DNA Nanomachines Research Articles

Cation-Activated Avidity for Rapid Reconfiguration of DNA Nanodevices.

The ability to design and control DNA nanodevices with programmed conformational changes has established a foundation for molecular-scale robotics with applications in nanomanufacturing, drug delivery, and controlling enzymatic reactions. The most commonly used approach for actuating these devices, DNA binding and strand displacement, allows devices to respond to molecules in solution, but this approach is limited to response times of minutes or greater. Recent advances have enabled electrical and magnetic control of DNA structures with sub-second response times, but these methods utilize external components with additional fabrication requirements. Here, we present a simple and broadly applicable actuation method based on the avidity of many weak base-pairing interactions that respond to changes in local ionic conditions to drive large-scale conformational transitions in devices on sub-second time scales. To demonstrate such ion-mediated actuation, we modified a DNA origami hinge with short, weakly complementary single-stranded DNA overhangs, whose hybridization is sensitive to cation concentrations in solution. We triggered conformational changes with several different types of ions including mono-, di-, and trivalent ions and also illustrated the ability to engineer the actuation response with design parameters such as number and length of DNA overhangs and hinge torsional stiffness. We developed a statistical mechanical model that agrees with experimental data, enabling effective interpretation and future design of ion-induced actuation. Single-molecule Förster resonance energy-transfer measurements revealed that closing and opening transitions occur on the millisecond time scale, and these transitions can be repeated with time resolution on the scale of one second. Our results advance capabilities for rapid control of DNA nanodevices, expand the range of triggering mechanisms, and demonstrate DNA nanomachines with tunable analog responses to the local environment.

Design and Synthesis of a Reconfigurable DNA Accordion Rack

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to ångström resolution, show great potential in various fields of nanotechnology such as the molecular reactors, drug delivery, and nanoplasmonic systems. The reconfigurable DNA accordion rack, which can collectively manipulate a 2D or 3D nanoscale network of elements, in multiple stages in response to the DNA inputs, is described. The platform has potential to increase the number of elements that DNA nanomachines can control from a few elements to a network scale with multiple stages of reconfiguration. In this protocol, we describe the entire experimental process of the reconfigurable DNA accordion rack of 6 by 6 meshes. The protocol includes a design rule and simulation procedure of the structures and a wet-lab experiment for synthesis and reconfiguration. In addition, analysis of the structure using TEM (transmission electron microscopy) and FRET (fluorescence resonance energy transfer) is included in the protocol. The novel design and simulation methods covered in this protocol will assist researchers to use the DNA accordion rack for further applications.

DNA Origami Nanomachines

DNA can assemble various molecules and nanomaterials in a programmed fashion and is a powerful tool in the nanotechnology and biology research fields. DNA also allows the construction of desired nanoscale structures via the design of DNA sequences. Structural nanotechnology, especially DNA origami, is widely used to design and create functionalized nanostructures and devices. In addition, DNA molecular machines have been created and are operated by specific DNA strands and external stimuli to perform linear, rotational, and reciprocating movements. Furthermore, complicated molecular systems have been created on DNA nanostructures by arranging multiple molecules and molecular machines precisely to mimic biological systems. Currently, DNA nanomachines, such as molecular motors, are operated on DNA nanostructures. Dynamic DNA nanostructures that have a mechanically controllable system have also been developed. In this review, we describe recent research on new DNA nanomachines and nanosystems that were built on designed DNA nanostructures.

Highly Ordered and Field-Free 3D DNA Nanostructure: The Next Generation of DNA Nanomachine for Rapid Single-Step Sensing.

Herein, by directly using Watson-Crick base pairing, a highly ordered and field-free three-dimensional (3D) DNA nanostructure is self-assembled by azobenzene (azo)-functionalized DNA nippers in a few minutes, which was applied as a 3D DNA nanomachine with an improved movement efficiency compared to traditional Au-based 3D nanomachines due to the organized and high local concentration of nippers on homogeneous DNA nanostructure. Once microRNA (miRNA) interacts with the 3D nanomachine, the nippers "open" to hybridize with the miRNA. Impressively, photoisomerization of the azo group induces dehybridization/hybridization of the nippers and miRNA under irradiation at different wavelengths, which easily solves one main technical challenge of DNA nanotechnology and biosensing: reversible locomotion in one step within 10 min. As a proof of concept, the described 3D machine is successfully applied in the rapid single-step detection of a biomarker, which gives impetus to the design of new generations of mechanical devices beyond the traditional ones with ultimate applications in sensing analysis and diagnostic technologies.

