Recent advancements in DNA nanotechnology-enabled extracellular vesicles detection and diagnosis:A mini review

Extracellular vesicles(EVs)are cell-derived nanosized vesicles widely recognized for their critical roles in various pathophysiological processes.Molecular analysis of EVs is currently being considered an emerging tool for diseases diagnosis.However,the small size and heterogeneity of EVs has staggered the EVs research for diseases diagnosis.DNA nanotechnology enables self-assembly of versatile DNA nanostructures and has shown enormous potential in assisting EVs biosensing.In this review,we briefly introduce the recent advances in DNA nanotechnology approaches for EVs detection.The approaches were categorized based on the dimension of DNA nanostructures.We provide critical evaluation of these approaches,and summarize the pros and cons of specific methods.Further,we discuss the challenges and future perspectives in this field.

DNA Nanotechnology for Multimodal Synergistic Theranostics

Over the past decade,DNA nanotechnology has developed rapidly due to its unique characteristics,such as excellent biocompatibility,high programmability,good predictability,automatically chemical synthesis,and so on.So far,a variety of DNA-based nanostructures,from small to large and simple to complex,have been designed and synthesized with controllable size and shape in one,two,or three dimensions.Therefore,DNA has become a kind of competitive materials for biosensing,bioimaging and biomedicine.In particular,the integration of DNA nanotechnology with multimodal synergistic theranostics can not only achieve accurate cancer diagnosis by the sensitive and accurate detection of cancer biomarkers,but also achieve enhanced anti-cancer therapeutic efficacy,which promote the development of DNA nanotechnology and nanomedicine.In this review,we first give a comprehensive introduction of DNA nanotechnology,and then summarize the DNA self-assembly and amplification strategies for the construction of functional nanoplatforms for multimodal synergistic theranostics.Finally,the challenges and opportunities faced by DNA nanotechnology in biomedicine are discussed.

Applications of DNA Nanotechnology in Synthesis and Assembly of Inorganic Nanomaterials

In addition to its inherited genetic function, DNA is one of the smartest and most flexible self-assembling na- nomaterials with programmable and predictable features, for which, more and more scientists combine DNA with nanomaterials and put them into designing, synthesizing and assembling. In this review, four modes of action of DNA molecules are introduced in a figurative and intuitive way, based on the four different roles it plays in synthe- sis and assembly of nanomaterials： （a） smart linkers to guide nanoparticle assembly, （b） 2D or 3D scaffold with well-designed binding sites, （c） nucleation sites to directly facilitate Au/Pd/Ag/Cu nanowires, nanoparticles, nano- arrays and （d） serving as capping agents to prevent crystal growth, and control size and morphology. To be sure, this state-of-the-art combination of functional DNA molecules and inorganic nanomaterials greatly encouraged step towards the development of analytical science, life science, environmental science, and other promising field they can address. DNA-guided nanofabrication will eventually exceed expectations far beyond our scope in the near fu- ture.

Gel electrophoresis as a nanoseparation tool serving DNA nanotechnology

The past years have witnessed a rapid development of DNA nanotechnology in nanomaterials science with a central focus on programmable material construction on the nanoscale. An efficient method is therefore highly desirable（but challenging） for analytical/preparative purification of DNA-conjugated nano-objects and their DNA-assemblies. In this regard, agarose gel electrophoresis, a traditional technique that has been invented for biomacromolecule separation, has found many innovative uses.This includes shape, size, charge, and ligand-valence separations of nanoparticle building blocks as well as monitoring a self-assembly process towards product identification and purification.

Hybridization chain reaction-based DNA nanomaterials for biosensing,bioimaging and therapeutics

DNA nanomaterials hold great promise in biomedical fields due to its excellent sequence programmability,molecular recognition ability and biocompatibility.Hybridization chain reaction(HCR)is a simple and efficient isothermal enzyme-free amplification strategy of DNA,generating nicked double helices with repeated units.Through the design of HCR hairpins,multiple nanomaterials with desired functions are assembled by DNA,exhibiting great potential in biomedical applications.Herein,the recent progress of HCR-based DNA nanomaterials for biosensing,bioimaging and therapeutics are summarized.Representative works are exemplified to demonstrate how HCR-based DNA nanomaterials are designed and constructed.The challenges and prospects of the development of HCR-based DNA nanomaterials are discussed.We envision that rationally designing HCR-based DNA nanomaterials will facilitate the development of biomedical applications.

