Simulation-Guided Rational Design of DNA Walker-Based Theranostic Platform.

Biomolecule-functionalized nanoparticles represent a type of promising biomaterials in biomedical applications owing to their excellent biocompatibility and versatility. DNA-based reactions on nanoparticles have enabled emerging applications including intelligent biosensors, drug delivery, and biomimetic devices. Among the reactions, strand hybridization is the critical step to control the sensitivity and specificity of biosensing, and the efficiency of drug delivery. However, a comprehensive understanding of DNA hybridization on nanoparticles is still lacking, which may differ from the process in homogeneous solutions. To address this limitation, coarse-grained model-based molecular dynamic simulation is harnessed to disclose the critical factors involved in intermolecular hybridization. Based on simulation guidance, DNA walker-based smart theranostic platform (DWTP) based on "on-particle" hybridization is developed, showing excellent consistency with simulation. DWTP is successfully applied for highly sensitive miRNA 21 detection and tumor-specific miRNA 21 imaging, driven by tumor-endogenous APE 1 enzyme. It enables the precise release of antisense oligonucleotide triggered by tumor-endogenous dual-switch miRNA 21 and APE 1, facilitating effective gene silencing therapy with high biosafety. The simulation of "on-particle" DNA hybridization has improved the corresponding biosensing performance and the release efficiency of therapeutic agents, representing a conceptually new approach for DNA-based device design.

Rapid detection of melamine by DNA Walker mediated SERS sensing technique based on signal amplification function.

A new method is proposed for detecting typical melamine dopants in food using surface-enhanced Raman scattering (SERS) biosensing technology. Melamine specific aptamer was used as the identification probe, and gold magnets (AuNPs@MNPs) and small gold nanoparticles (AuNPs@MBA) were used as the basis for Raman detection. The Raman signal of the detection system can directly detect melamine quantitatively. Under optimized conditions, the detection of melamine was carried out in the low concentration range of 0.001-500 mg/kg, the enhancement factor (EF) was 2.3 × 107, and the detection limit was 0.001 mg/kg. The method is sensitive and rapid, and can be used for the rapid detection of melamine in the field environment.

DNA nanosensor based on bipedal 3D DNA walker-driven proximal catalytic hairpin assembly for sensitive and fast TK1 mRNA detection.

Hyperproliferative  diseases are the first step for tumor formation; thymidine kinase 1 (TK1) mRNA is closely related to cell proliferation. Therefore, the risk of malignant proliferation can be identified by sensitively detecting the variance in TK1 mRNA concentration, which can be used for tumor auxiliary diagnosis and monitoring tumor treatment. Owing to the low abundance and instability of TK1 mRNA in real samples, the development of a sensitive and fast mRNA detection method is necessary. A DNA nanosensor that can be used for detecting TK1 mRNA based on bipedal 3D DNA walker-driven proximal catalytic hairpin assembly (P-CHA) was developed. P-CHA hairpins were hybridized to a linker DNA strand coupled with magnetic nanoparticles to increase their local concentrations. The bipedal DNA walking on the surface of NPs accelerates reaction kinetics using the proximity effect. Taking advantage of the signal amplification of P-CHA as well as the rapid reaction rate of the DNA walker in 80 min, the proposed sensor detects TK1 mRNA with a low detection limit of 14 pM and may then be applied to clinical diagnosis.

High-affinity truncated aptamers for detection of Cronobacter spp with magnetic separation-assisted DNAzyme-driven 3D DNA walker.