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.

Exploration of the Kinetics of Toehold-Mediated Strand Displacement via Plasmon Rulers.

DNA/RNA strand displacement is one of the most fundamental reactions in DNA and RNA circuits and nanomachines. In this work, we reported an exploration of the dynamic process of the toehold-mediated strand displacement via core-satellite plasmon rulers at the single-molecule level. Applying plasmon rulers with unlimited lifetime, single-strand displacement triggered by the invader that resulted in stepwise leaving of satellite from the core was continuously monitored by changes of scattering signal for hours. The kinetics of strand displacement in vitro with three different toehold lengths have been investigated. Also, the study revealed the difference in the kinetics of strand displacement between DNA/RNA and DNA/DNA duplexes. For the kinetics study in vivo, influence from the surrounding medium has been evaluated using both phosphate buffer and cell lysate. Applying core-satellite plasmon rulers with high signal/noise ratio, kinetics study in living cells proceeded for the first time, which was not possible by conventional methods with a fluorescent reporter. The plasmon rulers, which are flexible, easily constructed, and robust, have proven to be effective tools in exploring the dynamical behaviors of biochemical reactions in vivo.

Accelerating DNA nanomotor by branched DNAzyme for ultrasensitive optical detection of thrombin

Although DNA assembly-based nanomachines or nanodevice have been intensively studied, the application of DNA nanomachines can be suffered from the slow kinetic of DNA assembly. Herein, we integrated a branched DNAzyme into synthetic DNA motors, thus significantly accelerating the operation of DNA motors. Compared with DNA motors built by a single DNAzyme, the branched DNAzyme DNA nanomotor has much faster operation features. For the application, it could detect as low as 100fM thrombin within 45min. The strategy endowed the DNA nanomotor a great potentiality to the analytical application in clinical diagnosis.

Quantifying Local Molecular Tension Using Intercalated DNA Fluorescence.

The ability to measure mechanics and forces in biological nanostructures, such as DNA, proteins and cells, is of great importance as a means to analyze biomolecular systems. However, current force detection methods often require specialized instrumentation. Here, we present a novel and versatile method to quantify tension in molecular systems locally and in real time, using intercalated DNA fluorescence. This approach can report forces over a range of at least ∼0.5–65 pN with a resolution of 1–3 pN, using commercially available intercalating dyes and a general-purpose fluorescence microscope. We demonstrate that the method can be easily implemented to report double-stranded (ds)DNA tension in any single-molecule assay that is compatible with fluorescence microscopy. This is particularly useful for multiplexed techniques, where measuring applied force in parallel is technically challenging. Moreover, tension measurements based on local dye binding offer the unique opportunity to determine how an applied force is distributed locally within biomolecular structures. Exploiting this, we apply our method to quantify the position-dependent force profile along the length of flow-stretched DNA and reveal that stretched and entwined DNA molecules—mimicking catenated DNA structures in vivo—display transient DNA–DNA interactions. The method reported here has obvious and broad applications for the study of DNA and DNA–protein interactions. Additionally, we propose that it could be employed to measure forces in any system to which dsDNA can be tethered, for applications including protein unfolding, chromosome mechanics, cell motility, and DNA nanomachines.

Three-dimensional structural dynamics of DNA origami Bennett linkages using individual-particle electron tomography

Scaffolded DNA origami has proven to be a powerful and efficient technique to fabricate functional nanomachines by programming the folding of a single-stranded DNA template strand into three-dimensional (3D) nanostructures, designed to be precisely motion-controlled. Although two-dimensional (2D) imaging of DNA nanomachines using transmission electron microscopy and atomic force microscopy suggested these nanomachines are dynamic in 3D, geometric analysis based on 2D imaging was insufficient to uncover the exact motion in 3D. Here we use the individual-particle electron tomography method and reconstruct 129 density maps from 129 individual DNA origami Bennett linkage mechanisms at ~ 6–14 nm resolution. The statistical analyses of these conformations lead to understanding the 3D structural dynamics of Bennett linkage mechanisms. Moreover, our effort provides experimental verification of a theoretical kinematics model of DNA origami, which can be used as feedback to improve the design and control of motion via optimized DNA sequences and routing.

