DNA Nanomachines Research Articles

A photo zipper locked DNA nanomachine with an internal standard for precise miRNA imaging in living cells.

DNA nanomachines are capable of converting tiny triggers into autonomous accelerated cascade hybridization reactions and they have been used as a signal amplification strategy for intracellular imaging. However, the "always active" property of most DNA nanomachines with an "absolute intensity-dependent" signal acquisition mode results in "false positive signal amplification" by extracellular analytes and impairs detection accuracy. Here we design a photo zipper locked miRNA responsive DNA nanomachine (PZ-DNA nanomachine) based on upconversion nanoparticles (UCNPs) with a photo-cleavable DNA strand to block the miRNA recognition region, which provided sufficient protection to the DNA nanomachine against nonspecific extracellular activation and allowed satisfactory signal amplification for sensitive miRNA imaging after intracellular photoactivation. Multiple emissions from the UCNPs were also utilized as an internal standard to self-calibrate the intracellular miRNA responsive fluorescence signal. The presented PZ-DNA nanomachine demonstrated the sensitive imaging of intracellular miRNA from different cell lines, which resulted in good accordance with qRT-PCR measurements, providing a universal platform for precise imaging in living cells with high spatial-temporal specificity.

A Janus 3D DNA nanomachine for simultaneous and sensitive fluorescence detection and imaging of dual microRNAs in cancer cells.

Herein, a Janus three-dimensional (3D) DNA nanomachine was constructed for the simultaneous and sensitive fluorescence detection and imaging of dual microRNAs (miRNAs) in cancer cells, which could effectively eliminate signal interference in a homogeneous nanoparticle-based 3D DNA nanostructure caused by the proximity of the two different signal probes to achieve accurate co-location in the same position of living cancer cells. In this system, the Janus nanoparticles were synthesized as the carrier for immobilizing two different oligonucleotides on two different functionalized hemispheres of the nanoparticles to form a Janus 3D DNA nanostructure, which could convert trace amounts of miRNA-21 and miRNA-155 targets into massive FAM and Cy5-labeled duplexes to induce two remarkable fluorescence emissions by the catalytic hairpin assembly (CHA) and 3D DNA walker cascade nucleic acid amplification strategy, realizing sensitive detection and imaging of miRNA targets in cancer cells. Impressively, in comparison with current miRNA imaging methods based on nanoparticle assemblies, the proposed strategy could efficiently eliminate “false positive” results obtained in single type miRNA detection and distinctly increase the immobilization concentration of two different signal probes using Janus nanoparticles as the carrier to further enhance fluorescence intensity, resulting in accurate co-location in the same position of living cells. Meanwhile, the proposed fluorescence imaging technology makes it possible to visualize low concentrations of miRNAs with tiny change associated with some cancers, which could significantly improve the accuracy and precision compared to those of the conventional fluorescence in situ hybridization (FISH) approach. Therefore, it could serve as persuasive evidence for supplying accurate information to better understand biological processes and investigate mechanisms of various biomolecules and subcellular organelles, resulting in the further validation of their function in tumor proliferation and differentiation. This strategy provided an innovative approach to design new generations of nanomachines with ultimate applications in bioanalysis and clinical diagnoses.

Target-activated DNA nanomachines for the ATP detection based on the SERS of plasmonic coupling from gold nanoparticle aggregation.

The self-assembly of plasmonic nanoparticles provides a powerful approach to generate surface-enhanced Raman scattering (SERS), which promotes the actual applications in chemical and biomolecular analyses. Herein, we developed a facile SERS sensing strategy for an ATP assay with a 3-D DNA nanomachine that walks by the Exo III cleavage, leading to the formation of AuNP aggregates, which resulted in the enhancement of the electromagnetic field. Depending on the target-activated Exo III cleavage, the 3-D nanomachine can walk along the 3-D track on the surface of AuNPs and generate self-assembled hot-spots to enhance the SERS signal of a Raman dye, allowing a homogenous assay of the ATP concentration with high sensitivity and reproducibility. Under optimized experimental conditions, the biosensor detected ATP with a widened dynamic range from 1 pM to 1 × 105 pM with a limit of detection of up to 0.29 pM. Hence, the novel strategy provides a useful and practical platform for the SERS assay of ATP with high sensitivity and repeatability. Besides, this platform shows great potential for applications in high-throughput assays for drug screening and clinical diagnostics.

