DNA Nanomachines Research Articles

A Membrane Tension-Responsive Mechanosensitive DNA Nanomachine.

Membrane curvature reflects physical forces operating on the lipid membrane, which plays important roles in cellular processes. Here, we design a mechanosensitive DNA (MSD) nanomachine that mimics natural mechanosensitive PIEZO channels to convert the membrane tension changes of lipid vesicles with different sizes into fluorescence signals in real time. The MSD nanomachine consists of an archetypical six-helix-bundle DNA nanopore, cholesterol-based membrane anchors, and a solvatochromic fluorophore, spiropyran (SP). We find that the DNA nanopore effectively amplifies subtle variations of the membrane tension, which effectively induces the isomerization of weakly emissive SP into highly emissive merocyanine isomers for visualizing membrane tension changes. By measuring the membrane tension via the fluorescence of MSD nanomachine, we establish the correlation between the membrane tension and the curvature that follows the Young-Laplace equation. This DNA nanotechnology-enabled strategy opens new routes to studying membrane mechanics in physiological and pathological settings.

Dual-layer 3D DNA nanostructure: The next generation of ultrafast DNA nanomachine for microRNA sensing and intracellular imaging

The working efficiency of traditional 3D DNA nanomachines is extremely restricted due to the complex DNA components modified on nanoparticles in the same spatial height. Herein, an ultrafast dual-layer 3D DNA nanomachine (UDDNM) based on catalytic hairpin assembly (CHA) was developed by assembling two different lengths of hairpin DNA on the surface of gold nanoparticles, the long hairpin 1 (H1), to capture the trigger, and the short hairpin 2 (H2), as the signal probe, to recycle the trigger. Compared to the traditional single-layer 3D DNA nanomachine, the dual-layer 3D DNA nanostructure greatly enhances the effective collision between trigger and targeted DNA probe, H1, since the H1 located in outer layer would react with the trigger, inhibiting the invalid collision between the trigger and residual DNA component, H2, and remarkably decreasing the steric hindrance associated with the nucleic acids layer around the nanoparticles. Especially, when the distance of two layers was fixed at 3 nm, the corresponding UDDNM could accomplish the overall reaction only in 3 min with a dramatically high initial rate of up to 5.93 × 10−7 M s−1, which was at least 5-fold beyond that of the typical single-layer 3D DNA nanomachines. As a proof of concept, the described UDDNM was successfully applied in ultrasensitive fluorescence detection and sensitive intracellular imaging of miRNA-21. Consequently, our strategy, based on the creation of dual-layer 3D DNA nanostructure, may create a new approach to designing the next generation of DNA nanomachine and has enormous potential for applications in bio-analysis, logic gate operations, and clinical diagnoses.

Endogenous Adenosine Triphosphate-Assisted Three-Dimensional DNA Walker Assembled on Soft Nanoparticles for Intracellular MicroRNA Imaging.

DNA walkers, a sophisticated type of nanomachines, exhibit intelligent application in biosensing with high programmability and flexibility but usually need additional auxiliary driving force, particularly when walking on hard surfaces. Herein, we construct a three-dimensional (3D) DNA walker on the soft surface of DNA nanospheres (DSs) by using a single-stranded DNA (ssDNA), which is powered by endogenous adenosine triphosphate (ATP) of live cells, so as to sensitively image microRNA (miRNA) in the tumor microenvironment. When the DS walker enters into live cells, miR-21, a general overexpressed biomarker in cancer cells, binds with the blocking strand (B), releasing the walking strand (W) and triggering an ATP-propelled walking reaction. The walking of the DS walker then generates an increasing Cy3 fluorescence signal that indicates the content of miR-21 with about 2.73-fold increase in sensitivity and about 157-fold decrease in the detection limit. Notably, the assembly of the DS walker on soft nanoparticles needs just an easy hybridization process, which facilitates the operation. Meanwhile, this endogenous ATP-powered 3D DNA walker walking on the soft surface performs real-time in situ imaging of miR-21 in live cells, which not only avoids the complex cell treatment and signal error induced by additional auxiliary factors, but also shows high promise of designing programmable DNA nanomachines.