After optimizing the original aptamer sequence by truncation strategy, a magnetic separation-assisted DNAzyme-driven 3D DNA walker fluorescent aptasensor was developed for detecting the food-borne pathogen Cronobacter species. Iron oxide magnetic nanoparticles (MNPs) modified with a hybrid of truncated aptamer probe and DNAzyme strand (AP-E1) denoted as MNPs@AP-E1, were employed as capture probes. Simultaneously, a DNAzyme-driven 3D-DNA walker was utilized as the signal amplification element. The substrate strand (Sub) was conjugated with the gold nanoparticles (AuNPs), resulting in the formation of AuNPs@Sub, which served as a 3D walking track. In the presence of the target bacteria and Mg2+, E1-DNAzyme was activated and moved along AuNPs@Sub, continuously releasing the signal probe. Under optimized conditions, a strong linear correlation was observed for Cronobacter sakazakii (C. sakazakii) in the concentration range 101 to 106 CFU mL-1, with a low detection limit of 2 CFU mL-1. The fluorescence signal responses for different Cronobacter species exhibited insignificant differences, with a relative standard deviation of 3.6%. Moreover, the aptasensor was successfully applied to determine  C. sakazakii in real samples with recoveries of 92.86%-108.33%. Therefore, the novel method could be a good candidate for ultra-sensitive and selective detection of Cronobacter species without complex manipulation.

Exonuclease III-propelled DNAzyme walker: an electrochemical strategy for microRNA diagnostics.

MicroRNA detection is crucial for early infectious disease diagnosis and rapid cancer screening. However, conventional techniques like reverse transcription-quantitative polymerase chain reaction, requiring specialized training and intricate procedures, are less suitable for point-of-care analyses. To address this, we've developed a straightforward amplifier based on an exonuclease III (exo III)-propelled DNAzyme walker for sensitive and selective microRNA detection. This amplifier employs a specially designed hairpin probe with two exposed segments for strand recognition. Once the target microRNA is identified by the hairpin's extended single-strand DNA, exo III initiates its digestion, allowing microRNA regeneration and subsequent hairpin probe digestion cycles. This cyclical process produces a significant amount of DNAzyme, leading to a marked reduction in electrochemical signals. The biosensor exhibits a detection range from 10 fM to 100 pM and achieves a detection limit of 5 fM (3σ criterion). Importantly, by integrating an "And logic gate," our system gains the capacity for simultaneous diagnosis of multiple microRNAs, enhancing its applicability in RNA-based disease diagnostics.

Dual Spatially Localized DNA Walker for Fast and Efficient RNA Detection.

DNA walkers, which are synthetic nanodevices that drive the processive movement of nucleic acids along a well-designed track, have emerged as a powerful tool in biosynthesis, biocomputing, and biosensing due to their exquisite programmability, good biocompatibility, and efficient signal amplification capacity. However, many existing approaches are still hindered by limited reaction kinetics. Herein, we designed a dual spatially localized DNA walker that utilized bipedal catalysts to drive high-speed stochastic movement along three-dimensional tracks via a proximity-driven catalytic hairpin assembly. We demonstrated that the dual colocalization of autocatalytic circuits significantly increased their local concentrations and accelerated reaction kinetics through proximity. We also showed that the use of bipedal catalysts further improved reaction rates compared with unipedal catalysts. Taking advantage of these unique features, we constructed an RNA-responsive PCHA walker for mRNA imaging in live cells, providing a novel and efficient tool for biomolecule detection and biological functions regulation.

A visual detection strategy for SARS-CoV-2 based on dual targets-triggering DNA walker.

SARS-CoV-2, a highly transmissible and mutagenic virus, made huge threats to global public health. The detection strategies, which are free from testing site requirements, and the reagents and instruments are portable, are vital for early screening and play a significant role in curbing the spread. This work proposed a silver-coated glass slide (SCGS)/DNA walker based on a dual targets-triggering mechanism, enzyme-catalyzed amplification, and smartphone data analysis, which build a portable visual detection strategy for the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) gene. By this method, the detection was reflected by the ultraviolet absorbance changes and visible color changes to the naked eye which was analyzed by Red-Green-Blue (RGB) data analysis via smartphone within 30 min, simplifying the detection process and shortening the detection time. Meanwhile, the dual targets-triggering mechanism and dual signal amplification strategy ensured detection specificity and sensitivity. Further, the practicability was verified by the detection of the real sample which provided this method an application potential in SARS-CoV-2 rapid detection.