Time-Resolved Activation of pH Sensing and Imaging in Vivo by a Remotely Controllable DNA Nanomachine.

Construction of probes or nanodevices capable of sensing pH with high spatial and temporal precision remains a challenge, despite their importance in monitoring of diverse physiological and pathological processes. Here we disclose the first remotely and noninvasively controlled DNA nanomachine that can monitor pH in live cells and animals in a temporally programmable manner. The nanomachine is designed by rational engineering of the DNA motif with a light-responsive element and further combination with an upconversion nanoparticle that works as a transducer to manipulate the nanomachine with the high precision of NIR light. The nanomachine not only allows for activated fluorescent imaging of intracellular pH, but it also can exert spatiotemporal control over its pH sensing activity in tumor-bearing mice by NIR light irradiation at a chosen time and place. This work illustrates the potential of combining DNA nanotechnology with upconversion tools to yield a precisely controlled nanomachine for temporally resolved pH sensing and imaging.

A pH-regulated stimuli-responsive strategy for RNA-cleaving DNAzyme

RNA-cleaving DNAzymes possess important roles in DNAzymes and have been widely used in the biosensors, DNA nanomachines owing to their ion-specific dependence. However, there are still challenges in constructing universal but versatile stimuli-responsive strategies of RNA-cleaving DNAzymes. Herein, a stimuli-responsive strategy for RNA-cleaving DNAzyme is proposed by the artful design of hairpin nanostructure, in which the activities of DNAzyme (Pb2+-dependent DNAzyme as a model) in the hairpin’s loop are pH-regulated by using the triplex stem as the “lock”. Upon introducing the “key”, pH values, the DNAzyme will be activated and fragment the substrate of it in the presence of Pb2+, accompanied by the turn-on of the fluorescence quenched by fluorescence resonance energy transfer (FRET). The regulation ability of pH can be controlled by the length and sequence of the triplex stem, and the wide pH regulation range may be helpful for the application of DNAzymes in biological medicine delivery systems.

Micro/Nano Technology for Next‐Generation Diagnostics

AbstractThere has been a constant and urgent need to upgrade the current diagnostic technologies to the next generation by utilizing innovative advanced technologies in order to meet new challenges. Micro/nano technology, a series of methods for design and manipulation materials at the micrometer and nanometer scales, which perfectly covers the key building blocks of life including cells, organelles, proteins, nucleic acids, and other components, has unique advantages over current diagnostic methods. For the applications of micro–nano technology in next‐generation diagnostics, three categories, including microchip‐based devices, DNA nanomachines, and DNA nanomaterials, are extensively reviewed. Specifically, for the microchip‐based devices, terahertz technology and surface acoustic wave technology show their ultrasensitivity of label‐free, amplification‐free diagnosis. For the DNA nanomachines, DNA tweezers, DNA robots, and DNA walkers exhibit a great potential for diagnosis with the arbitrary structures and controllable motion behaviors. For the DNA nanomaterials, DNA aptamers, DNAzymes, and hybrid DNA nanomaterials provide sensitive biorecognition elements and versatile signal transductions, which are demonstrated as promising material candidates for the next‐generation diagnostics. Through this review, it is envisioned that micro/nano technologies will play an important role in the next‐generation diagnostics.

Biomimetic 3D DNA Nanomachine via Free DNA Walker Movement on Lipid Bilayers Supported by Hard SiO2@CdTe Nanoparticles for Ultrasensitive MicroRNA Detection.