Allosteric DNAzyme-based encoder for molecular information transfer

Dynamic DNA nanotechnology plays a significant role in nanomedicine and information science due to its high programmability based on Watson-Crick base pairing and nanoscale dimensions.Intelligent DNA machines and networks have been widely used in various fields,including molecular imaging,biosensors,drug delivery,information processing,and logic operations.Encoders serve as crucial components for information compilation and transfer,allowing the conversion of information from diverse application scenarios into a format recognized and applied by DNA circuits.However,there are only a few encoder designs with DNA outputs.Moreover,the molecular priority encoder is hardly designed.In this study,we introduce allosteric DNAzyme-based encoders for information transfer.The design of the allosteric domain and the recognition arm allows the input and output to be independent of each other and freely programmable.The pre-packaged mode design achieves uniformity of baseline dynamics and dynamics controllability.We also integrated non-nucleic acid molecules into the encoder through the aptamer design of the allosteric domain.Furthermore,we developed the 2^(n)-n encoder and the EndoⅣ-assisted priority encoder inspired by immunoglobulin's molecular structure and effector patterns.To our knowledge,the proposed encoder is the first enzyme-free DNA encoder with DNA output,and the priority encoder is the first molecular priority encoder in the DNA reaction network.Our encoders avoid complex operations on a single molecule,and their simple structure facilitates their application in complex DNA circuits and biological scenarios.

A DNA micro-complex containing polyaptamer for exosome separation and wound healing

Exosomes(EXOs)have showed great potential in regenerative medicine.The separation of EXOs from complex biological media is essential for the down-stream applications.Herein,we report a deoxyribonucleic acid(DNA)-based micro-complex(DMC)containing polyaptamers,which realized the specific separation of EXOs from cell culture media and the significant promotion of wound healing.The synthesis of DMCs was based on a biomineralization process via rolling circle amplification(RCA)under the catalysis of phi29 DNA polymerase.To endow DMCs with the ability to capture EXOs,the DNA template of RCA was integrated with complementary sequence of aptamer that specifically recognized the CD63 proteins on EXOs.The obtained DMCs contained polyaptamers that can specifically capture the EXOs in cell culture media.The EXOs-capturing DMCs were collected by centrifugation,achieving the separation of EXOs.Mesenchymal stem cell(MSC)-derived EXOs(MSC-EXOs)were separated by this DMC-based strategy,and the separated MSC-EXOs significantly enhanced the migration ability of cells.In particular,the significant therapeutic efficacy of the DMCs with MSC-EXOs was verified in full-thickness wound excision mouse models,in which the wounds completely healed in 10 days.We envision that this DMC-based separation strategy can be a promising route to promote the development of EXOs in biomedicine.

Self-assembly multifunctional DNA tetrahedron for efficient elimination of antibiotic-resistant bacteria

Antibiotic resistance is a major challenge in the clinical treatment of bacterial infectious diseases.Herein,we constructed a multifunctional DNA nanoplatform as a versatile carrier for bacteria-specific delivery of clinical antibiotic ciprofloxacin(CIP)and classic nanoantibiotic silver nanoparticles(AgNP).In our rational design,CIP was efficiently loaded in the self-assembly double-bundle DNA tetrahedron through intercalation with DNA duplex,and single-strand DNA-modified AgNP was embedded in the cavity of the DNA tetrahedron through hybridization.With the site-specific assembly of targeting aptamer in the well-defined DNA tetrahedron,the bacteria-specific dual-antibiotic delivery system exhibited excellent combined bactericidal properties.With enhanced antibiotic accumulation through breaking the out membrane of bacteria,the antibiotic delivery system effectively inhibited biofilm formation and promoted the healing of infected wounds in vivo.This DNAbased antibiotic delivery system provides a promising strategy for the treatment of antibiotic-resistant infections.

Light-Mediated Spatiotemporally Dynamic Assembly of DNA Nanostructures in Living Cells Regulates Autophagy

The assembly of exogenous artificial architectures inside cells can regulate a series of biological events,which heavily relies on the development of spatiotemporally controlled molecular assembly systems.We herein report a designer deoxyribonucleic acid(DNA)nanostructure that enables light-mediated spatiotemporally dynamic assembly in living cells and consequently achieves efficient regulation of cell autophagy.The DNA nanostructure was constructed from i-motif moiety-containing branched DNA,photocleavable bond-containing linker,and tumor cell-targeting aptamer.After cellular uptake mediated by aptamers,under the spatiotemporal control of both UV light and late endosomal/lysosomal acidic environments,disassembly/reassembly of DNA nanostructure occurred via two rationally designed routes,generating microsized DNA assembly.As a result,autophagy was significantly enhanced with the increase of DNA assembly size.The enhanced autophagy showed an impact on related biological effects.Our system is expected to be a powerful tool for the regulation of intracellular events and cellular behaviors.

