A DNA Nanodevice-Based Platform with Diverse Capabilities.

Social biotic colonies often perform intricate tasks by interindividual communication and cooperation. Inspired by these biotic behaviors, a DNA nanodevice community is proposed as a universal and scalable platform. The modular nanodevice as the infrastructure of platform contains a DNA origami triangular prism framework and a hairpin-swing arm machinery core. By coding and decoding a signal domain on the shuttled output strand in different nanodevices, an orthogonal inter-nanodevice communication network is established to connect multi-nanodevices into a functional platform. The nanodevice platform enables implementation of diverse tasks, including signal cascading and feedback, molecular input recording, distributed logic computing, and modeling of simulation for virus transmission. The nanodevice platform with powerful compatibility and programmability presents an elegant example of the combination of the distributed operation of multiple devices and the complicated interdevice communication network, and may become a new generation of intelligent DNA nanosystems.

Stimuli-Responsive DNA Origami Nanodevices and Their Biological Applications.

DNA origami nanotechnology has provided predictable static nanoarchitectures and dynamic nanodevices with rationally designed geometries, precise spatial addressability, and marked biocompatibility. Multiple functional elements, such as peptides, aptamers, nanoparticles, fluorescence probes, and proteins, etc. can be easily integrated into DNA origami templates with nanoscale precision, leading to a variety of promising applications. Triggered by chemical/physical stimuli, dynamic DNA origami nanodevices can switch between defined conformations or translocate autonomously, providing powerful tools for intelligent biosensing and drug delivery. In this minireview, we summarize the recent progress of dynamic DNA origami nanodevices with desired reconfigurability and feasibility to perform multiple biological tasks. We introduce varieties of DNA nanodevices that can be controlled by different molecular triggers and external stimuli. Subsequently, we highlight the recent advances in employing DNA nanodevices as biosensors and drug delivery vehicles. At last, future possibilities and perspectives are also discussed.

A Multivariate-Gated DNA Nanodevice for Spatioselective Imaging of Pro-metastatic Targets in Extracellular Microenvironment.

Probing pro-metastatic biomarkers is of significant importance to evaluate the risk of tumor metastasis, but spatially selective imaging of such targets in extracellular microenvironment is particularly challenging. By introducing the bilinguality of PNA/peptide hybrid that can speak both peptide substrate and nucleobase-pairing languages to combine with aptamer technology, we designed a smart DNA nanodevice programmed to respond sequentially to dual pro-metastatic targets, MMP2/9 and ATP, in extracellular tumor microenvironment (TME). The DNA nanodevice is established based on the combination of an ATP-responsive aptamer sensor and a MMP2/9-hydrolyzable PNA/peptide copolymer with a cell membrane-anchoring aptamer module. Taking 4T1 xenograft as a highly aggressive tumor model, the robustness of the DNA nanodevice in spatioselective imaging of MMP2/9 and ATP in TME is demonstrated. We envision that this design will enable the simultaneous visualization of multiple pro-metastatic biomarkers, which allows to gain insights into their pathological roles in tumor metastasis.

A Redox-Activatable DNA Nanodevice for Spatially-Selective, AND-Gated Imaging of ATP and Glutathione in Mitochondria.

Design of biosensors capable of imaging ATP and glutathione (GSH) in mitochondria remains a challenge, despite their importance in elucidating their correlated pathophysiological events. Here, we report a new strategy that uses redox-activatable aptamer sensor design combined with nanoparticle-based targeting capability to achieve spatially controlled, AND-gated imaging of ATP and GSH in mitochondria. The DNA nanodevice was designed by the controlled assembly of the redox-responsive ATP aptamer probe on the nanoparticles and further decorated with mitochondria-targeting signals. We demonstrate that the system allows for mitochondria-specific, correlated imaging of ATP and GSH in living cells and in vivo. Furthermore, because the system can be lighted up only when meeting the "dual keys" (overexpressed ATP and GSH in mitochondria) simultaneously, the DNA nanodevice enables specific imaging of tumors in vivo with improved tumor-to-normal tissue ratio. This work illustrates the potential of the DNA nanodevices in the imaging of mitochondrial multivariate targets.

Multifunctional Clip Strand for the Regulation of DNA Strand Displacement and Construction of Complex DNA Nanodevices.