Herein, a novel three-dimensional (3D) DNA nanomachine with high walking efficiency via free DNA walker movement on biomimetic lipid bilayers supported by hard silica@CdTe quantum dots (SiO2@CdTe) was constructed for ultrasensitive fluorescence detection of microRNA. The synthesized SiO2@CdTe nanoparticles were adopted as the fluorescence indicator and spherical carrier of lipid bilayers, and then the DNA substrates were anchored on lipid bilayers with biomimetic fluidity through the cholesterol-lipid interaction. Once target microRNA-141 interacted with the 3D DNA nanomachine to release cholesterol labeled arm (Chol-arm), the Chol-arm could generate a series of strand displacement reactions by moving freely on the lipid bilayers, resulting in the releasement of numerous quenchers from the SiO2@CdTe nanoparticles and inducing a strong fluorescence signal. Impressively, compared with traditional 3D DNA nanomachine conjugating DNA substrates on hard surfaces (such as gold or silica) with limited reactivity, the proposed biomimetic 3D DNA nanomachine not only immobilized DNA substrates rapidly and effectively but also kept it with a favorable fluidity, which significantly enhanced the walking efficiency. As expected, the biomimetic 3D DNA nanomachine for fluorescence detection of microRNA-141 exhibited an excellent performance with a detection limit of 0.21 pM and presented promising properties in cell lysate detection and intracellular imaging. Thus, the described biomimetic 3D DNA nanomachine provided a novel avenue for sensitive detection of biomolecules, which could be useful for bioanalysis and early clinical diagnoses of disease.

Deoxyribozyme-Based DNA Machines for Cancer Therapy.

Soon after their discovery, RNA-cleaving deoxyribozymes (RCDZ) were explored as anticancer gene therapy agents. Despite low toxicity found in clinical trials, there is no clinically significant anticancer RCDZ-based therapy. Some of the reported disadvantages of RCDZ agents include poor accessibility to folded nucleic acids, low catalytic efficiency inside cells, and problems of intracellular delivery. On the other hand, structural DNA nanotechnology provides an opportunity to build multifunctional nano-associations that can address some of these problems. Herein we discuss the possibility of building RCDZ-based multifunctional DNA nanomachines equipped with RNA unwinding, cancer marker recognition, and RCDZ-based RNA-cleavage functions. An important advantage of such "nanomachines" is the possibility to cleave a housekeeping gene mRNA in a cancer-cell-specific manner. The proposed design could become a starting point for building sophisticated DNA-based nanodevices for cancer treatment.

Highly ordered 3D electrochemical DNA biosensor based on dual orientation controlled rolling motor and graftable tetrahedron DNA

Herein, a robust and highly ordered three-dimensional electrochemical DNA (3D E-DNA) biosensor was proposed, and its orientation was controlled from top down by poly adenine oligonucleotides (polyA-ODNs)-mediated rolling motor (PRM) and graftable tetrahedron DNA (GTD). The GTD with a grafting domain was immobilized on the electrode surface to construct a well-organized sensing interface and controlled the orientation and distribution of the whole system at the “bottom” of this biosensor. The polyA-ODNs regulated the direction and density of the leg DNA attached on PRM at the “top” of the biosensor. The motion was achieved through the target induced cyclic cleaving, which triggered the motor rolling rather than walk. Impressively, the duplex strand DNA (dsDNA) formed after grafting, as a girder, provided a stable support to the soft long single strand (ssDNA), which facilitated the formation of the catalytic center, elevated the efficiency of the rolling cleavage. Under the optimal conditions, the designed biosensor exhibited a lower limit of 0.17 nM and wide linear range from 0.5 nM to 1.5 μM for adenosine rapid detection. Unique dual orientation regulated characteristics of the system increased the probability hybridization enormously and improved the motion efficiency significantly, which offered new avenue of DNA nanomachines development in biosensor platform.

Rational Design of pH-Responsive DNA Motifs with General Sequence Compatibility.

Reconfigurable molecular events are key to molecular machines. In response to external cues, molecular machines rearrange/change their structures to perform certain functions. Such machines exist in nature, for example cell surface receptors, and have been artificially engineered. To be able to build sophisticated and efficient molecular machines for an increasing range of applications, constant efforts have been devoted to developing new mechanisms of controllable structural reconfiguration. Herein, we report a general design principle for pH-responsive DNA motifs for general DNA sequences (not limited to triplex or i-motif forming sequences). We have thoroughly characterized such DNA motifs by polyacrylamide gel electrophoresis (PAGE) and fluorescence spectroscopy and demonstrated their applications in dynamic DNA nanotechnology. We expect that it will greatly facilitate the development of DNA nanomachines, biosensing/bioimaging, drug delivery, etc.