A novel ternary Y-DNA walker amplification strategy designed fluorescence aptasensor based on Au@SiO2@Fe3O4 nanomaterials for ochratoxin A detection.

A novel ternary Y-DNA walker amplification strategy designed fluorescence aptasensor based on Au@SiO2@Fe3O4 nanomaterials for ultrasensitive and specific ochratoxin A detection in food samples is presented. Au@SiO2@Fe3O4 nanomaterials provide the loading platform as well as separation and recovery properties for the ternary Y-DNA walker. The ternary Y-DNA walker is designed to be driven by Nb.BbvCI cleaving a large number of FAM probes to achieve signal amplification. Since Ochratoxin A (OTA) can bind to the constituent aptamer in the ternary Y-DNA walker, adding OTA will destroy the structure of the ternary Y-DNA walker, thereby inhibiting the driving process of the walker. After optimization of various parameters, a standard curve was obtained from 100 to 0.05 ng·mL-1 of OTA with the limit of determination of 0.027 ng·mL-1. The spiked recovery of peanut samples by this method was 82.00-93.30%, and the aptasensor showed excellent specificity and long-term stability. This simple, robust, and scalable oligonucleotide chain-based ternary Y-DNA walker can provide a general signal amplification strategy for trace analysis.

Multiorbital DNA walker nanoprobe for portable photothermal detection based on H2S etching of cubic Prussian blue.

In the developed assay, multiorbital 3D DNA walker (MO DNA walker) was applied as signal amplified protocol for enhancing the detection signal of the photothermal biosensor, which was designed for sensitive detection of miRNA based on the H2S triggered conversation of photothermal reagent. When the target molecule is present, the DNA walking strand was released and then hybridize with track strands. The landing of walking particles (WPT) on the tracking particles (TPT) promotes the relative movement of the WPT around TPT, thus releasing large amount of horseradish peroxidase (HRP) with the aid of DNAzyme. After reacting with Na2S2O3 and H2O2, multiple H2S can be generated in situ based on the catalytic ability of HRP. Meanwhile, cubic Prussian blue (CPB) was synthesized and exhibited superior photothermal response, which can be served as efficient photothermal reagent and H2S responsive acceptor. Significantly, the photothermal signal of CPB could be obviously reduced after challenged with H2S ascribed to synchronous reaction between the ferric ion (Fe3+) and H2S. The improved walking area and freedom enable significant signal amplification, enhancing the biosensor's performance. Under ideal circumstances, the proposed photothermal assay demonstrated excellent performance for determination of miRNA-21.

Advances in Designer DNA Nanorobots Enabling Programmable Functions.

The advent of DNA nanotechnology has paved the way for the development of nanoscale robotics capable of executing smart and sophisticated tasks in a programmed and automatic manner. The programmability and customizable functionality of designer DNA nanorobots interfacing with biology would offer great potential for basic and applied research in the interdisciplinary fields of chemistry, biology, and medicine. This review aims to summarize the latest progress in designer DNA nanorobotics enabling programmable functions. We first describe the state-of-art engineering principles and the functional modules used in the rational design of a dynamic DNA nanorobot. Subsequently, we summarize the distinct types of DNA nanorobots performing sensing tasks, sensing-and-actuation, or continuous actuation, highlighting the versatility of designer DNA nanorobots in accurate biosensing, targeted drug delivery, and autonomous molecular operations to promote desired cellular behavior. Finally, we discuss the challenges and opportunities in the development of functional DNA nanorobotics for biomedical applications. We envision that significant progress in DNA-enabled nanorobotics with programmable functions will improve precision medicine in the future.

Three-dimensional self-powered DNA walking machine based on catalyzed hairpin assembly energy transfer strategy.