Programmable Modulation of Copper Nanoclusters Electrochemiluminescence via DNA Nanocranes for Ultrasensitive Detection of microRNA

The DNA nanocrane with functionalized manipulator and fixed-size base offered a programmable approach to modulate the luminous efficiency of copper nanoclusters (Cu NCs) for achieving remarkable electrochemiluminescence (ECL) enhancement, further the Cu NCs as signal label was constructed in biosensor for ultrasensitive detection of microRNA-155. Herein, the DNA nanocrane was first constructed by combining binding-induced DNA assembly as manipulator and tetrahedral DNA nanostructure (TDN) as base, which harnessed a small quantity of specific target (microRNA (miRNA)-155) binding to trigger assembly of separate DNA components for producing numerous AT-rich double-stranded DNA (dsDNA) on the vertex of TDN. Upon the incubation of Cu2+ on the AT-rich dsDNA, each DNA-stabilized Cu NCs probe could be in situ electrochemically generated on an individual TDN owing to the A-Cu2+-T bond. Thus, the generation of Cu NCs was highly regulated with AT-rich dsDNA as the template, and its lateral distance was tuned by the TDN size, which were two key factors to influence the luminous efficiency of Cu NCs. By coordinate modulation, the detection limit of the ultrasensitive biosensor for miRNA-155 down to 36 aM and the programmable modulation strategy paved the way for comprehensive applications of DNA nanomachines and metal nanoclusters in biosensing and clinical diagnosis.

Real Time Actuation of a DNA Based Robotic Arm

Since the early days of nanotechnology, scientists have dreamt of nanoscale mechanical systems that resemble macroscale industrial assembly lines. Molecular self-assembly with DNA molecules, used for the bottom-up creation of complex molecular constructs, has been shown to hold large potential to push towards a realization of this vision. Starting out from simple mechanical mechanisms, increasingly intricate systems have been demonstrated in the past decade. A wide collection of mechanical elements, potentially used as components for even more elaborate systems have been developed over the past years. These elements were based on a variety of different concepts to facilitate conformational changes, sliding motion and rotation. We here demonstrate a DNA-based molecular robotic arm integrated on a square base plate created with the DNA origami technique, expanding the repertoire of available building components for DNA assembled nanomachines. A flexible connection between base plate and arm allows rotation of the arm with respect to the specifically addressable base plate. Based on a one pot folding approach, the structure efficiently self-assembles. With the help of single-molecule fluorescence techniques, diffusive arm rotation and temporary fixation at different docking positions on the base plate is observed on a millisecond time scale. Utilizing the high intrinsic charge of our DNA nanoconstruct we also demonstrate the use of electrical fields to actively control the arm's angle relative to the base plate. Actuation of the robot arm only requires milliseconds, which is many orders of magnitude faster than established methods for the operation of synthetic DNA nanomachines. For future work, the system promises to act a as versatile rapid prototyping platform for the development of components for molecular assembly lines and highly parallelizable nanoscale force spectroscopy.

A nonenzymatic DNA nanomachine for biomolecular detection by target recycling of hairpin DNA cascade amplification

Synthetic enzyme-free DNA nanomachine performs quasi-mechanical movements in response to external intervention, suggesting the promise of constructing sensitive and specific biosensors. Herein, a smart DNA nanomachine biosensor for biomolecule (such as nucleic acid, thrombin and adenosine) detection is developed by target-assisted enzyme-free hairpin DNA cascade amplifier. The whole DNA nanomachine system is constructed on gold nanoparticle which decorated with hundreds of locked hairpin substrate strands serving as DNA tracks, and the DNA nanomachine could be activated by target molecule toehold-mediated exchange on gold nanoparticle surface, resulted in the fluorescence recovery of fluorophore. The process is repeated so that each copy of the target can open multiplex fluorophore-labeled hairpin substrate strands, resulted in amplification of the fluorescence signal. Compared with the conventional biosensors of catalytic hairpin assembly (CHA) without substrate in solution, the DNA nanomachine could generate 2–3 orders of magnitude higher fluorescence signal. Furthermore, the DNA nanomachine could be used for nucleic acid, thrombin and adenosine highly sensitive specific detection based on isothermal, and homogeneous hairpin DNA cascade signal amplification in both buffer and a complicated biomatrix, and this kind of DNA nanomachine could be efficiently applied in the field of biomedical analysis.

