DNA Motor Research Articles

Energetically biased DNA motor containing a thermodynamically stable partial strand displacement state.

Current work in tuning DNA kinetics has focused on changing toehold lengths and DNA concentrations. However, kinetics can also be improved by enhancing the completion probability of the strand displacement process. Here, we execute this strategy by creating a toehold DNA motor device with the inclusion of a synthetic nucleotide, inosine, at selected sites. Furthermore, we found that the energetic bias can be tuned such that the device can stay in a stable partially displaced state. This work demonstrates the utility of energetic biases to change DNA strand displacement kinetics and introduces a complementary strategy to the existing designs.

A bipedal DNA motor that travels back and forth between two DNA origami tiles.

In this work, the successful operation of a dynamic DNA device constructed from two DNA origami building blocks is reported. The device includes a bipedal walker that strides back and forth between the two origami tiles. Two different DNA origami tiles are first prepared separately; they are then joined together in a controlled manner by a set of DNA strands to form a stable track in high yield as confirmed by single-molecule fluorescence (SMF). Second, a bipedal DNA motor, initially attached to one of the two origami units and operated by sequential interaction with "fuel" and "antifuel" DNA strands, moves from one origami tile to another and then back again. The operational yield, measured by SMF, was similar to that of a motor operating on a similar track embedded in a single origami tile, confirming that the transfer across the junction from one tile to the other does not result in dissociation that is any more than that of steps on a single tile. These results demonstrate that moving parts can reliably travel from one origami unit to another, and it demonstrates the feasibility of dynamic DNA molecular machines that are made of more than a single origami building block. This study is a step toward the development of motors that can stride over micrometer distances.

Regulation of DNA pairing in homologous recombination.

Homologous recombination (HR) is a major mechanism for eliminating DNA double-strand breaks from chromosomes. In this process, the break termini are resected nucleolytically to form 3' ssDNA (single-strand DNA) overhangs. A recombinase (i.e., a protein that catalyzes homologous DNA pairing and strand exchange) assembles onto the ssDNA and promotes pairing with a homologous duplex. DNA synthesis then initiates from the 3' end of the invading strand, and the extended DNA joint is resolved via one of several pathways to restore the integrity of the injured chromosome. It is crucial that HR be carefully orchestrated because spurious events can create cytotoxic intermediates or cause genomic rearrangements and loss of gene heterozygosity, which can lead to cell death or contribute to the development of cancer. In this review, we will discuss how DNA motor proteins regulate HR via a dynamic balance of the recombination-promoting and -attenuating activities that they possess.

DNA nanotechnology based on i-motif structures.

CONSPECTUS: Most biological processes happen at the nanometer scale, and understanding the energy transformations and material transportation mechanisms within living organisms has proved challenging. To better understand the secrets of life, researchers have investigated artificial molecular motors and devices over the past decade because such systems can mimic certain biological processes. DNA nanotechnology based on i-motif structures is one system that has played an important role in these investigations. In this Account, we summarize recent advances in functional DNA nanotechnology based on i-motif structures. The i-motif is a DNA quadruplex that occurs as four stretches of cytosine repeat sequences form C·CH(+) base pairs, and their stabilization requires slightly acidic conditions. This unique property has produced the first DNA molecular motor driven by pH changes. The motor is reliable, and studies show that it is capable of millisecond running speeds, comparable to the speed of natural protein motors. With careful design, the output of these types of motors was combined to drive micrometer-sized cantilevers bend. Using established DNA nanostructure assembly and functionalization methods, researchers can easily integrate the motor within other DNA assembled structures and functional units, producing DNA molecular devices with new functions such as suprahydrophobic/suprahydrophilic smart surfaces that switch, intelligent nanopores triggered by pH changes, molecular logic gates, and DNA nanosprings. Recently, researchers have produced motors driven by light and electricity, which have allowed DNA motors to be integrated within silicon-based nanodevices. Moreover, some devices based on i-motif structures have proven useful for investigating processes within living cells. The pH-responsiveness of the i-motif structure also provides a way to control the stepwise assembly of DNA nanostructures. In addition, because of the stability of the i-motif, this structure can serve as the stem of one-dimensional nanowires, and a four-strand stem can provide a new basis for three-dimensional DNA structures such as pillars. By sacrificing some accuracy in assembly, we used these properties to prepare the first fast-responding pure DNA supramolecular hydrogel. This hydrogel does not swell and cannot encapsulate small molecules. These unique properties could lead to new developments in smart materials based on DNA assembly and support important applications in fields such as tissue engineering. We expect that DNA nanotechnology will continue to develop rapidly. At a fundamental level, further studies should lead to greater understanding of the energy transformation and material transportation mechanisms at the nanometer scale. In terms of applications, we expect that many of these elegant molecular devices will soon be used in vivo. These further studies could demonstrate the power of DNA nanotechnology in biology, material science, chemistry, and physics.