Herein, catalyzed hairpin assembly is implemented as an automated strategy, which can respond in living cells to detect specific target DNA. Using the principle of catalyzed hairpin assembly (CHA), the auxiliary chain connects the fuel and starting chain to form a triple-stranded DNA to complete such a single system. Hundreds of single systems are modified on gold nanoparticles as DNA orbitals. Through the specific recognition of base complementation, the target DNA can realize the automatic walking of the three-dimensional fluorescence machine. This is a novel walking nanomachine that has a simple structure and can independently exist in cells to achieve automatic operation.

Rational engineering the DNA tetrahedrons of dual wavelength ratiometric electrochemiluminescence biosensor for high efficient detection of SARS-CoV-2 RdRp gene by using entropy-driven and bipedal DNA walker amplification strategy.

Fast and effective detection of epidemics is the key to preventing the spread of diseases. In this work, we constructed a dual-wavelength ratiometric electrochemiluminescence (ECL) biosensor based on entropy-driven and bipedal DNA walker cycle amplification strategies for detection of the RNA-dependent RNA polymerase (RdRp) gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The entropy-driven cyclic amplification reaction was started by the SARS-CoV-2 RdRp gene to generate a bandage. The bandage could combine with two other single-stranded S1 and S2 to form a bipedal DNA walker to create the following cycle reaction. After the bipedal DNA walker completed the walking process, the hairpin structures at the top of the DNA tetrahedrons (TDNAs) were removed. Subsequently, the PEI-Ru@Ti3C2@AuNPs-S7 probes were used to combine with the excised hairpin part of TDNAs on the surface of Au-g-C3N4, and the signal change was realized employing electrochemiluminescence resonance energy transfer (ECL-RET). By combining entropy-driven and DNA walker cycle amplification strategy, the ratiometric ECL biosensor exhibited a limit of detection (LOD) as low as 7.8 aM for the SARS-CoV-2 RdRp gene. As a result, detecting the SARS-CoV-2 RdRp gene in human serum still possessed high recovery so that the dual-wavelength ratiometer biosensor could be used in early clinical diagnosis.

A Self-Serviced-Track 3D DNA Walker for Ultrasensitive Detection of Tumor Exosomes by Glycoprotein Profiling.

Sensitive and accurate analysis of low-concentration of tumor-derived exosomes (Exos) in biofluids is essential for noninvasive cancer diagnosis but is still challenging due to the lack of high-sensitive methods with low-cost and easy-operation. Herein, exploiting target Exos as a three-dimensional (3D) track for the first time, we developed a self-serviced-track DNA walker (STDW) for wash-free detection of tumor Exos using exosomal glycoprotein, which was enabled by split aptamer-recognition-initiated autonomous running powered by a catalytic hairpin assembly (CHA). Benefiting from high selectivity and sensitivity of the STDW assay, direct detection of tumor Exos in cell culture medium and serum could also be realized. Furthermore, this method exhibited high accuracy in clinical sample analysis, offering the potential for early cancer diagnosis and postoperative response prediction.

Electrochemiluminescence aptasensor for Siglec-5 detection based on MoS2@Au nanocomposites emitter and exonuclease III-powered DNA walker.

Lectins are highly specific binding proteins for glycoproteins which widely exist in living organisms, playing a vital role in exploring the biological evolution process, such as cellular proliferation, differentiation, carcinogenesis and apoptosis. Therefore, the content monitoring of lectin becomes particularly significant and urgent in the bioanalytical application. In this work, we fabricated an aptasensor, majorly capitalizing the eminent affinity between sialic acid-binding immunoglobulin (Ig)-like lectin 5 (Siglec-5) and nucleic acids aptamer (K19), with nontoxic MoS2@Au nanocomposites as electrochemiluminescence (ECL) emitters based on exonuclease III (Exo III)-powered DNA walker for the bioassays of Siglec-5. The DNA track was constructed on the emitters' surface, providing a reliable platform for the DNA walker's autonomous move. In the assay, the primer DNA in the DNA duplex was replaced by Siglec-5 due to the aptamer interactions and repeatedly released to participate in the movement of the DNA walker, further triggering cascade signal amplification. Finally, our aptasensor indicates significant potential for assays of Siglec-5 with a detection limit of 8.9 pM.