Autonomous DNA nanomachine based on cascade amplification of strand displacement and DNA walker for detection of multiple DNAs

DNA can be modified to function as a scaffold for the construction of a DNA nanomachine, which can then be used in analytical applications if the DNA nanomachine can be triggered by the presence of a diagnostic DNA or some other analyte. We herein propose a novel and powerful DNA nanomachine that can detect DNA via combining the tandem strand displacement reactions and a DNA walker. Three different DNA sensing platforms are described, where the whole DNA machine was constructed on a gold electrode (GE). This cascade multiple amplification strategy exhibited an excellent sensitivity. Under optimal conditions, the electrochemical sensor could achieve a detection limit of 36 fM with a linear range from 50 to 500 fM. In particular, the electrochemical sensor could easily distinguish the base mutations. More interestingly, the DNA nanomachine could be used to construct analog AND and OR logic gates. We demonstrate that electrochemical signals generated from the different input combinations can be used to distinguish multiple target DNAs. The practical applicability of the present biosensor is demonstrated by the detection of target DNA in human serum with satisfactory results, which holds great potential for a future application in clinical diagnosis.

Comprehensive profiling of the ligand binding landscapes of duplexed aptamer families reveals widespread induced fit

Duplexed aptamers (DAs) are ligand-responsive constructs engineered by hybridizing an aptamer with an aptamer-complementary element (ACE, e.g., a DNA oligonucleotide). Although DAs are commonly deployed, the binding dynamics of ternary ACE-aptamer–ligand systems remain underexplored, having been conventionally described by a conformational selection framework. Here we introduce aptamer-complementary element scanning (ACE-Scan) as a method to generate comprehensive hybridization, spontaneous off-rate, and induced fit ligand-binding landscapes for entire DA families. ACE-Scan reveals induced fit in DAs engineered from small molecule- and protein-binding DNA and RNA aptamers, as well as DAs engineered from the natural add riboswitch aptamer. To validate ACE-Scan, we engineer solution-phase ATP-specific DAs from 5 ACEs with varying spontaneous and induced fit off-rates, generating aptasensors with 8-fold differences in dynamic range consistent with ACE-Scan. This work demonstrates that ACE-Scan can readily map induced fit in DAs, empowering aptamers in biosensing, synthetic biology, and DNA nanomachines.

Bioderived DNA Nanomachines for Potential Uses in Biosensing, Diagnostics, and Therapeutic Applications.

Beside its genomic properties, DNA is also recognized as a novel material in the field of nanoengineering. The specific bonding of base pairs can be used to direct the assembly of highly structured materials with specific nanoscale features such as periodic 2D arrays, 3D nanostructures, assembly of nanomaterials, and DNA nanomachines. In recent years, a variety of DNA nanomachines are developed because of their many potential applications in biosensing, diagnostics, and therapeutic applications. In this review, the fuel-powered motors and secondary structure motors, whose working mechanisms are inspired or derived from natural phenomena and nanomachines, are discussed. The combination of DNA motors with other platforms is then discussed. In each section of these motors, their mechanisms and their usage in the biomedical field are described. Finally, it is believed that these DNA-based nanomachines and hybrid motifs will become an integral point-of-care diagnostics and smart, site-specific therapeutic delivery.

How Well Can DNA Rupture DNA? Shearing and Unzipping Forces inside DNA Nanostructures.

A purely DNA nanomachine must support internal stresses across short DNA segments with finite rigidity, producing effects that can be qualitatively very different from experimental observations of isolated DNA in fixed-force ensembles. In this article, computational simulations are used to study how well the rigidity of a driving DNA duplex can rupture a double-stranded DNA target into single-stranded segments and how well this stress can discriminate between unzipping or shearing geometries. This discrimination is found to be maximized at an optimal length but deteriorates as the driving duplex is either lengthened or shortened. This differs markedly from a fixed-force ensemble and has implications for the design parameters and limitations of dynamic DNA nanomachines.