Strand displacement reactions are important bricks for the construction of various DNA nanodevices, among which the toehold-mediated strand displacement reaction is the most prevalently adopted. However, only a limited number of tools could be used to finely regulate the toehold reaction, thus restricting DNA nanodevices from being more multifunctional and powerful. Herein, we developed a regulation tool, Clip, and achieved multiple regulatory functions, including subtle adjustment of the reaction rates, allosteric strand displacement, selective activation, and resetting of the reaction. Taking advantages of the multiple functions, we constructed Clip-toehold-based DNA walking machines. They showed behaviors of controllable walking, concatenation, and programmable pathways. Furthermore, we built Clip-toehold-based AND and OR logic gates and integrated those logic gates to construct multilayer circuits, which could be reset and reused to process different input signals. We believe that the proposed Clip tool has expanded the functionality of DNA strand displacement-based nanodevices to a much more complex and diverse level and anticipate that the tool will be widely adopted in DNA nanotechnology.

A Cascade Signal Amplification Based on Dynamic DNA Nanodevices and CRISPR/Cas12a Trans-cleavage for Highly Sensitive MicroRNA Sensing.

The variations of microRNA (miRNA) expression can be valuable biomarkers in disease diagnosis and prognosis. However, current miRNA detection techniques mainly rely on reverse transcription and template replication, which suffer from slowness, contamination risk, and sample loss. To address these limitations, here we introduce a cascade toehold-mediated strand displacement reaction (CTSDR) and CRISPR/Cas12a trans-cleavage for highly sensitive fluorescent miRNA sensing, namely CTSDR-Cas12a. In this work, the target miRNA hybridizes with the terminal toehold site of a rationally designed probe and subsequently initiates dynamic CTSDR, leading to enzyme-free target recycling and the production of multiple programmable DNA duplexes. The obtained DNA duplex acts as an activator to trigger Cas12a trans-cleavage, generating significantly amplified fluorescence readout for highly sensitive detection of the miRNA target. Under the optimal conditions, the developed sensing method can detect target miRNA down to 70.28 fM with a wide linear range from 100 fM to 100 pM. In particular, by designing a set of probes and crRNAs, we demonstrate its broad applicability for the detection of six kinds of miRNAs with high sequence specificity. Furthermore, the method can be satisfactorily applied to monitor miR-21 in total RNA extracted from cells and clinical serum samples. Considering the high sensitivity, specificity, universality, and ease of handling, this strategy provides a great potential platform for the detection of miRNA biomarkers in molecular diagnostic practice.

Aptamer-Programmed DNA Nanodevices for Advanced, Targeted Cancer Theranostics.

DNA has been demonstrated to be a versatile material for construction at the nanoscale. DNA nanodevices are highly programmable and allow functionalization with multiple entities such as imaging modalities (fluorophores), targeting entities (aptamers), drug conjugation (chemical linkers), and triggered release (photoresponsive molecules). These features enhance the use of DNA nanodevices in biological applications, catalyzing the rapid growth of this domain of research. In this review, we focus on recent progress in the development and use of aptamer-functionalized DNA nanodevices as theranostic agents, their characterization, applications as delivery platforms, and advantages. We provide a brief background on the development of aptamers and DNA nanodevices in biomedical applications, and we present specific applications of these entities in cancer diagnosis and therapeutics. We conclude with a perspective on the challenges and possible solutions for the clinical translation of aptamer-functionalized DNA nanodevices in the domain of cancer therapeutics.

Spatiotemporal Dynamics of Endocytic Pathways Adapted by Small DNA Nanocages in Model Neuroblastoma Cell-Derived Differentiated Neurons.

Multiple endocytic pathways operate on the plasma membrane of cells at any moment with diverse but specific cellular functions. Knowledge of uptake of synthetic nanoparticles and ligands with respect to endocytic pathways is crucial to device the appropriate ligands for therapeutic delivery into differentiated neurons for targeting neuronal diseases. We herein explore the mechanisms of cellular uptake of 3D tetrahedral DNA nanocages at different stages of differentiating neurons. We monitored the uptake, kinetics, and dynamics of DNA cages of different geometries, and interestingly we find a specific pattern and adaptability of the uptake of DNA devices with respect to the geometry of the ligand and specific endocytic pathways. We find that tetrahedral DNA nanocages get endocytosed mostly via clathrin-mediated endocytosis in fully mature neurons. This endocytic uptake and intracellular choreography of DNA nanodevices will help us design the smartly targeted biotherapeutics for targeting neuronal disorders.

In situ Analysis of Cancer Cells Based on DNA Signal Amplification and DNA Nanodevices.

