Engineering Biomimetic Biosensor Using Dual-Targeting Multivalent Aptamer Regulated 3D DNA Walker Enables High-Performance Detection of Heterogeneous Circulating Tumor Cells.

The phenotypic heterogeneity of circulating tumor cells (CTCs) and the nonspecific adsorption of background cells impede the effective and sensitive detection of rare CTCs. Although leukocyte membrane coating approach has a good antileukocyte adhesion ability and holds great promise for addressing the challenge of capture purity, its limited specificity and sensitivity prevent its use in the detection of heterogeneous CTCs. To overcome these obstacles, a biomimetic biosensor that integrated dual-targeting multivalent aptamer/walker duplex functionalized biomimetic magnetic beads and an enzyme-powered DNA walker signal amplification strategy is designed. As compared to conventional leukocyte membrane coating, the biomimetic biosensor achieves efficient and high purity enrichment of heterogeneous CTCs with different epithelial cell adhesion molecule (EpCAM) expression while minimizing the interference of leukocytes. Meanwhile, the capture of target cells can trigger the release of walker strands to activate an enzyme-powered DNA walker, resulting in cascade signal amplification and the ultrasensitive and accurate detection of rare heterogeneous CTCs. Importantly, the captured CTCs remained viable and can be recultured in vitro with success. Overall, this work provides a new perspective for the efficient detection of heterogeneous CTCs by biomimetic membrane coating and paves the way for early cancer diagnosis.

A DNA Molecular Robot that Autonomously Walks on the Cell Membrane to Drive Cell Motility.

Synthetic molecular robots can execute sophisticated molecular tasks at nanometer resolution. However, a molecular robot capable of controlling cellular behavior remains unexplored. Herein, we report a self-propelled DNA robot operating on the cell membrane to control the migration of a cell. Driven by DNAzyme catalytic activity, the DNA robot could autonomously and stepwise move on the membrane-floating cell-surface receptors in a stochastic manner and simultaneously trigger the receptor-dimerization to activate downstream signaling for cell motility. The cell membrane-associated continuous motion and operation of a DNA robot allowed for the ultrasensitive regulation of MET/AKT signaling and cytoskeleton remodeling to enhance cell migration. Finally, we designed distinct conditional DNA robots to orthogonally manipulate the cell migration in a coculture of mixed cell populations. We have developed a novel strategy to engineer a cell-driving molecular robot, representing a promising avenue for precise cell manipulation with nanoscale resolution.

Highly Polyvalent DNA Motors Generate 100+ pN of Force via Autochemophoresis.

Motor proteins such as myosin, kinesin, and dynein are essential to eukaryotic life and power countless processes including muscle contraction, wound closure, cargo transport, and cell division. The design of synthetic nanomachines that can reproduce the functions of these motors is a longstanding goal in the field of nanotechnology. DNA walkers, which are programmed to "walk" along defined tracks via the burnt bridge Brownian ratchet mechanism, are among the most promising synthetic mimics of these motor proteins. While these DNA-based motors can perform useful tasks such as cargo transport, they have not been shown to be capable of cooperating to generate large collective forces for tasks akin to muscle contraction. In this work, we demonstrate that highly polyvalent DNA motors (HPDMs), which can be viewed as cooperative teams of thousands of DNA walkers attached to a microsphere, can generate and sustain substantial forces in the 100+ pN regime. Specifically, we show that HPDMs can generate forces that can unzip and shear DNA duplexes (∼12 and ∼50 pN, respectively) and rupture biotin-streptavidin bonds (∼100-150 pN). To help explain these results, we present a variant of the burnt-bridge Brownian ratchet mechanism that we term autochemophoresis, wherein many individual force generating units generate a self-propagating chemomechanical gradient that produces large collective forces. In addition, we demonstrate the potential of this work to impact future engineering applications by harnessing HPDM autochemophoresis to deposit "molecular ink" via mechanical bond rupture. This work expands the capabilities of synthetic DNA motors to mimic the force-generating functions of biological motors. Our work also builds upon previous observations of autochemophoresis in bacterial transport processes, indicating that autochemophoresis may be a fundamental mechanism of pN-scale force generation in living systems.

