temporal delays through concentration thresholding coupled to the production, degradation, activation, or inhibition of downstream effectors. In this way, the stochastic dynamics of many individual molecules yield essentially deterministic bulk behavior through ensemble averaging. As a result, their temporal evolution is governed by ensemble dynamics rather than by the behavior of an individual molecule or complex. Here, we present a general approach for the design of single-molecule clocks that permits quasi-deterministic control over the lifetime of single molecular interactions without any external synchronization. By coupling the dissociation of a bimolecular complex to a series of irreversible chemical steps, we interpose a well-defined time delay between binding and dissociation. The number and speed of irreversible steps can be varied to systematically tune both the lifetimes of complexes and the precision of the time delay, raising the prospect of localized timekeeping in nanoscale systems and devices.

Molecular robots guided by prescriptive landscapes.

Traditional robots rely for their function on computing, to store internal representations of their goals and environment and to coordinate sensing and any actuation of components required in response. Moving robotics to the single-molecule level is possible in principle, but requires facing the limited ability of individual molecules to store complex information and programs. One strategy to overcome this problem is to use systems that can obtain complex behaviour from the interaction of simple robots with their environment. A first step in this direction was the development of DNA walkers, which have developed from being non-autonomous to being capable of directed but brief motion on one-dimensional tracks. Here we demonstrate that previously developed random walkers-so-called molecular spiders that comprise a streptavidin molecule as an inert 'body' and three deoxyribozymes as catalytic 'legs'-show elementary robotic behaviour when interacting with a precisely defined environment. Single-molecule microscopy observations confirm that such walkers achieve directional movement by sensing and modifying tracks of substrate molecules laid out on a two-dimensional DNA origami landscape. When using appropriately designed DNA origami, the molecular spiders autonomously carry out sequences of actions such as 'start', 'follow', 'turn' and 'stop'. We anticipate that this strategy will result in more complex robotic behaviour at the molecular level if additional control mechanisms are incorporated. One example might be interactions between multiple molecular robots leading to collective behaviour; another might be the ability to read and transform secondary cues on the DNA origami landscape as a means of implementing Turing-universal algorithmic behaviour.

Discovering anomalous hybridization kinetics on DNA nanostructures using single-molecule fluorescence microscopy.

DNA nanostructures are finding diverse applications as scaffolds for molecular organization. In general, components such as nucleic acids, proteins, and nanoparticles are attached to addressable DNA nanostructures via hybridization, and there is interest in exploiting hybridization for localized computation on DNA nanostructures. This report details two fluorescence microscopy methods, single-particle fluorescence resonance energy transfer (spFRET) and DNA-PAINT (points accumulation for imaging in nanoscale topography), that have been successfully used to detect anomalies of hybridization reactions on individual DNA nanostructures. We compare and contrast the two techniques, highlighting their respective strengths in studying equilibrium and non-equilibrium hybridization as well as assessing the variability of behaviors within individual nanostructures and across a population of nanostructures.

Multifactorial modulation of binding and dissociation kinetics on two-dimensional DNA nanostructures.

We use single-particle fluorescence resonance energy transfer (FRET) to show that organizing oligonucleotide probes into patterned two-dimensional arrays on DNA origami nanopegboards significantly alters the kinetics and thermodynamics of their hybridization with complementary targets in solution. By systematically varying the spacing of probes, we demonstrate that the rate of dissociation of a target is reduced by an order of magnitude in the densest probe arrays. The rate of target binding is reduced less dramatically, but to a greater extent than reported previously for one-dimensional probe arrays. By additionally varying target sequence and buffer composition, we provide evidence for two distinct mechanisms for the markedly slowed dissociation: direct hopping of targets between adjacent sequence-matched probes and nonsequence-specific, salt-bridged, and thus attractive electrostatic interactions with the DNA origami pegboard. This kinetic behavior varies little between individual copies of a given array design and will have significant impact on hybridization measurements and overall performance of DNA nanodevices as well as microarrays.