Dynamic DNA nanomachines for amplification imaging of diseased cells based on stimuli-responsive mechanism

As a basic functional unit, living cell with sophisticated structures play an indispensable role in life activities. Since the abnormality of important molecules inside cells is closely related to diseases, the dynamic analysis and spatio-temporal monitoring of specific molecules in living cells can provide precious information for the diagnosis and treatment of diseases. More recently, DNA has not only been recognized as the carrier of genetic information, but has also used as a robust building block for the assembly of multitudinous nanoscale structures due to the intrinsic advantages of high programmability of classic Watson–Crick base-pairing rule. Intensive study promotes the rapid progress of nanotechnology in various fields, such as bioimaging, diagnosis, and therapeutics. Among numerous well-defined DNA nanomaterials, DNA nanomachines have been widely exploited in cell imaging owing to their desirable ability to achieve high-resolution temporal and spatial images in response to endogenous or exogenous stimuli. In brief, elaborate DNA nanomachines can undergo structural changes upon the stimuli of target analytes or environmental factors, resulting in rapid increase or reduction of output signals and thereby indirectly reflecting the expression level of targets. DNA nanomachines with high sensitivity and specificity contribute to the recognition of diseased tissues. In this review, we introduce the basic assembly modules of DNA nanomachines and summarize the recent advances in dynamic DNA nanomachines for diseased-cell imaging. Finally, the current challenges and future directions of DNA nanomachines for bioimaging are discussed.

An intelligent DNA nanomachine for amplified MicroRNA imaging and MicroRNA-Guided efficient gene silencing

The DNA nanomachines as excellent synthetic biological tools have been widely used for the sensitive detection of intracellular microRNA (miRNA) and DNAzyme-involved gene silencing. However, intelligent DNA nanomachines which have the ability to sense intracellular specific biomolecules and respond to external information in complex environments still remain challenging. Herein, we develop a miRNA-responsive DNAzyme cascaded catalytic (MDCC) nanomachine to perform multilayer cascade reactions, enabling the amplified intracellular miRNA imaging and miRNA-guided efficient gene silencing. The intelligent MDCC nanomachine is designed based on multiple DNAzyme subunit-encoded catalyzed hairpin assembly (CHA) reactants sustained by the pH-responsive Zeolitic imidazolate framework-8 (ZIF-8) nanoparticles. After cellular uptake, the MDCC nanomachine degrades in acidic endosome and releases three hairpin DNA reactants and Zn2+, and the latter can act as an effective cofactor for DNAzyme. In the presence of miRNA-21, a catalytic hairpin assembly (CHA) reaction is triggered, which produces a large number of Y-shaped fluorescent DNA constructs containing three DNAzyme modules for gene silencing. The construction of Y-shaped DNA modified with multisite fluorescence and the circular reaction realizes ultrasensitive miRNA-21 imaging of cancer cells. Moreover, miRNA-guided gene silencing inhibits the cancer cell proliferation through the DNAzyme-specific recognition and cleavage of target EGR-1 (Early Growth Response-1) mRNA, which is one key tumor-involved mRNA. The strategy may provide a promising platform for highly sensitive determination of biomolecules and accurate gene therapy of cancer cells.

A novel AD theranostic platform with NIR-II laser controlled drug release and real-time monitoring of therapeutic outcomes

There is a long-standing lack of monitoring of Alzheimer's disease (AD) treatment. Imaging-guided dynamic monitoring of neurological disease treatment can provide a basis for sustained treatment, reflecting the outcomes of treatment and guiding the adjustment of therapy strategies. Here, by ingeniously using a second near-infrared (NIR-II) laser twice in tandem, we designed an AD theranostic nanoplatform with real-time monitoring of AD therapeutic outcomes. Upon first being irradiated with a 1064 nm laser, it realizes the light-controlled delivery of nanoparticles for valid antioxidant therapy. Notably, with secondary irradiation, the presence of AβO and Ca2+ triggered the DNA nanomachine, causing the fluorescent signal to recover. Using a two-step series switch based on thermodynamics, the platform can not only differentiate AD mice from wild-type mice but also provide guidance for the treatment of AD via monitoring the degree of intracellular calcium overload in the brain. Results suggest that the degree of calcium overload is negatively correlated with treatment outcome and can be used as a prognostic indicator for precision medicine. This method provides an image-based non-invasive strategy for early prognosis prediction of AD treatment, which is helpful for future precision medicine treatment planning and prognosis stratification.