A novel acylaminoimidazole derivative, WN1316, alleviates disease progression via suppression of glial inflammation in ALS mouse model.

Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron degenerative disease. Given that oxidative stress and resulting chronic neuronal inflammation are thought to be central pathogenic, anti-oxidative agents and modulators of neuronal inflammation could be potential therapies for ALS. We report here that the novel small molecular compound, 2-[mesityl(methyl)amino]-N-[4-(pyridin-2-yl)-1H-imidazol-2-yl] acetamide trihydrochloride (WN1316) selectively suppresses oxidative stress-induced cell death and neuronal inflammation in the late-stage ALS mice. WN1316 has high blood-brain-barrier permeability and water solubility, and boosts both neuronal apoptosis inhibitory protein (NAIP) and NF-E2-related factor 2 (Nrf2) which governed glutathione (GSH)-related anti-oxidation pathway protecting motor neurons against oxidative injuries. Post-onset oral administration of low dose (1–100 µg/kg/day) WN1316 in ALS(SOD1H46R) and ALS(SOD1G93A) mice resulted in sustained improved motor function and post onset survival rate. Immunohistochemical analysis revealed less DNA oxidative damage and motor neuronal inflammation as well as repression of both microgliosis and astrocytosis, concomitant down regulation of interleukin-1β and inducible nitric oxide synthase, and preservation of the motoneurons in anterior horn of lumbar spinal cord and skeletal muscle (quadriceps femoris). Thus, WN1316 would be a novel therapeutic agent for ALS.

Human Replication Protein a (RPA) Can Diffuse Along Single Stranded DNA

Replication Protein A (RPA) is a hetero-trimeric protein that plays critical roles in many cellular processes. The main function of RPA is to bind to single stranded (ss) DNA and to regulate its availability during DNA metabolic processes. RPA is also known to interact with an array of other proteins in DNA replication, repair and recombination processes in eukaryotic organisms. RPA binds to the transient ssDNA that forms during nearly all aspects of DNA metabolism and protects ssDNA from nucleases. Although RPA binds to ssDNA with a very high affinity, it must be dissociated from or redistributed along ssDNA during DNA replication. To probe this re-arrangement of RPA along ssDNA, ensemble and single molecule studies have been used as complementary techniques to investigate human RPA diffusion along fluorescently labeled ssDNA oligomers. The dynamics of human RPA (hRPA) along fluorescently labeled DNA oligomers was also studied with fluorophore - labeled hRPA. These experiments illustrate that hRPA can spontaneously re-arrange along ssDNA by diffusing while remaining tightly bound. In addition, hRPA can also transiently melt DNA hairpin structures by diffusing in from ssDNA that is adjacent to the DNA hairpin. This ability of hRPA to diffuse along ssDNA means that directional DNA motor proteins such as polymerases or translocases can push RPA and re-organize it along ssDNA. This diffusion property of RPA is shared with the bacterial analogue of RPA, the E. coli SSB protein that also has previously been shown to diffuse along ssDNA. (GM030498 (TML), GM098509 (RG), GM044721 (MSW)).