Super-resolution fingerprinting detects chemical reactions and idiosyncrasies of single DNA pegboards.

We employ the single-particle fluorescence nanoscopy technique points accumulation for imaging in nanoscale topography (PAINT) using site-specific DNA probes to acquire two-dimensional density maps of specific features patterned on nanoscale DNA origami pegboards. We show that PAINT has a localization accuracy of ~10 nm that is sufficient to reliably distinguish dense (>10(4) features µm(-2)) sub-100 nm patterns of oligonucleotide features. We employ two-color PAINT to follow enzyme-catalyzed modification of features on individual origami and to show that single nanopegboards exhibit stable, spatially heterogeneous probe-binding patterns, or "fingerprints." Finally, we present experimental and modeling evidence suggesting that these fingerprints may arise from feature spacing variations that locally modulate the probe binding kinetics. Our study highlights the power of fluorescence nanoscopy to perform quality control on individual soft nanodevices that interact with and position reagents in solution.

Ultrasensitive amplification-free quantification of a methyl CpG-rich cancer biomarker by single-molecule kinetic fingerprinting.

The most well-studied epigenetic marker in humans is the 5-methyl modification of cytosine in DNA, which has great potential as a disease biomarker in liquid biopsies of cell-free DNA. Currently, quantification of DNA methylation relies heavily on bisulfite conversion followed by PCR amplification and NGS or microarray analysis. PCR is subject to potential bias in differential amplification of bisulfite-converted methylated versus unmethylated sequences. Here, we combine bisulfite conversion with single-molecule kinetic fingerprinting to develop an amplification-free assay for DNA methylation at the branched-chain amino acid transaminase 1 (BCAT1) promoter. Our assay selectively responds to methylated sequences with a limit of detection below 1 fM and a specificity of 99.9999%. Evaluating complex genomic DNA matrices, we reliably distinguish 2-5% DNA methylation at the BCAT1 promoter in whole blood DNA from completely unmethylated whole-genome amplified DNA. Taken together, these results demonstrate the feasibility and sensitivity of our amplification-free, single-molecule quantification approach to improve the early detection of methylated cancer DNA biomarkers.

Highly sensitive protein detection by aptamer-based single-molecule kinetic fingerprinting.

Sensitive assays of protein biomarkers play critical roles in clinical diagnostics and biomedical research. Such assays typically employ immunoreagents such as monoclonal antibodies that suffer from several drawbacks, including relatively tedious production, significant batch-to-batch variability, and challenges in site-specific, stoichiometric modification with fluorophores or other labels. One proposed alternative to such immunoreagents, nucleic acid aptamers generated by systematic evolution of ligand by exponential enrichment (SELEX), can be chemically synthesized with much greater ease, precision, and reproducibility than antibodies. However, most aptamers exhibit relatively poor affinity, yielding low sensitivity in the assays employing them. Recently, single molecule recognition through equilibrium Poisson sampling (SiMREPS) has emerged as a platform for detecting proteins and other biomarkers with high sensitivity without requiring high-affinity detection probes. In this manuscript, we demonstrate the applicability and advantages of aptamers as detection probes in SiMREPS as applied to two clinically relevant biomarkers, VEGF165 and IL-8, using a wash-free protocol with limits of detection in the low femtomolar range (3-9 fM). We show that the kinetics of existing RNA aptamers can be rationally optimized for use as SiMREPS detection probes by mutating a single nucleotide in the conserved binding region or by shortening the aptamer sequence. Finally, we demonstrate the detection of endogenous IL-8 from human serum at a concentration below the detection limit of commercial ELISAs.

Attomolar Sensitivity in Single Biomarker Counting upon Aqueous Two-Phase Surface Enrichment.