Visual Detection of Stem-Loop Primer Amplification (SPA) Products without Denaturation Using Peroxidase-like DNA Machines (PxDM).

Rapid, inexpensive, and accurate determination of nucleic acids is a decisive factor in evaluating population's health and monitoring treatment at point-of-care (POC) settings. Testing systems with visual outputs can provide instrument-free signal detection. Isothermal amplification technologies can substitute conventional polymerase chain reaction (PCR) testing due to compatibility with the POC diagnostic. Here, we have visually detected DNA fragments obtained by stem-loop-primer-assisted isothermal amplification (SPA), but not those obtained by PCR or LAMP amplification using DNA nanomachines with peroxidase-like activity (PxDM) with sensitivity to a single nucleotide substitution. Compared to the diagnostics with conventional loop-mediated isothermal amplification (LAMP), the PxDM method produces no false positive signals with the non-specific amplification products. The study suggests that PxDM, in conjunction with SPA isothermal amplification, can become a valid platform for POC testing systems.

A label-free and ratiometric fluorescent immunosensor by integrating CRISPR/Cas 12a and 3D DNA nanomachine

Sensitive, selective, especially label-free detection of disease relevant protein biomarkers is highly valuable for clinical diagnosis and disease process monitoring but remains a challenge. Herein, with prostate specific antigen (PSA) as a model target, we have designed a label-free ratiometric fluorescent immunosensor based on three-dimensional (3D) DNA walker and the powerful clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system, with poly(thymine) templated Cu nanoclusters and DAPI as signals. The aptamer-linked activator DNA was designed to convert target recognition event into activate deoxyribonuclease activity of CRISPR/Cas12a system and further cleave oligonucleotides. The cleavage behavior leads to the quenching of copper nanoclusters and stagnation of the cascade amplification progress of 3D DNA nanomachine. The DNA machine is constructed from primer DNA and silica nanospheres loaded by double-stranded DNA (dsDNA) with prominent 3′ end, and movement of the primer DNA is powered by exonuclease (Exo) III via a burnt-bridge mechanism. Without primer DNA, the dyes (DAPI) were embedded in the complementary DNA and emit strong fluorescence. The practical applicability of the biosensor was demonstrated by assay of PSA in human serum with satisfactory result. In addition, this completely label-free method, reduced consumption of antibody, greatly simplify the procedures and saving cost.

Magnetic DNA Nanomachine for On-Particle Cascade Amplification-Based Ferromagnetic Resonance Detection of Plant MicroRNA.

Plant microRNAs play critical roles in post-transcriptional gene regulation of many processes, thus motivating the development of accurate and user-friendly microRNA detection methods for better understanding of, e.g., plant growth, development, and abiotic/biotic stress responses. By integrating the capture probe, fuel strand, primer, and template onto the surface of a magnetic nanoparticle (MNP), we demonstrated a magnetic DNA nanomachine that could conduct an on-particle cascade amplification reaction in response to the presence of target microRNA. The cascade amplification consists of an exonuclease III-assisted target recycling step and a rolling circle amplification step, leading to changes in the MNP arrangement that can be quantified by ferromagnetic resonance spectroscopy. After a careful investigation of the exonuclease III side reaction, the biosensor offers a detection limit of 15 fM with a total assay time of ca. 70 min. Moreover, our magnetic DNA nanomachine is capable of discriminating the target microRNA from its family members. Our biosensor has also been tested on total endogenous microRNAs extracted from Arabidopsis thaliana leaves, with a performance comparable to qRT-PCR.

Shell penetration/free energy-driven crosstalk between intelligent DNA nanomachines for ultrasensitive detection of circulating tumor cells

Because of the extraordinary rarity, CTC detection remains a technological challenge. In this contribution, a stimuli-responsive intelligent DNA nanomachine system is demonstrated for ultrasensitive and specific CTC detection via a shell penetration/free energy-driven autonomous circuit. The responsive toeholds designed in linear probes are shielded by protective DNA self-assembled outer shell and hidden inwards from the attack of fuel strands, proposing an innovative concept different from conventional DNA hairpin-based catalytic reactions. DNA machines are very stable in the ‘OFF’ state, almost completely suppressing the background fluorescence. A linear trigger can permeate through outer shell and ignite the inner responsive toeholds, achieving an amplification fluorescent signal. CTC is specifically screened in a linear response range of three orders of magnitude with a detection limit of 2 cells/200 μL with almost 100% accuracy, indicating 3.4～50-fold improvement in assay ability.