Mechanical Characterization of a Fast DNA Motor: SpoIIIE

DNA translocation is a fundamental process in biology, required for essential cellular processes including recombination, replication, and chromosome segregation. The SpoIIIE motor is a homo-hexameric dsDNA translocase, part of the ASCE [Additional Strand Conserved E (glutamate)] superfamily of oligomeric ring NTPases. SpoIIIE is found in the bacterium Bacillus subtilis, its biological role is to actively translocate DNA to ensure the proper segregation of sister chromatids. SpoIIIE accomplishes this task by coupling the chemical energy provided by ATP to generate mechanical work, pumping the DNA across a cellular membrane at a rate of 4 kbp/s. While generating mechanical work is a common function of ring NTPases, the timing and order of individual power strokes applies this work in a nuanced manner to better suit particular biological tasks. Understanding the mechanical aspects of fast DNA translocation provides a unique insight into the diverse mechanical strategies employed by these large class of ring NTPases. Using single-molecule optical trapping techniques, we present real-time measurements of SpoIIIE DNA translocation. By challenging the motor with different lengths of modified DNA substrates, we have determined that SpoIIIE makes critical phosphate contacts with the DNA backbone during translocation and characterized the periodicity of motor-DNA interactions, suggesting a fundamental step size of 2 bp. Finally, velocity dependence on an applied external load and ATP analogues has given insight into an emerging mechanochemical model of DNA translocation, suggesting a unique partially coordinated mechanism of hydrolysis between the individual subunits of SpoIIIE.

A synthetic DNA motor that transports nanoparticles along carbon nanotubes

Intracellular protein motors have evolved to perform specific tasks critical to the function of cells such as intracellular trafficking and cell division. Kinesin and dynein motors, for example, transport cargoes in living cells by walking along microtubules powered by adenosine triphosphate hydrolysis. These motors can make discrete 8nm centre-of-mass steps and can travel over 1µm by changing their conformations during the course of adenosine triphosphate binding, hydrolysis and product release. Inspired by such biological machines, synthetic analogues have been developed including self-assembled DNA walkers that can make stepwise movements on RNA/DNA substrates or can function as programmable assembly lines. Here, we show that motors based on RNA-cleaving DNA enzymes can transport nanoparticle cargoes-CdS nanocrystals in this case-along single-walled carbon nanotubes. Our motors extract chemical energy from RNA molecules decorated on the nanotubes and use that energy to fuel autonomous, processive walking through a series of conformational changes along the one-dimensional track. The walking is controllable and adapts to changes in the local environment, which allows us to remotely direct 'go' and 'stop' actions. The translocation of individual motors can be visualized in real time using the visible fluorescence of the cargo nanoparticle and the near-infared emission of the carbon-nanotube track. We observed unidirectional movements of the molecular motors over 3µm with a translocation velocity on the order of 1nmmin(-1) under our experimental conditions.

Conformational transitions during FtsK translocase activation of individual XerCD– dif recombination complexes

Three single-molecule techniques have been used simultaneously and in tandem to track the formation in vitro of single XerCD-dif recombination complexes. We observed the arrival of the FtsK translocase at individual preformed synaptic complexes and demonstrated the conformational change that occurs during their activation. We then followed the reaction intermediate transitions as Holliday junctions formed through catalysis by XerD, isomerized, and were converted by XerC to reaction products, which then dissociated. These observations, along with the calculated intermediate lifetimes, inform the reaction mechanism, which plays a key role in chromosome unlinking in most bacteria with circular chromosomes.

Simulating a burnt-bridges DNA motor with a coarse-grained DNA model

We apply a recently-developed coarse-grained model of DNA, designed to capture the basic physics of nanotechnological DNA systems, to the study of a `burnt-bridges' DNA motor consisting of a single-stranded cargo that steps processively along a track of single-stranded stators. We demonstrate that the model is able to simulate such a system, and investigate the sensitivity of the stepping process to the spatial separation of stators, finding that an increased distance can suppress successful steps due to the build up of unfavourable tension. The mechanism of suppression suggests that varying the distance between stators could be used as a method for improving signal-to-noise ratios for motors that are required to make a decision at a junction of stators.