From longstanding techniques like enzyme-linked immunosorbent assay (ELISA) to modern next-generation sequencing, many of the most sensitive and specific biomarker detection assays require capture of the analyte at a surface. While surface-based assays provide advantages, including the ability to reduce background by washing away excess reagents and/or increase specificity through analyte-specific capture probes, the limited efficiency of capture from dilute solution often restricts assay sensitivity to the femtomolar-to-nanomolar range. Although assays for many nucleic acid analytes can decrease limits of detection (LODs) to the subfemtomolar range using polymerase chain reaction, such amplification may introduce biases, errors, and an increased risk of sample cross-contamination. Furthermore, many analytes cannot be amplified easily, including short nucleic acid fragments, epigenetic modifications, and proteins. To address the challenge of achieving subfemtomolar LODs in surface-based assays without amplification, we exploit an aqueous two-phase system (ATPS) to concentrate target molecules in a smaller-volume phase near the assay surface, thus increasing capture efficiency compared to passive diffusion from the original solution. We demonstrate the utility of ATPS-enhanced capture via single molecule recognition through equilibrium Poisson sampling (SiMREPS), a microscopy technique previously shown to possess >99.9999% detection specificity for DNA mutations but an LOD of only ∼1-5 fM. By combining ATPS-enhanced capture with a Förster resonance energy transfer (FRET)-based probe design for rapid data acquisition over many fields of view, we improve the LOD ∼ 300-fold to <10 aM for an EGFR exon 19 deletion mutation. We further validate this ATPS-assisted FRET-SiMREPS assay by detecting endogenous exon 19 deletion molecules in cancer patient blood plasma.

The shape-shifting quasispecies of RNA: one sequence, many functional folds.

E Unus pluribum, or "Of One, Many", may be at the root of decoding the RNA sequence-structure-function relationship. RNAs embody the large majority of genes in higher eukaryotes and fold in a sequence-directed fashion into three-dimensional structures that perform functions conserved across all cellular life forms, ranging from regulating to executing gene expression. While it is the most important determinant of the RNA structure, the nucleotide sequence is generally not sufficient to specify a unique set of secondary and tertiary interactions due to the highly frustrated nature of RNA folding. This frustration results in folding heterogeneity, a common phenomenon wherein a chemically homogeneous population of RNA molecules folds into multiple stable structures. Often, these alternative conformations constitute misfolds, lacking the biological activity of the natively folded RNA. Intriguingly, a number of RNAs have recently been described as capable of adopting multiple distinct conformations that all perform, or contribute to, the same function. Characteristically, these conformations interconvert slowly on the experimental timescale, suggesting that they should be regarded as distinct native states. We discuss how rugged folding free energy landscapes give rise to multiple native states in the Tetrahymena Group I intron ribozyme, hairpin ribozyme, sarcin-ricin loop, ribosome, and an in vitro selected aptamer. We further describe the varying degrees to which folding heterogeneity impacts function in these RNAs, and compare and contrast this impact with that of heterogeneities found in protein folding. Embracing that one sequence can give rise to multiple native folds, we hypothesize that this phenomenon imparts adaptive advantages on any functionally evolving RNA quasispecies.

Rapid kinetic fingerprinting of single nucleic acid molecules by a FRET-based dynamic nanosensor.

Biofluid-derived cell-free nucleic acids such as microRNAs (miRNAs) and circulating tumor-derived DNAs (ctDNAs) have emerged as promising disease biomarkers. Conventional detection of these biomarkers by digital PCR and next generation sequencing, although highly sensitive, requires time-consuming extraction and amplification steps that also increase the risk of sample loss and cross-contamination. To achieve the direct, rapid, and amplification-free detection of miRNAs and ctDNAs with near-perfect specificity and single-molecule level sensitivity, we herein designed a single-molecule kinetic fingerprinting assay, termed intramolecular single-molecule recognition through equilibrium Poisson sampling (iSiMREPS). iSiMREPS exploits a dynamic DNA nanosensor comprising a surface anchor and a pair of fluorescent detection probes: one probe captures a target molecule onto the surface, while the other transiently interrogates the target to generate kinetic fingerprints by intramolecular single-molecule Förster resonance energy transfer (smFRET) that are recorded by single-molecule fluorescence microscopy and identify the target after kinetic filtering and data analysis. We optimize the sensor design, use formamide to further accelerate the fingerprinting kinetics, and maximize sensitivity by removing non-target-bound probes using toehold-mediated strand displacement to reduce background. We show that iSiMREPS can detect, in as little as 10 s, two distinct, promising cancer biomarkers-miR-141 and a common EGFR exon 19 deletion-reaching a limit of detection (LOD) of ~3 fM and a mutant allele fraction among excess wild-type as low as 1 in 1 million, or 0.0001%. We anticipate that iSiMREPS will find utility in research and clinical diagnostics based on its features of rapid detection, high specificity, sensitivity, and generalizability.