DNA Nanomachine (DNM) Biplex Assay for Differentiating Bacillus cereus Species

Conventional methods for the detection and differentiation of Bacillus cereus group species have drawbacks mostly due to the complexity of genetic discrimination between the Bacillus cereus species. Here, we describe a simple and straightforward assay based on the detected unamplified bacterial 16S rRNA by DNA nanomachine (DNM). The assay uses a universal fluorescent reporter and four all-DNA binding fragments, three of which are responsible for "opening up" the folded rRNA while the fourth stand is responsible for detecting single nucleotide variation (SNV) with high selectivity. Binding of the DNM to 16S rRNA results in the formation of the 10-23 deoxyribozyme catalytic core that cleaves the fluorescent reporter and produces a signal, which is amplified over time due to catalytic turnover. This developed biplex assay enables the detection of B. thuringiensis 16S rRNA at fluorescein and B. mycoides at Cy5 channels with a limit of detection of 30 × 103 and 35 × 103 CFU/mL, respectively, after 1.5 h with a hands-on time of ~10 min. The new assay may simplify the analysis of biological RNA samples and might be useful for environmental monitoring as a simple and inexpensive alternative to amplification-based nucleic acid analysis. The DNM proposed here may become an advantageous tool for detecting SNV in clinically significant DNA or RNA samples and can easily differentiate SNV under broadly variable experimental conditions and without prior amplification.

Versatile fluorescence detection of T4 PNK and mRNA based on unique DNA nanomachine amplification

The development of DNA nanomachines provides a new strategy for the detection of tumor markers. In this work, an intelligent three-dimensional (3D) DNA walking machine with polynucleotide kinase (PNK) activator was designed, which was coupled with unique nanomachine formed by DNA nanowire cascade amplification reaction for versatile fluorescence detection of T4 PNK activity and messenger RNA (mRNA). When PNK exists, the free DNA walker was formed by hydrolysis cleavage of exonuclease, then the fluorophore-labeled report probe on the Au nanoparticles (NPs) was sheared during cycling cleavage reaction, thus the fluorescence signal was recovered for detection of PNK. Moreover, the DNA nanowires were produced by rolling ring amplification, then target mRNA sequentially initiated interval hybridization of hairpin probes through DNA nanowire, thus realizing DNA cascade reaction (DCR) with high “on” signal of DNA nanomachine for mRNA assay. This developed novel fluorescence nanomachine reported a new assay method with promising application for versatile targets and showed great potential for molecular-target therapies, and clinic diagnostics.

Self-powered DNA nanomachines for fluorescence detection of lead.

A versatile DNA nanomachine detection system has been developedvia the combination ofDNAzyme with catalytic hairpin assembly (CHA) technology for achieving accurate and sensitive detection of lead ions (Pb2+). In the presence of target Pb2+, capture DNA nanomachine formed by AuNP and DNAzyme recognized and reacted with Pb2+, which yielded an "active" DNAzyme, that induced the cleavage of substrate strand, and then released the initiator DNA (TT) for CHA. With the help of the initiator DNA TT, self-powered CHA was activated to achieve the signal amplification reaction in the detection of DNA nanomachine. Meanwhile, the initiator DNA TT was released and hybridized with the other H1 strand to initiate another CHA, replacement, and turnovers, producing enhanced fluorescence signal of fluorophore FAM (excitation 490nm/emission 520nm) for sensitive determination of Pb2+. Under the optimized conditions, the DNA nanomachine detection system revealed high selectivity toward Pb2+ in the concentration range 50-600pM, with the limit of detection (LOD) of 31pM. Recovery tests demonstrated that the DNA nanomachine detection system has excellent detection capability in real samples. Therefore, the proposed strategy can be extended and act as a basic platform for highly accurate and sensitive detection of various heavy metal ions.

Ultrasensitive Electrochemiluminescence Biosensor Based on 2D Co3O4 Nanosheets as a Coreaction Accelerator and Highly Ordered Rolling DNA Nanomachine as a Signal Amplifier for the Detection of MicroRNA.