Cancer is a global disease which has been disturbing researchers in medicine and seriously threatens patients' health and lifetime around the world in the past several decades. Due to the characteristics of cancer cells, such as uncontrollable cell proliferation, cell invasion and metastasis to surrounding tissues, lower grade of differentiation, higher telomerase activity and others, it has been one of the most usual lethal factors, next to heart disease in incidence. Cancer mortality can be decreased by early diagnosis, and the people who with treatment at an early stage have an obvious improved survival rate. Consequently, early detection is significant for better understanding the pathogenesis of cancer and improving the prognosis of patients. In situ detection technique is a vital tool for imaging and cellular pathology research, which can provide effective information about tumor markers in the early cancer detection. In view of low expression of most tumor markers in the early stage of cancers, detection techniques based on DNA signal amplification and DNA nanodevices can provide a strong support for the diagnosis and detection of cancers. In this review, we summarize the research progress of different analytical techniques for detecting various tumor markers that have been reported in recent years. We compare different DNA amplification and nanodevices, then provide guidance and suggestions for better understanding in situ analysis of cancer cells.

Recent advances on functional nucleic acid-based biosensors for detection of food contaminants.

It has seen increasing development of reliable, robust, and flexible biosensors for rapid food-safety analysis in the past few decades. Recently, functional nucleic acid-based biosensors have attracted attention because of their programmability, bottom-up characteristics, and structural switches. However, few systematic reviews devoted to categorizing the potential of DNA nanostructures and devices were found for detecting food contaminants. Hence, the applications of functional nucleic acid-based biosensors were reviewed for analyzing food contaminants, including foodborne pathogen bacteria, biotoxins, heavy metals, and et al. In addition to categorizing the various biosensors, multiple signal readout strategies, such as optical, electrochemical, and mass-based signals were also examined. Finally, the future changes and potential opportunities, as well as practical applications of functional nucleic acid-based biosensors were discussed.

Processing DNA-Based Molecular Signals into Graphical Displays.

DNA is now well-established as a nanoscale building material with applications in fields such as biosensing and molecular computation. Molecular processes such as logic gates, nucleic acid circuits, and multiplexed detection have used different readout strategies to measure the output signal. In biosensing, this output can be the diagnosis of a disease biomarker, whereas in molecular computation, the output can be the result of a mathematical operation carried out using DNA. Recent developments have shown that the output of such processes can be displayed graphically as a macroscopic symbol or an alphanumeric character on multiwell plates, microarray chips, gels, lateral flow devices, and DNA origami surfaces. This review discusses the concepts behind such graphical readouts of molecular events, available display platforms, and the advantages and challenges in adapting such methods for practical use. Graphical display systems have the potential to be used in the creation of intelligent computing and sensing devices by which nanoscale binding events are translated into macroscopic visual readouts.

DNA Nanodevices for Base Excision Repair Regulates ATP In Situ Imaging and Tumor Therapy.

The design of DNA nanodevices has attracted broad attention in detecting specific targets and targeted drug delivery capacities of tumor cells. Here, we report the facile fluorometric method of dual-targeting DNA nanodevices for base excision repair (BER) regulates adenosine triphosphate (ATP) in situ imaging and tumor therapy that can counteract the mutagenic effects of uracil (U) on ATP aptamer based on the binding of U-containing damaged ATP aptamer. We prove that the DNA nanodevices not only effectively deliver the aptamer probe and tumor therapy but also able to analyze the overexpression of APE1 and uracil-DNA glycosylases (UDG) in the BER pathway via ATP in situ imaging in tumor cells. Therefore, the DNA nanodevices of the BER pathway provide the potential for tumor theranostics.

Dynamic DNA nanostructures in biomedicine: Beauty, utility and limits.

DNA composite materials are at the forefront, especially for biomedical science, as they can increase the efficacy and safety of current therapies and drug delivery systems. The specificity and predictability of the Watson-Crick base pairing make DNA an excellent building material for the production of programmable and multifunctional objects. In addition, the principle of nucleic acid hybridization can be applied to realize mobile nanostructures, such as those reflected in DNA walkers that sort and collect cargo on DNA tracks, DNA robots performing tasks within living cells and/or DNA tweezers as ultra-sensitive biosensors. In this review, we present the diversity of dynamic DNA nanostructures functionalized with different biomolecules/functional units, imaging smart biomaterials capable of sensing, interacting, delivery and performing complex tasks within living cells/organisms.