Supramolecular Polymerization of DNA Double-Crossover-Like Motifs in Various Dimensions

Double-crossover-like(DXL)molecules are a series of DNA motifs containing two strands with identical or different sequences.These homo-or hetero-dimers can further polymerize into bulk structures through specific hydrogen bonding between sticky ends.DXL molecules have high designability,predictivity and sequence robustness;and their supramolecular polymerization products would easily achieve controllable morphology.In addition,among all available DNA nanomotifs,DXL molecules are small in size so that the cost of DXL-based nanostructures is low.These properties together make DXL-based nanostructures good candidates for patterning,templating,information and matter storage,etc.Herein,we will discuss DXL motifs in terms of the detailed molecular design,and their supramolecular polymerization in various dimensions,and related applications.

DNA-based supramolecular hydrogels:From construction strategies to biomedical applications

DNA-based supramolecular hydrogels are important and promising biomaterials for various applications due to their inherent biocompatibility and tunable physicochemical properties.The three-dimensional supramolecular matrix of DNA formed by non-covalently dynamic cross-linking provides exceptional adaptability,self-healing,injectable and responsive properties for hydrogels.In addition,DNA hydrogels are also ideal bio-scaffold materials owing to their tissue-like mechanics and intrinsic biological functions.Technically,DNA can assemble into supramolecular networks by pure complementary base pairing;it can also be combined with other building blocks to construct hybrid hydrogels.This review focuses on the development and construction strategies of DNA hydrogels.Assembly and synthesis methods,diverse responsiveness and biomedical applications are summarized.Finally,the challenges and prospects of DNA-based supramolecular hydrogels are discussed.

DNA Reaction Network Mimicking the Basic Steps of Synaptic Communication

Biological systems use intricate networks of chemical reactions to exchange information. How to simulate complex systems with simple strand-displacement reactions is crucial to broaden the application scenario of the DNA reaction network. Here, we report the artificial DNA reaction network to mimic the operation and function of biological information transfer via strand-displacement reaction. DNA is used as simple artificial analogs to schematize structures and transmit information. Using chemical synapses in neural networks as an example, we show that the proposed network enables core functions of biological systems, such as the long-term potential of synapses, which underpin learning and memory. Also, we performed the simple “silicon mimetic” to link electronic circuits to chemical network-based biological structures. As such, synaptic communication simulated by the DNA reaction network provides a complete demonstration for designing artificial reaction networks based on the essence of information interaction.

Locked nucleic acids based DNA circuits with ultra-low leakage

DNA circuits based on toehold-mediated DNA strand displacement reaction are powerful tools owing to their programmability and predictability.However,performance and practical application of the circuits are greatly restricted by leakage,which refers to the fact that there is no input(invading strand)in the circuit,and the output signal is still generated.Herein,we constructed locked nucleic acids-based DNA circuits with ultra-low leakage.High binding affinity of LNA(locked nucleic acid)-DNA/LNA suppressed the leakage by inhibiting the breathing effect.Based on the strategy,we have built various low-leakage DNA circuits,including translator circuit,catalytic hairpin assembly(CHA)circuit,entropy-driven circuit(EDC),and seesaw circuit.More importantly,our strategy would not affect the desired main reactions:The output signal remained above 85%for all tested circuits,and the signalto-noise ratios were elevated to 148.8-fold at the most.We believe our strategy will greatly promote the development and application of DNA circuits-based DNA nanotechnology.

Construction of Polymeric DNA Network and Application for Cell Manipulation

Comprehensive Summary Deoxyribonucleic acid(DNA)is a biomacromolecule,as well as a polymeric material,whose sequences with different manipulative structures enable them to implement a series of functions,such as reorganization,target,and catalysis.Compared to existing traditional materials incapable of multifunctional integration,the polymeric DNA network is a form of material that can achieve functional integration while maintaining specific DNA properties.Furthermore,precise target enabled by DNA network is one of the most essential components of cellular manipulation.Hence,the DNA network is indispensable and irreplaceable to cell manipulation that it is a versatile tool for the understanding of basic laws of living life and treatments of diseases,such as cell isolation,cell delivery,and cell interference.Herein,the construction of polymeric DNA network is briefly introduced from the aspects of assembly modules,construction methods,and properties.

Aptamer-based Membrane Protein Analysis and Molecular Diagnostics

Membrane proteins are vital components of the cell membrane and play crucial roles in various cellular activities.Analysis of membrane proteins is of paramount importance for studying molecular events inside cells and organisms and holds promising prospects for early disease diagnosis and treatment assessment.Benefiting from obvious merits including high affinity,high specificity and ease of modification,aptamers have been regarded as ideal molecular recognition elements in membrane protein analysis and molecular diagnostics strategies.This review summarised recent advances in membrane protein-specific aptamer screening,aptamer-based static and dynamic membrane protein analysis,and aptamer-based molecular diagnostic techniques.Prospects and challenges were also discussed.