A novel ultrasensitive electrochemiluminescence (ECL) biosensor was constructed using two-dimensional (2D) Co3O4 nanosheets as a novel coreaction accelerator of the luminol/H2O2 ECL system for the detection of microRNA-21 (miRNA-21). Impressively, coreaction accelerator 2D Co3O4 nanosheets with effective mutual conversion of the Co2+/Co3+ redox pair and abundant active sites could promote the decomposition of coreactant H2O2 to generate more superoxide anion radicals (O2•-), which reacted with luminol for significantly enhancing ECL signals. Furthermore, the trace target miRNA-21 was transformed into a large number of G-wires through the strand displacement amplification (SDA) process to self-assemble the highly ordered rolling DNA nanomachine (HORDNM), which could tremendously improve the detection sensitivity of biosensors. Hence, on the basis of the novel luminol/H2O2/2D Co3O4 nanosheet ternary ECL system, the biosensor implemented ultrasensitive detection of miRNA-21 with a detection limit as low as 4.1 aM, which provided a novel strategy to design an effective ECL emitter for ultrasensitive detection of biomarkers for early disease diagnosis.

High-Efficiency 3D DNA Walker Immobilized by a DNA Tetrahedral Nanostructure for Fast and Ultrasensitive Electrochemical Detection of MiRNA.

Herein, by directly limiting the reaction space, an ingenious three-dimensional (3D) DNA walker (IDW) with high walking efficiency is developed for rapid and sensitive detection of miRNA. Compared with the traditional DNA walker, the IDW immobilized by the DNA tetrahedral nanostructure (DTN) brings stronger kinetic and thermodynamic favorability resulting from its improved local concentration and space confinement effect, accompanied by a quite faster reaction speed and much better walking efficiency. Once traces of target miRNA-21 react with the prelocked IDW, the IDW could be largely activated and walk on the interface of the electrode to trigger the cleavage of H2 with the assistance of Mg2+, resulting in the release of amounts of methylene blue (MB) labeled on H2 from the electrode surface and the obvious decrease of the electrode signal. Impressively, the IDW reveals a conversion efficiency as high as 9.33 × 108 in 30 min with a much fast reaction speed, which is at least five times beyond that of typical DNA walkers. Therefore, the IDW could address the inherent challenges of the traditional DNA walker easily: slow walking speed and low efficiency. Notably, the IDW as a DNA nanomachine was utilized to construct a sensitive sensing platform for rapid miRNA-21 detection with a limit of detection (LOD) of 19.8 aM and realize the highly sensitive assay of biomarker miRNA-21 in the total RNA lysates of cancer cell. The strategy thus helps in the design of a versatile nucleic acid conversion and signal amplification approach for practical applications in the areas of biosensing assay, DNA nanotechnology, and clinical diagnosis.

Thresholding DNA Nanomachine as a Highly Cooperative and Efficient Enzyme-Like System for Controlled RNA Cleavage.

Therapeutic nucleic acid agents (TNA) can be activated by a marker RNA sequence followed by initiation of targeted RNA cleavage. This property can be used in conditional cell suppression, e. g., cancer marker-dependent cell death. However, healthy cells often express lower levels of cancer markers, thus jeopardizing TNA activation exclusively in cancer cells. Earlier, we developed a conditionally activated split deoxyribozyme construct (DNA thresholder or DTh) that can be activated by high but not by low concentrations of cancer markers. It's activity, however, was suppressed by very high marker concentrations. Herein, we combine the DTh functional units in a single DNA association (Thresholding DNA nanomachine or Th-DNM). Th-DNM maintains a high level of RNA cleavage activity in the presence of marker concentrations above the threshold level. Th-DNM differentiated fully complementary miR17 markers sequence from double base mismatched miR-20. Th-DNM can become a building block of DNA nanorobots for cancer treatment.

A DNA Finite-State Machine Based on the Programmable Allosteric Strategy of DNAzyme.