Dynamic DNA Structures.

Dynamic DNA structures, a type of DNA construct built using programmable DNA self-assembly, have the capability to reconfigure their conformations in response to environmental stimulation. A general strategy to design dynamic DNA structures is to integrate reconfigurable elements into conventional static DNA structures that may be assembled from a variety of methods including DNA origami and DNA tiles. Commonly used reconfigurable elements range from strand displacement reactions, special structural motifs, target-binding DNA aptamers, and base stacking components, to DNA conformational change domains, etc. Morphological changes of dynamic DNA structures may be visualized by imaging techniques or may be translated to other detectable readout signals (e.g., fluorescence). Owing to their programmable capability of recognizing environmental cues with high specificity, dynamic DNA structures embody the epitome of robust and versatile systems that hold great promise in sensing and imaging biological analytes, in delivering molecular cargos, and in building programmable systems that are able to conduct sophisticated tasks.

Stochastic DNA Walkers in Droplets for Super-Multiplexed Bacterial Phenotype Detection.

Pathogen detection is growing in importance in the global health arena because of the high morbidity and mortality associated with bacterial blood stream infections. In this work, we present stochastic DNA walkers in droplets (SDwalker-Drop), a one-step, rapid, and super-multiplex method for ultrahigh-throughput bacterial detection. The SDwalkers, by exploiting cascade signal amplification, endow our analytical platform with fast analysis times and single-cell analysis ability. The autonomous and multiple-step walking behavior of the SDwalkers provides a super-multiplex droplet-encoding strategy by embedding intensity coded barcodes into a sequence of color-multiplexed barcodes. We realized a theoretical coding capacity of 83 -1=511 and achieved 20 distinct patterns for bacterial phenotype detection and identification. Moreover, our SDwalker-Drop platform could be readily integrated with a flow cytometer to afford a general approach for super-multiplexed, high-throughput biological assays and screening.

Exonuclease-catalyzed recycling and annular four-footed DNA walking amplification-assisted "on-off-super on" signal transitions for photoelectrochemical biosensing of kanamycin.

Photoelectrochemical (PEC) biosensors have exhibited a promising potential for assays of a large variety of analytes; however, how to realize their low background-based "super on" signal output is still a great challenge. Herein, we report a novel multiple nucleic acid amplification-assisted "on-off-super on" signal transition mechanism for the PEC biosensing of kanamycin antibiotics. The biosensing platform was constructed on a perylene-3,4,9,10-tetracarboxylic dianhydride-based photoelectrode, and its strong photocurrent could be well inhibited by an anchored ferrocene (Fc)-labeled hairpin DNA to produce a low background signal. Two target biorecognition-triggered exonuclease III-catalytic reactions were adopted to produce an annular four-footed DNA walker (AFW) and a methylene blue (MB)-labeled DNA strand. By using their synergistic effect to release Fc quenchers and simultaneously capture MB sensitizers, a "super on" signal output was realized. As a result, a very wide linear range from 10 fg mL-1 to 10 ng mL-1 and an ultra-low detection limit of 7.8 fg mL-1 were obtained. Meanwhile, the aptamer recognition-based homogeneous reaction and AFW-based multiple nucleic acid amplification effectively simplified the assay manipulation and well ensured the repeatability of the method. The satisfactory sample experiment results indicated its good reliability and accuracy for the antibiotic residue analysis application.

Stochastic bipedal dual-DNA walkers for fast and sensitive detection of apurinic/apyrimidinic endonuclease1 and inhibitor screening.