MnO2-Nanosheet-Powered Protective Janus DNA Nanomachines Supporting Robust RNA Imaging.

Both biomarker and probe degradations cause serious false assay results. However, protecting a target or a target and a probe simultaneously has rarely been explored. Herein, MnO2-nanosheet-powered target- and probe-protective Janus DNA nanomachines are reported. It is formed in living cells by an RNA-responsive assembly of two chemically modified DNA partzymes and one substrate probe. MnO2 nanosheets are used to facilitate the cellular uptake of DNA reagents and generate Mn2+, which are indispensable DNAzyme cofactors for efficient catalytic cleavage. We find that DNA partzymes with modified sugar moieties (e.g., LNA or ones with 2'-O-methylation) protect the RNA of RNA-DNA hybrids from RNase H degradation. LNA blocks RNase H recruitment on the hybrid best because of its 2'-O, 4'-C methylene bridge structure. In contrast, modifications at DNA phosphate moieties fail to protect the RNA. RNA protection can exclude target-degradation-induced false negative results. In addition, the phosphorothioate-modified substrate probe is known to resist nuclease degradation, which minimizes false positive interference. Compared to canonical DNA systems without chemical modifications, the protective Janus nanomachine avoids false results and supports robust RNA imaging.

Rational construction of a DNA nanomachine for HIV nucleic acid ultrasensitive sensing.

HIV nucleic acids, one kind of significant biomarker, play an important role in fundamental studies and clinical diagnosis. Importantly, the early accurate diagnosis for HIV nucleic acids at ultralow concentrations can potentially extend the life of patients. In the current work, we developed a DNA nanomachine on gold nanoparticles (AuNPs) coupling rolling circle amplification and DNA walker cascade amplification for ultrasensitive detection of HIV nucleic acids. This DNA nanomachine sensing strategy exhibits a significantly low detection limit down to 1.46 fM. Furthermore, this DNA nanomachine biosensor is capable of detecting target DNA in real samples because of its high selectivity and sensitivity. Moreover, the DNA nanomachine biosensor is capable of discriminating single-base mismatch lower than 3.5 pM. The results showed that this DNA nanomachine biosensor has the potential for biomedical studies and clinical applications.

A highly integrated DNA nanomachine operating in living cells powered by an endogenous stimulus.

Synthetic molecular machines have received increasing attention because of their great ability to mimic natural biological motors and create novel modes of motion. However, very few examples have been implemented with real autonomous movement inside living cells, due to the challenges of the driving force and highly integrated system design. In this work, we report an elegant, highly integrated DNA nanomachine that can be powered by endogenous ATP molecules and autonomously operated inside living cells without any auxiliary additives. It assembles all components on a single gold nanoparticle (AuNP) including a hairpin-locked swing arm encoding a start triggered by an intracellular target molecule and a two-stranded DNA track responding to the motion of the swing arm. When the intracellular target activates the nanomachine via the unlocking swing arm, the machine autonomously and progressively operates on the established DNA track via intramolecular toehold-mediated strand migration and internal ATP binding. This paper also demonstrates the machine's bioanalytical application for specific microRNA (miRNA) imaging in living cells.

Simulation-guided engineering of an enzyme-powered three dimensional DNA nanomachine for discriminating single nucleotide variants.

Single nucleotide variants (SNVs) are important both clinically and biologically because of their profound biological consequences. Herein, we engineered a nicking endonuclease-powered three dimensional (3D) DNA nanomachine for discriminating SNVs with high sensitivity and specificity. Particularly, we performed a simulation-guided tuning of sequence designs to achieve the optimal trade-off between device efficiency and specificity. We also introduced an auxiliary probe, a molecular fuel capable of tuning the device in solution via noncovalent catalysis. Collectively, our device produced discrimination factors comparable with commonly used molecular probes but improved the assay sensitivity by ∼100 times. Our results also demonstrate that rationally designed DNA probes through computer simulation can be used to quantitatively improve the design and operation of complexed molecular devices and sensors.