Rational Design of DNA Motors: Fuel Optimization through Single-Molecule Fluorescence

While numerous DNA-based molecular machines have been developed in recent years, high operational yield and speed remain a major challenge. To understand the reasons for the limited performance, and to find rational solutions, we applied single-molecule fluorescence techniques and conducted a detailed study of the reactions involved in the operation of a model system comprised of a bipedal DNA walker that strides on a DNA origami track powered by interactions with fuel and antifuel strands. Analysis of the kinetic profiles of the leg-lifting reactions indicates a pseudo-first-order antifuel binding mechanism leading to a rapid and complete leg-lifting, indicating that the fuel-removal reaction is not responsible for the 1% operational yield observed after six steps. Analysis of the leg-placing reactions showed that although increased concentrations of fuel increase the reaction rate, they decrease the yield by consecutively binding the motor and leading to an undesirable trapped state. Recognizing this, we designed asymmetrical hairpin-fuels that by regulating the reaction hierarchy avoid consecutive binding. Motors operating with the improved fuels show 74% yield after 12 consecutive reactions, a dramatic increase over the 1% observed for motors operating with nonhairpin fuels. This work demonstrates that studying the mechanisms of the reactions involved in the operation of DNA-based molecular machines using single-molecule fluorescence can facilitate rationally designed improvements that increase yield and speed and promote the applicability of DNA-based machines.

Nonequilibrium Distributions and Hydrodynamic Coupling Distort the Measurement of Nanoscale Forces near Interfaces

We calculate the displacement of a single spherical particle from the minimum of a harmonic well positioned near a plane wall and immersed in a uniform flow. A failure to account for the fluctuations in particle position orthogonal to the plane (leading to fluctuations in hydrodynamic drag) results in large discrepancies, with the naive displacement calculated by assuming no fluctuations in the balance of forces. The chief criterion for neglecting such fluctuations is that the stiffness of the harmonic potential exceeds the thermal stresses on the particle by at least two orders of magnitude. For micrometer-diameter particles typically employed in force spectroscopy of DNA, macromolecules, and molecular motors, this can lead to errors of up to 100% in the measured properties. The Supporting Material to the article provides an implementation of this model intended to fit experimental measurements for the stiffness of the harmonic potential constraining the particle.

An Active Gel based on DNA and DNA-Associated Motor Proteins

Biological systems continuously acquire and use energy sources to perform various functions. This energy is, in part, transduced to generate the forces that control the mechanical behavior of the cell (e.g. cell shape and motion). In this case, the system is in the non-equilibrium state and the material may be called “Active Soft Matter”.To investigate the mechanical properties of soft, active systems, we have synthesized an active gel with a well-known semi-flexible biopolymer, DNA, and DNA-associated motor proteins. We study the mechanics of this system using two kinds of microrheological techniques. First, we use a passive measurement in which the intrinsic fluctuation of embedded particles gives information on gel mechanics. Second, we use an active measurement utilizing the forced oscillating motion of embedded particles by an external magnetic field. We discuss these results in comparison to cytoskeletal systems, and seek to establish universal principles of motor-driven active gels.