Living organisms can produce corresponding functions by responding to external and internal stimuli, and this irritability plays a pivotal role in nature. Inspired by such natural temporal responses, the development and design of nanodevices with the ability to process time-related information could facilitate the development of molecular information processing systems. Here, we proposed a DNA finite-state machine that can dynamically respond to sequential stimuli signals. To build this state machine, a programmable allosteric strategy of DNAzyme was developed. This strategy performs the programmable control of DNAzyme conformation using a reconfigurable DNA hairpin. Based on this strategy, we first implemented a finite-state machine with two states. Through the modular design of the strategy, we further realized the finite-state machine with five states. The DNA finite-state machine endows molecular information systems with the ability of reversible logic control and order detection, which can be extended to more complex DNA computing and nanomachines to promote the development of dynamic nanotechnology.

A novel nanoparticle surface-constrained CRISPR-Cas12a 3D DNA walker-like nanomachines for sensitive and stable miRNAs detection

The CRISPR-Cas system has broad prospects as a new type of nucleic acid signal amplification technology based on the trans-cleavage activity of Cas12a to single-stranded DNA, but the trans-cleavage reaction efficiency is relatively low in solution. In order to overcome this negative factor, a new 3D DNA nanomachine whose CRISPR-Cas12a is limited to the surface of nanoparticles is used for sensitive and stable detection of miRNA. By loading Cas12a activator onto spherical nucleic acid (SNA), the CRISPR-Cas12a activator system on the surface of Au nanoparticles (AuNPs) acts as a walker to carry out continuous recognition-walking-cutting reaction on the surface of AuNPs, which enhances the trans-cleavage activity of Cas12a to SNAs. Benefiting from the confinement effect of spherical nucleic acids surface, a 3D DNA nanomachine has been developed for the detection of miRNA-21, which has achieved high sensitivity and accuracy, and the detection limit is able to reach 8.0 pM. This new 3D DNA walker-like nanomachine provided another insight for future bioanalysis and early clinical diagnoses of disease and liquid biopsy.

Electrochemiluminescence detection of miRNA-21 based on dual signal amplification strategies: Duplex-specific nuclease -mediated target recycle and nicking endonuclease-driven 3D DNA nanomachine

DNA nanomachines have shown potential application in the construction of various biosensors. Here, an electrochemiluminescence biosensor for the sensitive detection of miRNA-21 were reported based on three-dimensional (3D) DNA nanomachine and duplex-specific nuclease (DSN)-mediated target recycle amplification strategy. First, the bipedal DNA walkers were obtained by DSN-mediated digestion reaction initiated by target miRNA-21.3D DNA tracks were prepared by modifying Fe3O4 magnetic beads (MBs) with ferrocene-labeled DNA (Fc-DNA). The produced DNA walkers autonomously moved along 3D DNA tracks powered by nicking endonuclease. During the movement, ferrocene-labeled DNA was cleaved, resulting in large amounts of Fc-labeled DNA fragments away from the MBs surface. Finally, the liberated Fc-labeled DNA fragments were dropped on the C-g-C3N4 modified electrode surface, leading to the quenching of C-g-C3N4 electrochemiluminescence (ECL). Benefiting from the dual amplification strategy of 3D DNA nanomachine and DSN-mediated target recycling, the developed ECL biosensor exhibited an excellent performance for miRNA-21 detection with a wide linear range of 10 fM to 10 nM and a low detection limit of 1.0 fM. This work offers a new thought for the application of DNA walkers in the construction of various biosensors.

Simultaneous Imaging and Visualizing the Association of Survivin mRNA and Telomerase in Living Cells by Using a Dual-Color Encoded DNA Nanomachine.

Simultaneous imaging and especially visualizing the association of survivin mRNA and telomerase in living cells are of great value for the diagnosis and prognosis of cancer because their co-expression facilitates the development of cancer and identifies patients at high risk of tumor-related death. The challenge is to develop methods that enable visualizing the association of multiplex targets and avoid the distorted signals due to the different delivery efficiency of probes. Herein, we engineered a DNA triangular prism nanomachine (DTPN) for simultaneous multicolor imaging of survivin mRNA and telomerase and visualizing their association in living cells. Two recognizing probes targeted survivin mRNA and telomerase, and the reporter probe was assembled on the DTP in equal amounts, ensuring the same delivery efficiency of the probes to the living cells. The results showed that this DTPN could quantify intracellular survivin mRNA expression and telomerase activity. Moreover, it also enabled us to visualize the effect of the down-regulation of one target on the expression of another target under different drug stimulations. The results implied that our DTPN provided a promising platform for cancer diagnosis, prognosis, drug screening, and related biological research.