Single-molecule mechanical fingerprinting with DNA nanoswitch calipers.

Decoding the identity of biomolecules from trace samples is a longstanding goal in the field of biotechnology. Advances in DNA analysis have substantially affected clinical practice and basic research, but corresponding developments for proteins face challenges due to their relative complexity and our inability to amplify them. Despite progress in methods such as mass spectrometry and mass cytometry, single-molecule protein identification remains a highly challenging objective. Towards this end, we combine DNA nanotechnology with single-molecule force spectroscopy to create a mechanically reconfigurable DNA nanoswitch caliper capable of measuring multiple coordinates on single biomolecules with atomic resolution. Using optical tweezers, we demonstrate absolute distance measurements with ångström-level precision for both DNA and peptides, and using multiplexed magnetic tweezers, we demonstrate quantification of relative abundance in mixed samples. Measuring distances between DNA-labelled residues, we perform single-molecule fingerprinting of synthetic and natural peptides, and show discrimination, within a heterogeneous population, between different posttranslational modifications. DNA nanoswitch calipers are a powerful and accessible tool for characterizing distances within nanoscale complexes that will enable new applications in fields such as single-molecule proteomics.

Automatic classification and segmentation of single-molecule fluorescence time traces with deep learning.

Traces from single-molecule fluorescence microscopy (SMFM) experiments exhibit photophysical artifacts that typically necessitate human expert screening, which is time-consuming and introduces potential for user-dependent expectation bias. Here, we use deep learning to develop a rapid, automatic SMFM trace selector, termed AutoSiM, that improves the sensitivity and specificity of an assay for a DNA point mutation based on single-molecule recognition through equilibrium Poisson sampling (SiMREPS). The improved performance of AutoSiM is based on accepting both more true positives and fewer false positives than the conventional approach of hidden Markov modeling (HMM) followed by hard thresholding. As a second application, the selector is used for automated screening of single-molecule Förster resonance energy transfer (smFRET) data to identify high-quality traces for further analysis, and achieves ~90% concordance with manual selection while requiring less processing time. Finally, we show that AutoSiM can be adapted readily to novel datasets, requiring only modest Transfer Learning.

Exploring the speed limit of toehold exchange with a cartwheeling DNA acrobat.

Dynamic DNA nanotechnology has yielded nontrivial autonomous behaviours such as stimulus-guided locomotion, computation and programmable molecular assembly. Despite these successes, DNA-based nanomachines suffer from slow kinetics, requiring several minutes or longer to carry out a handful of operations. Here, we pursue the speed limit of an important class of reactions in DNA nanotechnology-toehold exchange-through the single-molecule optimization of a novel class of DNA walker that undergoes cartwheeling movements over a field of complementary oligonucleotides. After optimizing this DNA 'acrobat' for rapid movement, we measure a stepping rate constant approaching 1 s-1, which is 10- to 100-fold faster than prior DNA walkers. Finally, we use single-particle tracking to demonstrate movement of the walker over hundreds of nanometres within 10 min, in quantitative agreement with predictions from stepping kinetics. These results suggest that substantial improvements in the operating rates of broad classes of DNA nanomachines utilizing strand displacement are possible.