Watching Individual DNA Helicases and Motor Proteins Behaving and Misbehaving

We can now watch individual proteins acting on single molecules of DNA. Using these approaches, we have been imaging the translocation of motor proteins, such as the ssDNA translocases and DNA helicases, RecBCD, RecQ, and Sgs1, and the dsDNA translocases and chromatin-remodeling enzymes, Rad54 and Tid1 proteins.The RecBCD enzyme comprises two motor subunits: RecB, a 3’→5’ SF1-helicase, and RecD, a 5’→3’ SF1-helicase. Although the mean unwinding rate of individual RecBCD enzyme molecules is close to ensemble measurements, the rates of individual enzyme molecules vary widely. Individual RecBCD molecules unwind DNA at constant rates, suggesting static, not dynamic, disorder. This apparent conformational heterogeneity is static on the experimental time scale of DNA unwinding. We discovered that transiently halting a single enzyme-DNA complex changes the rates of the RecBCD molecule. We will demonstrate that the behavior of individual RBCD enzymes manifests a basic tenet of the ergodic hypothesis, suggesting that ligand binding kinetically traps a single conformer that is capable of redistributing to all other states within the population of molecules at equilibrium.Using TIRF microscopy, the helicase activity of RecQ was visualized on single molecules of DNA using a fluorescent ssDNA sensor. By monitoring the formation and progression of individual unwinding forks, we observe that both the frequency of initiation and rate of unwinding are highly dependent on RecQ concentration. We establish that unwinding forks can initiate internally by melting into dsDNA and can proceed in both directions. The findings suggest that initiation requires a RecQ dimer, and continued unwinding involves the repeated cooperative action of multiple monomers at the DNA unwinding fork. We propose a distinctive model wherein RecQ melts dsDNA internally to initiate unwinding, and subsequently unwinds DNA as a dynamic assembly of proteins cooperating at the fork.

Simultaneous High-Resolution Optical Trapping and Single Molecule Fluorescence Measurements of the UvrD Helicase: DNA Motor Protein Structure and Function Observed Simultaneously

We present single-molecule in vitro measurements of the DNA unwinding activity of the Superfamily 1 UvrD helicase that reveal directly how helicase stoichiometry and conformation correspond to motor activity. Although a single UvrD molecule can translocate along ssDNA, it has been postulated that only a dimer is capable of unwinding duplex DNA. UvrD is known to exhibit two different conformations: the ‘closed' and ‘open' orientation of the 2B regulatory subdomain. The function of these conformations has remained elusive. using a new instrument combining high-resolution optical tweezers with simultaneous single-molecule fluorescence microscopy (Comstock, Ha, and Chemla, Nature Methods 2011), we obtain an unprecedented detailed view of helicase motor dynamics by recording single base pair-scale DNA unwinding activity (optical tweezers) simultaneously with helicase stoichiometry and conformation (fluorescence). We observe two classes of DNA unwinding by UvrD: long distance continuous unwinding (∼100 bp) and repetitive, non-processive (∼12 bp) unwinding. We determine directly the stoichiometry of UvrD molecules participating in unwinding by quantifying the fluorescence intensity using singly-labeled UvrD molecules. We observe that pairs of UvrD molecules are required for long distance unwinding but individual molecules exhibit limited non-processive unwinding activity. Furthermore, we observe directly the conformation of the 2B subdomain of individual UvrD molecules during unwinding by measuring the fluorescence of FRET-labeled proteins. We observe that UvrD is in the ‘closed' conformation during DNA unwinding but surprisingly switches to the ‘open' conformation when UvrD translocates on the opposing ssDNA strand away from the junction with the DNA junction re-annealing behind it. These measurements may explain UvrD helicase's remarkable ability to switch translocation strands. We hypothesize that the UvrD 2B subdomain acts as a switch that controls DNA unwinding vs. re-annealing.

Targeting of XJB-5-131 to Mitochondria Suppresses Oxidative DNA Damage and Motor Decline in a Mouse Model of Huntington’s Disease

Oxidative damage and mitochondrial dysfunction are implicated in aging and age-related neurodegenerative diseases, including Huntington's disease (HD). Many naturally occurring antioxidants have been tested for their ability to correct for deleterious effects of reactive oxygen species, but often they lack specificity, are tissue variable, and have marginal efficacy in human clinical trials. To increase specificity and efficacy, we have designed a synthetic antioxidant, XJB-5-131, to target mitochondria. We demonstrate in a mouse model of HD that XJB-5-131 has remarkably beneficial effects. XJB-5-131 reduces oxidative damage to mitochondrial DNA, maintains mitochondrial DNA copy number, suppresses motor decline and weight loss, enhances neuronal survival, and improves mitochondrial function. The findings poise XJB-5-131 as a promising therapeutic compound.