Rational Design of pH‐Responsive DNA Motifs with General Sequence Compatibility

AbstractReconfigurable molecular events are key to molecular machines. In response to external cues, molecular machines rearrange/change their structures to perform certain functions. Such machines exist in nature, for example cell surface receptors, and have been artificially engineered. To be able to build sophisticated and efficient molecular machines for an increasing range of applications, constant efforts have been devoted to developing new mechanisms of controllable structural reconfiguration. Herein, we report a general design principle for pH‐responsive DNA motifs for general DNA sequences (not limited to triplex or i‐motif forming sequences). We have thoroughly characterized such DNA motifs by polyacrylamide gel electrophoresis (PAGE) and fluorescence spectroscopy and demonstrated their applications in dynamic DNA nanotechnology. We expect that it will greatly facilitate the development of DNA nanomachines, biosensing/bioimaging, drug delivery, etc.

Molecular Switching of a Self-Assembled 3D DNA Nanomachine for Spatiotemporal pH Mapping in Living Cells.

DNA nanomachines have received great interest due to their potential to mimic various natural biomolecular machines. Intracellular pH sensing and imaging are of great significance to understand cellular behaviors and disease diagnostics. In this work, we report the novel molecular switching of a self-assembled 3D DNA triangular prism nanomachine (TPN) for pH sensing and imaging in living cells. The TPN was self-assembled in quantitative yields by hybridization with two DNA triangles and three I-strands (containing i-motif sequences). At acidic conditions, the TPN was compressed due to the I-strand that formed an intramolecular i-tetraplex, which was in between the fluorophores Cy3 and Cy5, resulting in a significant fluorescence resonance energy transfer (FRET) signal. At neutral or weakly alkaline conditions, the TPN adopted an extended state due to the random coil form of the I-strand, leading to spatial separation of the two fluorophores and the FRET being blocked. The TPN was fully reversible and could rapidly respond to the pH changes, entered into living cells automatically via an endocytic pathway, monitored spatiotemporal pH changes during endocytosis, maintained its structural integrity after escape from lysosomes, still had the ability for pH sensing, and also visualized pH fluctuations under varying stimuli in living cells. We foresee that this TPN can become a generic platform for a pH-related cell biology study and in disease diagnostics.

Homogeneous label-free protein binding assay using small-molecule-labeled DNA nanomachine with DNAzyme-Based chemiluminescence detection

Detecting the interactions between small molecules and proteins was critical for disease theranostics and drug development. Here we propose a novel universal assay strategy for monitoring small molecule-protein interactions in solution using strand displacement amplification (SDA) mediated by protein binding to small molecule with DNAzyme-based chemiluminescence detection. The DNA polymerase and nicking enzyme assisted SDA could yield a great amount of peroxidase-mimicking DNAzyme sequences which cause significantly chemiluminescence signals, while protein binding to the small molecule label would prevent DNA polymerase from extending nick site and DNAzyme sequence, and thus the chemiluminescence signals would obviously decrease. This strategy was demonstrated using folate and its binding protein (folate receptor), and the results revealed that the developed strategy enable offer a label-free, homogeneous, and highly sensitive chemiluminescence detection of folate receptor with a detection limit of 1pM. At the same time, it has been successfully used for folate receptor detection in human serum. The proposed chemiluminescence sensing method might provide a generic, robust, and high-throughput platform for detecting various small molecule-protein interactions for biological applications.

DNA Cascade Reaction with High-Efficiency Target Conversion for Ultrasensitive Electrochemiluminescence microRNA Detection.

DNA amplification strategy has been a valuable tool for improving the sensitivity of biosensors. However, the freely diffusing reactants in most DNA amplification strategies limit the rate of DNA reaction, which further affects the amplification efficiency with unsatisfactory sensitivity. In the present work, a novel localized DNA cascade reaction (LDCR) in a DNA nanomachine was designed for high-efficiency target conversion to construct an electrochemiluminescence (ECL) biosensor for ultrasensitive microRNA-21 detection. The DNA nanomachine was constructed by using three-footholds DNA scaffold to immobilize two metastable hairpins and reporter probe and confine them in a localized space. In the presence of microRNA-21, it initiated the LDCR and produced large amounts of mimic target (ferrocene labeled DNA, Fc-DNA) due to the locality effect. Thus, sensitive detection of microRNA-21 could be realized since Fc could effectively quench the ECL intensity of graphitic carbon nitride nanosheets (CNNS) due to the energy and electron transfer from the excited state of CNNS to oxidized species of Fc. Moreover, compared with the other two developed DNA cascade reactions with freely diffusing reactants, the proposed LDCR benefits by shortening the reaction time and improving the amplification efficiency with enhanced sensitivity of the biosensor. Therefore, the proposed LDCR could be used as a highly efficient amplification strategy for ultrasensitive determination of biomarkers with low abundance, which may promote the diagnostic efficiency of disease.