DNA walkers have emerged as a powerful tool in various biosensors, enabling the detection of low-abundance analytes with their precise programmability and efficient signal amplification capacity. However, many existing approaches are hampered by limited reaction kinetics. Herein, we designed a stochastic bipedal dual-DNA walkers (SBDW) that can traverse at high speed on AuNP-based three-dimensional (3D) tracks powered by Exo III. The SBDW exhibited superior reaction kinetics and are up to least 2.25 times faster than traditional DNA walkers, reaching a plateau within 40 min. This advancement allows for rapid and highly sensitive fluorescence detection of a significant base excision repair enzyme of APE1 with a detection limit of 0.001 U/mL. In comparison to traditional DNA walkers, this platform enables highly sensitive and specific APE1 assays in cell lysate and facilitates rapid and accurate screening of APE1 inhibitors. Given its rapid, sensitive, specific, and reliable analysis features, the strategy shows great promise in drug discovery and clinical diagnosis.

Dual cascade DNA walking-induced "super on" photocurrent response for constructing a novel antibiotic biosensing method.

The construction of effective methods for the convenient testing of antibiotic residues in real samples has attracted considerable interest. Herein, we designed a dual cascade DNA walking amplification strategy and combined it with the controllable photocurrent regulation of a photoelectrode to develop a novel photoelectrochemical (PEC) biosensing method for antibiotic detection. The photoelectrode was prepared through the surface modification of a glassy carbon electrode with the TiO2/CdS QDs nanocomposite synthesized by an in situ hydrothermal deposition method. The strong anodic PEC response of the nanocomposite could be well inhibited by the introduction of a silver nanoclusters (Ag NCs)-labeled DNA hairpin onto its surface. Upon the target biorecognition reaction, an Mg2+-dependent DNAzyme (MNAzyme)-driven DNA walking was triggered to release another MNAzyme strand-linked streptavidin (SA) complex. As this SA complex could serve as a four-legged DNA walker, its cascade walking on the electrode surface not only released Ag NCs but also caused the linking of Rhodamine 123 with the electrode to realize the "super on" photocurrent output. By using kanamycin as the model analyte, this method showed a very wide linear range from 10 fg mL-1 to 1 ng mL-1 and a very low detection limit of 0.53 fg mL-1. Meanwhile, the simple photoelectrode preparation and the aptamer recognition-based autonomous DNA walking resulted in the convenient manipulation and excellent repeatability. These unique performances determine the great potential of the proposed method for practical applications.

DNA Walkers for Biosensing Development.

The ability to design nanostructures with arbitrary shapes and controllable motions has made DNA nanomaterials used widely to construct diverse nanomachines with various structures and functions. The DNA nanostructures exhibit excellent properties, including programmability, stability, biocompatibility, and can be modified with different functional groups. Among these nanoscale architectures, DNA walker is one of the most popular nanodevices with ingenious design and flexible function. In the past several years, DNA walkers have made amazing progress ranging from structural design to biological applications including constructing biosensors for the detection of cancer-associated biomarkers. In this review, the key driving forces of DNA walkers are first summarized. Then, the DNA walkers with different numbers of legs are introduced. Furthermore, the biosensing applications of DNA walkers including the detection- of nucleic acids, proteins, ions, and bacteria are summarized. Finally, the new frontiers and opportunities for developing DNA walker-based biosensors are discussed.

Dual CHA-mediated high-efficient formation of a tripedal DNA walker for constructing a novel proteinase-free dual-mode biosensing strategy.

DNA walkers have been recognized as a type of powerful signal amplification tool for biosensors, but how to adopt a proper strategy to increase their amplification efficiency is still highly desirable. Herein we design a dual-catalytic hairpin assembly (CHA)-mediated strategy for the high-efficient formation of a tripedal Mg2+-dependent DNAzyme (MNAzyme)-DNA walker, and thus develop a novel proteinase-free dual-mode biosensing method for the kanamycin (Kana) antibiotic assay. The first CHA is initiated by a target-biorecognition reaction, which can produce the DNA walker and also induce the target recycling. The second CHA is initiated by a special base sequence designed as a one-half substrate of the MNAzyme. Upon the first CHA-triggered DNA walking at a magnetic bead (MB) track, this "pseudo-target" sequence can be released to induce another CHA-cycle for the formation of the same DNA walker. Meanwhile, the other one-half substrate strand exposed on the MB surface will trigger the quantitative hybridization chain reaction (HCR)-assembly of a G-quadruplex DNAzyme (G-DNAzyme)-enriched double-stranded DNA polymer. So the enzymatic reaction of G-DNAzymes enabled the convenient colorimetric and photoelectrochemical dual-mode signal transduction of the method. Due to the dual-CHA facilitation to the tripedal and three-dimensional DNA walking and synergetic signal amplification of HCR, this method exhibits very low detection limits of 9.4 and 0.55 fg mL-1, respectively. In combination with its wide linear range, automated manipulation, and excellent selectivity, repeatability and reliability, the proposed method is expected to be used for the convenient semiquantitative screening and accurate determination of possible antibiotic residues in complicated matrices.