Electronic Activation of a DNA Nanodevice Using a Multilayer Nanofilm.

A method to control activation of a DNA nanodevice by supplying a complementary DNA (cDNA) strand from an electro-responsive nanoplatform is reported. To develop functional nanoplatform, hexalayer nanofilm is precisely designed by layer-by-layer assembly technique based on electrostatic interaction with four kinds of materials: Hydrolyzed poly(ß-amino ester) can help cDNA release from the film. A cDNA is used as a key building block to activate DNA nanodevice. Reduced graphene oxides (rGOs) and the conductive polymer provide conductivity. In particular, rGOs efficiently incorporate a cDNA in the film via several interactions and act as a barrier. Depending on the types of applied electronic stimuli (reductive and oxidative potentials), a cDNA released from the electrode can quantitatively control the activation of DNA nanodevice. From this report, a new system is successfully demonstrated to precisely control DNA release on demand. By applying more advanced form of DNA-based nanodevices into multilayer system, the electro-responsive nanoplatform will expand the availability of DNA nanotechnology allowing its improved application in areas such as diagnosis, biosensing, bioimaging, and drug delivery.

Dissecting the hybridization of oligonucleotides to structured complementary sequences.

BACKGROUND: When oligonucleotides hybridize to long target molecules, the process is slowed by the secondary structure in the targets. The phenomenon has been analyzed in several previous studies, but many details remain poorly understood. METHODS: I used a spectrofluorometric strategy, focusing on the formation/breaking of individual base pairs, to study the kinetics of association between a DNA hairpin and >20 complementary oligonucleotides ('antisenses'). RESULTS: Hybridization rates differed by over three orders of magnitude. Association was toehold-mediated, both for antisenses binding to the target's ends and for those designed to interact with the loop. Binding of these latter, besides being consistently slower, was affected to variable, non-uniform extents by the asymmetric loop structure. Divalent metal ions accelerated hybridization, more pronouncedly when nucleation occurred at the loop. Incorporation of locked nucleic acid (LNA) residues in the antisenses substantially improved the kinetics only when LNAs participated to the earliest hybridization steps. The effects of individual LNAs placed along the antisense indicated that the reaction transition state occurred after invading at least the first base pair of the stem. CONCLUSIONS: The experimental approach helps dissect hybridization reactions involving structured nucleic acids. Toehold-dependent, nucleation-invasion models appear fully appropriate for describing such reactions. Estimating the stability of nucleation complexes formed at internal toeholds is the major hurdle for the quantitative prediction of hybridization rates. GENERAL SIGNIFICANCE: While analyzing the mechanisms of a fundamental biochemical process (hybridization), this work also provides suggestions for the improvement of technologies that rely on such process.

Designer DNA Architectures: Applications in Nanomedicine.

DNA has been used as a material for the construction of nanoscale objects. These nanostructures are programmable and allow the conjugation of biomolecular guests to improve their functionality. DNA nanostructures display a wide variety of characteristics, such as cellular permeability, biocompatibility and stability, and responsiveness to external stimuli, making them excellent candidates for applications in nanomedicine.

Decoding the conformation-linked functional properties of nucleic acids by the use of computational tools.

DNA and RNA are large and flexible polymers selected by nature to transmit information. The most common DNA three-dimensional structure is represented by the double helix, but this biopolymer is extremely flexible and polymorphic, and can easily change its conformation to adapt to different interactions and purposes. DNA can also adopt singular topologies, giving rise, for instance, to supercoils, formed because of the limited free rotation of the DNA domain flanking a replication or transcription complex. Our understanding of the importance of these unusual or transient structures is growing, as recent studies of DNA topology, supercoiling, knotting and linking have shown that the geometric changes can drive, or strongly influence, the interactions between protein and DNA, so altering its own metabolism. On the other hand, the unique self-recognition properties of DNA, determined by the strict Watson-Crick rules of base pairing, make this material ideal for the creation of self-assembling, predesigned nanostructures. The construction of such structures is one of the main focuses of the thriving area of DNA nanotechnology, where several assembly strategies have been employed to build increasingly complex DNA nanostructures. DNA nanodevices can have direct applications in biomedicine, but also in the materials science field, requiring the immersion of DNA in an environment far from the physiological one. Crucial help in the understanding and planning of natural and artificial nanostructures is given by modern computer simulation techniques, which are able to provide a reliable structural and dynamic description of nucleic acids.