Self-assembled multifunctional DNA nanoflowers for the circumvention of multidrug resistance in targeted anticancer drug delivery

Cancer chemotherapy has been limited by its side effects and multidrug resistance （MDR）, the latter of which is partially caused by drug efflux from cancer cells. Thus, targeted drug delivery systems that can circumvent MDR are needed. Here, we report multifunctional DNA nanoflowers （NFs） for targeted drug delivery to both chemosensitive and MDR cancer cells that circumvented MDR in both leukemia and breast cancer cell models. NFs are self-assembled via potential co-precipitation of DNA and magnesium pyrophosphate generated by rolling circle replication, during which NFs are incorporated using aptamers for specific cancer cell recognition, fluorophores for bioimaging, and doxorubicin （Dox）- binding DNA for drug delivery. NF sizes are tunable （down to N200 nm in diameter）, and the densely packed drug-binding motifs and porous intrastructures endow NFs with a high drug-loading capacity （71.4%, wt/wt）. Although the Dox- loaded NFs （NF-Dox） are stable at physiological pH, drug release is facilitated under acidic or basic conditions. NFs deliver Dox into target chemosensitive and MDR cancer cells, preventing drug efflux and enhancing drug retention in MDR cells. NF-Dox induces potent cytotoxicity in both target chemosensitive cells and MDR cells, but not in nontarget cells, thus concurrently circumventing MDR and reducing side effects. Overall, these NFs are promising tools for circumventing MDR in targeted cancer therapy.

Self-Assembled DNA Nanostructures for Drug Delivery

Due to the uniform nanoscale sizes, well-defined shapes, precise spatial addressability and prominent biocom- patibility, self-assembled DNA nanostructures have been intensively studied for their biomedical applications. This review summarizes the recent development ofDNA nanotechnology in cancer therapy, and discusses the challenges and potential strategies to advance the methodologies of cancer treatments.

A smart DNA nanodevice for ATP-activatable bioimaging and photodynamic therapy

Rational design of activatable photosensitizers for controlled generation of singlet oxygen remains a challenge for precise photodynamic therapy(PDT).Herein,we present an aptamer-based nanodevice for adenosine 5′-triphosphate(ATP)-activatable bioimaging and PDT.The nanodevice is constructed by modifying ATP-responsive duplex DNA units and polyethylene glycol on the surface of a gold nanoparticle(AuNP)through the thiolate-Au chemistry.The DNA units were designed by the hybridization of the ATP aptamer strand with a methylene blue(MB)-modified complementary DNA(cDNA).The close proximity of MB to the surface of AuNP results in the low photodynamic activity of MB(OFF state).Once internalized into cancer cells,the ATP-binding induced conformation switch of aptamer strand leads to the release of the MB-bearing DNA strand from AuNPs,resulting in the activatable generation of singlet oxygen under light irradiation(ON state).We demonstrate that the DNA nanodevice represents a promising platform for ATP-responsive bioimaging and specific PDT in vitro and in vivo.This work highlights a potential way for specific tumor diagnosis and therapy.

Construction and applications of DNA-based nanomaterials in cancer therapy

As a biologically active macromolecule, deoxyribonucleic acid(DNA) has the advantages of sequence programmability and structure controllability and can accurately transmit sequence information to specific biological functions. Facing the complex internal microenvironment and heterogeneity in tumor treatment, the construction and applications of DNA-based nanomaterials have become a focus point of research. In particular, the hybridization of DNA molecules with other materials endows DNA-based nanomaterials with multiple functions such as targeting, stimulus responsiveness and regulations of biological activities, making DNA nanostructures great potential in the treatment of major human diseases.In this review, the construction and characteristics of DNA-based nanomaterials are introduced. Then,the functions and applications of DNA-based nanomaterials in the delivery of chemotherapy drugs and gene drugs, stimulus-responsive release and regulation of cell homeostasis are reviewed. Finally, the future development and challenges of DNA-based nanomaterials are prospected. We envision that DNAbased nanomaterials can enrich the nanomaterial system by rational design and synthesis and address the growing demands on biological and biomedical applications in the real world.

Supramolecular Wireframe DNA Polyhedra： Assembly and Applications

Wireframe, polyhedral, supramolecular complexes made of DNA have uniform sizes, defined three-dimensional shapes, porous facets, hollow interiors, good biocompatibilities, and chemical functionalizability. They confer great potentials in bottom-up nanoengineering towards various applications. In this review, we summarize recent ad- vances in the rational design and programmed assembly of DNA wireframe polyhedra. Their assembly is based on three distinctively different strategies： individual strands-based assembly, tile-based assembly, and scaffolded DNA origami. Applications of these polyhedral structures in templated nanomaterial assembly and in-vivo cargo delivery are discussed. In the future, expanding the structural complexity and exploring their applications, especially in na- nomaterials science and biomedicines, should be a primary focus of this rapidly developing and evolving activity of structural DNA nanotechnology.