Electrochemiluminescence biosensor based on molybdenum disulfide-graphene quantum dots nanocomposites and DNA walker signal amplification for DNA detection.

Based on the prominent electrochemiluminescence (ECL) performances of molybdenum disulfide-graphene quantum dots (MoS2-GQDs) nanocomposite and combined with enzyme-assisted recycling DNA walker signal amplification, an "on-off" switch ECL biosensor was proposed for sensitive assay of specific DNA sequences. Noticeably, MoS2 with two-dimensional nanosheet structure increased the loading capacity of GQDs to support abundant hairpin DNA (H). The composites of MoS2 and GQDs facilitated the charge transfer in ECL process, which significantly improved the ECL signal to achieve an "on" state. Then, the DNA walker cyclic amplification was performed by adding the target DNA and exonuclease III (Exo III). Finally, the DNA2-Fc-DNA1 was introduced into the system as ECL signal quencher, turning the ECL signal into an "off" state. The sensitive assay of ultra-low concentration specific DNA sequences was realized according to the variation of ECL signal strength before and after the existence of target DNA. The proposed ECL biosensor showed a good linear relationship ranging from 1 nM to 100 aM with a detection limit of 25.1 aM, providing a powerful strategy for biomedical research and clinical analysis.

Graphene-based fluorometric determination of agrD gene transcription in methicillin-resistant Staphylococcus aureus using exonuclease III-aided target recycling and DNA walker cascade amplification.

A graphene-based bioassay is described for the fluorometric determination of agrD gene transcription (mRNA) in methicillin-resistant Staphylococcus aureus (MRSA). This method includes exonuclease III (Exo III)-assisted target recycling and DNA walker cascade amplification. Hairpin1 (HP1) consists of a capture probe (CP) and DNA walker sequence. In the absence of the target, 5'-amino modified hairpin2 (HP2) labeled with carboxyfluorescein (FAM) at its 3' terminus is covalently linked to graphene via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) catalysis, resulting in the quenching of the FAM signal. The stem-loop structure of HP1 opens when the target is added to form partially complementary DNA/RNA hybrids. Exo III then initiates the target recycling process by cleaving the CP and DNA walker cascade reaction by automatic walking. This iterative reaction causes the FAM to dissociate from the graphene, and the fluorescence can be measured at excitation/emission wavelengths of 480/514 nm. Therefore, the target can be assayed by fluorescence. This method has a linear relationship with the concentration of target within the range 1 fM to 100 pM with a detection limit of 1 fM. The developed bioassay was used to monitor biofilm formation and investigate the mechanism of drug action with satisfactory results. Schematic representation of the graphene-based fluorescent bioassay for agrD gene transcription in methicillin-resistant Staphylococcus aureus by using exonuclease III-aided target recycling and DNA walker cascade amplification.

Mechanisms, Methods of Tracking and Applications of DNA Walkers: A Review.

In recent years, DNA nanotechnology expanded its scope from structural DNA nanoarchitecture towards designing dynamic and functional nanodevices. This progress has been evident in the development of an advanced class of DNA nanomachines, the so-called DNA walkers. They represent an evolution of basic switching between distinctly defined states into continuous motion. Inspired by the naturally occurring walkers such as kinesin, research on DNA walkers has focused on developing new ways of powering them and investigating their walking mechanisms and advantages. New techniques allowing the visualization of walkers as single molecules and in real time have provided a deeper insight into their behavior and performance. The construction of novel DNA walkers bears great potential for applications in therapeutics, nanorobotics or computation. This review will cover the various examples and breakthrough designs of recently reported DNA walkers that pushed the limits of their performance. It will also mention the techniques that have been used to investigate walker nanosystems, as well as discuss the applications that have been explored so far.