An Acidic-Microenvironment-Driven DNA Nanomachine Enables Specific ATP Imaging in the Extracellular Milieu of Tumor.

Extracellular ATP is an emerging target for cancer treatment because it is a key messenger for shaping the tumor microenvironment (TME) and regulating tumor progression. However, it remains a great challenge to design biochemical probes for targeted imaging of extracellular ATP in the TME. A TME-driven DNA nanomachine (Apt-LIP) that permits spatially controlled imaging of ATP in the extracellular milieu of tumors with ultrahigh signal-to-background ratio is reported. It operates in response to the mild acidity in the TME with the pH (low) insertion peptide (pHLIP) module, thus allowing the specific anchoring of the structure-switching signaling aptamer unit to the membrane of tumor cells for "off-on" fluorescence imaging of the extracellular ATP. Apt-LIP allows for acidity driven visualization of different extracellular concentrations of exogenous ATP, as well as the monitoring of endogenous ATP release from cells. Furthermore, it is demonstrated that Apt-LIP represents a promising platform for the specific imaging of the extracellular ATP in both primary and metastatic tumors. Ultimately, since diverse aptamers are obtained through in vitro selection, this design strategy can be further applied for precise detection of various extracellular targets in the TME.

Simply Constructed and Highly Efficient Classified Cargo-Discharge DNA Robot: A DNA Walking Nanomachine Platform for Ultrasensitive Multiplexed Sensing.

In this work, a classified cargo-discharge DNA robot with only two DNA strands was designed and driven by an analogous proximity ligation assay (aPLA)-based enzyme cleaving for fast walk to construct a novel electrochemical biosensor for simultaneously ultrasensitive detection of microRNA-155 (miRNA-155) and miRNA-21. Compared with traditional DNA nanomachines, the multifunctional DNA robot possessed simple structure, high self-assembling efficiency and walking efficiency. Once it interacted with target miRNAs, this DNA robot could walk fast on the electrode surface and realize the classified cargoes discharging including beacons methylene blue (MB) and ferrocene (Fc), respectively labeled in the double-stranded DNA (A1-A2) for ultrasensitive detection of multiple miRNAs simultaneously. As a result, the wide linearity ranging from 100 aM to 100 pM and low detection limits of 42.7 and 51.1 aM were obtained for miRNA-155 and miRNA-21 detection, respectively. As a proof of concept, the present strategy initiates a novel and highly efficient walking platform to realize the ultrasensitive detection of biomarkers and possesses potential applications in the clinical diagnosis of disease.

Construction of a Robust Entropy-Driven DNA Nanomachine for Single-Molecule Detection of Rare Cancer Cells.

Accurate and sensitive detection of rare cancer cells such as circulating tumor cells (CTCs) has attracted great interest in the fields of clinical diagnosis and cancer research. However, the reported methods for cancer cell detection often involve a complicated platform and laborious procedures with a limited sensitivity. Herein, we construct a new entropy-driven DNA nanomachine for single-molecule detection of rare cancer cells. This assay employs the entropy-driven DNA nanomachine for efficient cancer cell recognition and signal amplification without the involvement of any expensive and unstable antibodies and enzymes, and it enables one-step detection of living cancer cells with high sensitivity and good specificity. This DNA nanomachine can be further applied for rare CTC detection in whole blood samples.