Advances in Electrochemiluminescence Biosensors Based on DNA Walkers.

Mediated by Watson-Crick base-pairing principle, DNA can be used to construct multi-functional molecular machines, such as DNA walkers, tweezers, logic gates and motors. It is noteworthy that DNA walkers with the advantages of programmability and diverse structures within the micro-nano scale have attracted intense attention in the field of biosensing, bioimaging, drug delivery, and genetic diagnosis. DNA walkers are comprised of driving power, walking strands and the tracks. The driving power acts as an external stimulus and the tracks as a platform for the walking strands to move autonomously. Under the specific external stimulus as driving power, such as strand displacement strategies, enzymatic reactions and environmental condition stimulus, DNA walkers could realize the generation and amplification of signals. Electrochemiluminescence (ECL) biosensors, combining ECL technology and bio-identification strategies, exhibit the virtue of high sensitivity, wide linear response range and low background interference. Recently, the construction of DNA walker-based ECL biosensors can amplify the targets and signals via multiple identification and recycles, achieving ultrasensitive detection for diverse targets. Herein, this review systematically summarizes the construction of different types of DNA walkers and their applications in ECL biosensors. Ultimately, this review summarizes and discusses the prospects for DNA walkers.

Sensitive biosensor for p53 DNA sequence based on the photothermal effect of gold nanoparticles and the signal amplification of locked nucleic acid functionalized DNA walkers using a thermometer as readout.

A convenient photothermal biosensor was constructed for p53 DNA sequence detection based on the high discrimination capability of locked nucleic acid and high efficiency of signal amplification strategy of DNA walkers and difference photothermal effect between aggregated and dispersed gold nanoparticles (AuNPs). The presence of target activated the DNA walkers via the high affinity between target and complementary locked nucleic acid in the probe strand, resulting in the hybridization of the walker strand and substrate strand to form a specific enzyme recognition site. Under the cleavage of the endonuclease, single-stranded DNA (ssDNA) was released to the solution. Then the walker strand bound to a new substrate strand, and the next round of cleavage was triggered. The released ssDNA enhanced the stability of AuNPs against salt-induced aggregation. Given difference photothermal effects of the aggregated AuNPs and dispersed AuNPs under the near-infrared laser, the change of the temperature was detected by a common thermometer easily, which had a linear relationship with the target concentration in the range of 2.0-120.0 pM, the detection limit was 1.4 pM (S/N = 3). The proposed photothermal assay has been applied to detect p53 DNA sequence spiked complex samples with satisfying results.

The Formal Language and Design Principles of Autonomous DNA Walker Circuits.

Simple computation can be performed using the interactions between single-stranded molecules of DNA. These interactions are typically toehold-mediated strand displacement reactions in a well-mixed solution. We demonstrate that a DNA circuit with tethered reactants is a distributed system and show how it can be described as a stochastic Petri net. The system can be verified by mapping the Petri net onto a continuous-time Markov chain, which can also be used to find an optimal design for the circuit. This theoretical machinery can be applied to create software that automatically designs a DNA circuit, linking an abstract propositional formula to a physical DNA computation system that is capable of evaluating it. We conclude by introducing example mechanisms that can implement such circuits experimentally and discuss their individual strengths and weaknesses.