DNAzyme-powered DNA walking machine for ultrasensitive fluorescence aptasensing of kanamycin.

A DNAzyme-powered DNA walking machine was constructed to develop the fluorescence aptasensing for sensitive detection of kanamycin. The aptamer for kanamycin is partially hybridized with complementary DNA (cDNA) modified on magnetic beads (MBs). The specific interaction of target and aptamer triggered the cDNA to be free tentatively, which captured walker DNA. Then the autonomous motion of DNA walker on MBs surface was propelled via DNAzyme digestion of recognition sites. The signal probe was separated, and the amplified fluorescence signal was achieved by the accumulation of the signal probe. Kanamycin was used as a model analyte, and the developed assay achieves a detection limit of 0.00039 ng·mL-1 (S/N = 3) within a linear detection range from 0.001 to 2000 ng·mL-1. This aptasensing strategy can be extended for detection of other antibiotics by adapting corresponding target recognition aptamer sequence. Graphical abstract The fluorescence aptasensing for sensitive detection of kanamycin based on DNAzyme-powered DNA walking machine was constructed.

Paper-based electrochemiluminescence determination of streptavidin using reticular DNA-functionalized PtCu nanoframes and analyte-triggered DNA walker.

A paper-based electrochemiluminescence (ECL) biosensor characterized by the signal amplification of reticular DNA-functionalized PtCu nanoframes (DNA-PtCuTNFs) and analyte-triggered DNA walker was developed for sensitive streptavidin assay. Silver microflower functionalized paper-based sensing platform was prepared to fix the hairpin strand (S1). With addition of the streptavidin, plenty of DNA walkers consisting of the walking strands (S2) labeled with biotin and streptavidin were established, which protected S2 from digestion via the terminal protection mechanism. The sequential introduction of the DNA walker and capture probe initiated the hairpin structure opening of S1 and strand displacement reaction (SDR) happening, causing the S2 release. Subsequently, S1 hybridized with S3. The free S2 further hybridized with adjacent S1 to trigger the next cycle. After multiple cycles, the DNA-PtCuTNFs, the fire-new signal enhancer, with remarkable peroxidase activity, were successfully attached onto the paper electrode via metal-catalyst-free click chemistry. Based on the SDR of the DNA walker and the catalysis of DNA-PtCuTNFs, a significantly boosted ECL signal of luminol was obtained. Under the optimal conditions, the developed sensor for streptavidin assay exhibited a low detection limit of 33.4 fM with a linear range from 0.1 pM to 0.1 µM. Graphical abstract.

Ratiometric fluorescent 3D DNA walker and catalyzed hairpin assembly for determination of microRNA.

Using 6-carboxyfluorescein (FAM) and tetramethyl rhodamine (TAMRA) as fluorescent signals a ratiometric fluorescent three-dimensional (3D) DNA walker based on a catalytic hairpin assembly (CHA) reaction for microRNA-122 detection was constructed. This method uses CHA reaction triggered indirectly by the target to mediate the 3D DNA walker operation to amplify the signal. The dual emission ratio fluorescent signal with a single excitation wavelength was used as the signal output. This strategy combines DNA walker with CHA reaction and proportional fluorescence signal output methods, which can effectively reduce the background fluorescence signal and the risk of generating false-positive signals. Thus, the impact of environmental factors on the experiment is reduced, thereby obtaining reliable and stable experimental results. It uses the fluorescence excitation wavelength of 488 nm and the maximum fluorescence emission wavelength of 520 nm and 580 nm, respectively. It has a good linear response at a microRNA concentration range of 156.0 pM ~ 7.00 nM and a detection limit of 42.94 pM. This strategy has been successfully applied to detect microRNAs in spiked serum samples. Graphical abstract Schematic representation of three-dimensional (3D) DNA walker constructed using catalytic hairpin self-assembly reaction (CHA)-assisted amplification and ratiometric fluorescence signal output for the detection of miRNA-122 closely related to hepatitis.