Cationic comb-type copolymer as an artificial chaperone

Accurate folding of biopolymers and assembly of complexes containing these molecules are essential for biological activities. We designed cationic comb-type copolymers that act as an artificial chaperone to assist in the folding of biopolymers such as nucleic acids and peptides. The copolymers consist of a polycation backbone grafted with a high density of hydrophilic chains that allow the formation of soluble and soft interpolyelectrolyte complexes between the copolymer and oppositely charged biopolymers. The copolymers can chaperone DNA assembly into duplex, triplex, and quadruplex structures and accelerate strand exchange reactions while stabilizing DNA complexes. This chaperone activity of the copolymer is useful in nucleic acid-based nanotechnological applications such as DNA nanomachines, DNA logic gates, and DNA enzymes. The cationic comb-type copolymers also assisted in the folding of functional peptides. The basic E5 peptide folds into an alpha-helical structure and exhibits a stronger membrane-disrupting activity in the presence of the copolymer than in its absence. The cationic comb-type copolymers, as an artificial chaperone, were shown to be beneficial for enhancing the functions and facilitating the application of biopolymers in nanotechnology and medicine. The cationic comb-type copolymers, which consisted of a polycation backbone grafted with high density of hydrophilic chains, form soluble and soft interpolyelectrolyte complexes with biopolymers and act as an artificial chaperone to assist in the folding of nucleic acids and peptides. The copolymers stabilize DNA duplex, triplex, and quadruplex structures and accelerate strand exchange reactions as well as assist in the folding of functional peptides into the active conformation.

Multifunctional Fe3O4@Polydopamine@DNA-Fueled Molecular Machine for Magnetically Targeted Intracellular Zn2+ Imaging and Fluorescence/MRI Guided Photodynamic-Photothermal Therapy.

For the precise treatment of tumors, it is necessary to develop a theranostic nanoplatform that has both diagnostic and therapeutic functions. In this article, we designed a new theranostic probe for fluorescence imaging of Zn2+ and fluorescence/MRI guided magnetically targeted photodynamic-photothermal therapy. The fluorescence imaging of Zn2+ was based on an endogenous ATP-driven DNA nanomachine that could perform repetitive stand displacement reaction. It modifies all units on a single PDA/Fe3O4 nanoparticle containing a hairpin-locked initiated strand activated by a target molecule in cells, a two-stranded fuel DNA triggered by ATP, and a two-stranded DNA track responding to an initiated strand and fuel DNA. After entering the cell, the intracellular target Zn2+ initiates the nanomachine via an autocatalytic cleavage reaction, and the machine programmatically and gradually runs on the assembled DNA track via fuel DNA driving and the intramolecular toehold-mediated stand displacement reaction. The Fe3O4 core first exhibits magnetic targeting, increasing the ability of nanoparticles to enter tumor cells at the tumor site. The Fe3O4 could also be employed as a powerful magnetic resonance imaging (MRI) contrast agent and guided therapy. Using 808 nm laser and 635 nm laser irradiation together at the tumor site, the PDA nanoshell produced an excellent photothermal effect and the TMPyP4 molecules entering the cell generated reactive oxygen species, followed by cell damage. A series of reliable experiments suggested that the Fe3O4@PDA@DNA nanoprobe showed superior fluorescence specificity, MRI, a remarkable photothermal/photodynamic therapy effect, and favorable biocompatibility. This theranostic nanoplatform offered a split-new insight into tumor fluorescence and MRI diagnosis as well as effective tumor therapy.

Cis‐On/trans‐Off of DNA Hybridization with Alkylthio‐azobenzene on L‐Threoninol Responding to Visible Light

AbstractAn azobenzene modified with a highly branched secondary alkylthio group incorporated into DNA via an L‐threoninol scaffold can serve as a photo‐switch to reversibly control DNA hybridization. In the system reported here, irradiation with visible light at 400 nm causes photo‐isomerization of trans‐azobenzene to cis and 520 nm light causes the switch to the trans form. The DNA duplex is formed when the azobenzene is in the cis form (upon irradiation with 400 nm light) and is dissociated when the azobenzene is in the trans form (upon irradiation with 520 nm light). In our previously reported system, the switch required UV light. Here, visible light was used to induce both isomerizations, and a smooth trans‐to‐cis isomerization was observed below the melting temperature. This new photo‐switch with visible light overcomes one of the limitations for the potential in vivo use of our method. Combining it with the previously reported photo‐switch will allow the construction of elaborate DNA nanomachines.