Studying the Structural Dynamics of Bipedal DNA Motors with Single-Molecule Fluorescence Spectroscopy

We present a test case example of a detailed single-molecule fluorescence study of one of the most sophisticated and complex DNA devices introduced to date, a recently published autonomous bipedal DNA motor. We used the diffusion-based single-molecule Förster resonance energy transfer technique, coupled to alternating laser excitation (sm-FRET-ALEX), to monitor the motor assembly and operation. The study included verification of the formation of the correct structures, and of the correct motor operation, determination of the formation and stepping reaction yields, and identification of side products. Finally, the mechanisms of the motor assembly and operation were elucidated by measuring the reaction kinetics profile of track-walker binding and of lifting of the walker's leg upon fuel addition. The profiles revealed a fast phase, in which about half of the reaction was completed, followed by a slow phase which adds somewhat to the yield, reflecting the incomplete motor assembly and operation identified in the equilibrium experiments. Although further study is needed to fully understand the reasons for the incomplete assembly and operation, this work demonstrates that single-molecule fluorescence, based on its ability to provide detailed in situ structural dynamics information, inaccessible for traditional methods, constitutes an excellent tool for chaperoning the development of DNA-based technology.

BIOBOARD

AUSTRALIA – Aussie Meteorite Yields Anti-Epileptic Amino Acid. AUSTRALIA – Novel Partnership Could Transform Breast Cancer Diagnosis. AUSTRALIA – University of Sydney Academics Laud Sunbed Ban. AUSTRALIA – Australian Universities Losing Appeal to Asians. INDIA – New Nanobiotechnology Lab in Gurgaon. INDIA – China, India, and Myanmar Collaborate to Develop Brahmaputra-Salween Ecosystem. INDIA – Incredible India – Now Polio-Free. INDIA – Increase Women's Participation in Science for Over-All Development of Nation. JAPAN – DNA Motor Now Can Navigate Networks. JAPAN – RIKEN's Diagnosis Technique Detects H1N1 at Record Speed. JAPAN – New Hotspot on Rice Chromosome Could Yield Improved Varieties. OTHER REGIONS — Delivering RNAi the Spongy Way.

Nanotechnology: Listening To Track Motion, Origami To Make DNA Motors

Nanotechnology is making it possible to detect sound waves at the microscopic level—in effect, to listen to microbial cells, according to physicists Alexander Ohlinger, Jochen Feldmann, and their collaborators at the Ludwig Maximilian University of Munich in Germany. Separately, a nano-scale, DNA-based motor was programmed to run on a series of tracks with multiple switches, according to Masayuki Endo and Hiroshi Sugiyama from Kyoto University in Japan and Andrew Tuberfield from Oxford University in the United Kingdom, and their collaborators. The report from Ohlinger appears in the January 6, 2012 Physical Review Letters 108, 018101, while the report on the DNA motor is in the January 2012 Nature Nanotechnology (doi:10.1038/nnano.2011.253).

Single-Molecule Imaging Reveals Mechanisms of Roadblock Clearance by DNA Motor Enzymes

In the cell, nucleic acid motor proteins act on substrates occupied by other proteins, yet little is known regarding the inevitable collisions that must occur. Using nanofabricated curtains of DNA and real-time, multi-color single-molecule microscopy we visualized collisions between two model translocases and DNA-bound obstacles. We show that both RecBCD, a helicase necessary for initiating homologous DNA recombination, and FtsK, a DNA pump involved in chromosome dimer resolution, actively disrupts nucleoprotein complexes, including RNA polymerase (RNAP) holoenzyme. RecBCD pushed and eventually displaces RNAP, Lac repressor, EcoRI(E111Q) and even nucleosomes. FtsK pushed RNAP but was able to either push or bypass EcoRI(E111Q). We conclude that RecBCD acts as a powerful stripase that overwhelms potential roadblocks. In contrast, FtsK is able to bypass some roadblocks, possibly by dissociation and reassembly ahead of the block.