Dynamic Assembly of DNA Nanostructures in Cancer Cells Enables the Coupling of Autophagy Activating and Real-Time Tracking.

Developing dynamic nanostructures for in situ regulation of biological processes inside living cells is of great importance in biomedical research. Herein we report the cascaded assembly of Y-shaped branched DNA nanostructure (YDN) during intracellular autophagy. YDN contains one arm with semi-i-motif sequence and Cy3-BHQ2, and another arm with an apurinic/apyrimidinic (AP) site and Cy5-BHQ3. Upon uptake by cancer cells, intermolecular i-motif structures are formed in response to lysosomal H+, causing the formation of YDN-dimer and the recovery of Cy3 fluorescence; when escapes occur from the lysosome to the cytoplasm, the YDN-dimer responds to the overexpressed APE1, leading to the assembly of YDN into the DNA network and the fluorescence recovery of Cy5. Simultaneously, the cascaded assembly activates autophagy, and thus the process of assembly of YDN and autophagy flux can be spatiotemporally coupled. This work illustrates the potential of DNA nanostructures for the in situ regulation of intracellular dynamic events with spatiotemporal control.

A DNA/Poly-(L-lysine) Hydrogel with Long Shelf-Time for 3D Cell Culture.

Deoxyribonucleic acid (DNA)-based hydrogels are emerging as promising functional materials for biomedical applications. However, the shelf-time of DNA hydrogels in biological media is severely shortened by nucleases, which limit the application of DNA hydrogels. Herein, a DNA hydrogel with long shelf-time is reported for 3D cell culture. Poly-(L-lysine) (PLL) is introduced as both a cross-linker and a protectant. The electrostatic interaction between PLL and DNA drove the formation of hydrogel. PLL coating on DNA increased the steric hindrance between DNA and nucleases, thus weakening the digestion of nucleases toward phosphodiester bond. As a result, the shelf-time of DNA/PLL hydrogel for 3D cell culture is extended from generally 1 day to longer than 15 days, which has not been achieved previously. Notably, poly-AS1411-aptamers are integrated to DNA/PLL hydrogels for anchoring U87 cells, and the cell encapsulation efficiency of the DNA/PLL hydrogels with aptamer is 4-time higher than that of the hydrogels without aptamer. DNA/PLL hydrogel provided a favorable microenvironment to support the proliferation of cells, which formed cell spheroid in 15 days. This protective coating strategy solves the long-standing problem on the shelf-time of DNA hydrogel, and is envisioned to promote the development of DNA hydrogel in more biomedical applications.

A DNA/Upconversion Nanoparticle Complex Enables Controlled Co-Delivery of CRISPR-Cas9 and Photodynamic Agents for Synergistic Cancer Therapy.

Photodynamic therapy (PDT) depends on the light-irradiated exciting of photosensitizer (PS) to generate reactive oxygen species (ROS), which faces challenges and limitations in hypoxia and antioxidant response of cancer cells, and limited tissue-penetration of light. Herein, a multifunctional DNA/upconversion nanoparticles (UCNPs) complex is developed which enables controlled co-delivery of CRISPR-Cas9, hemin, and protoporphyrin (PP) for synergistic PDT. An ultralong single-stranded DNA (ssDNA) is prepared via rolling circle amplification (RCA), which contains recognition sequences of single guide RNA (sgRNA) for loading Cas9 ribonucleoprotein (RNP), G-quadruplex sequences for loading hemin and PP, and linker sequences for combining UCNP. Cas9 RNP cleaves the antioxidant regulator nuclear factor E2-related factor 2 (Nrf2), improving the sensitivity of cancer cells to ROS, and enhancing the synergistic PDT effect. The G-quadruplex/hemin DNAzyme mimicks horseradish peroxidase (HRP) to catalyze the endogenous H2O2 to O2, overcoming hypoxia condition in tumors. The introduced UCNP converts NIR irradiation with deep tissue penetration to light with shorter wavelength, exciting PP to transform the abundant O2 to 1O2. The integration of gene editing and PDT allows substantial accumulation of 1O2 in cancer cells for enhanced cell apoptosis, and this synergistic PDT has shown remarkable therapeutic efficacy in a breast cancer mouse model.

Telomerase-Mediated Self-Assembly of DNA Network in Cancer Cells Enabling Mitochondrial Interference.

The precise control of the artificially induced reactions inside living cells is emerging as an effective strategy for the regulation of cell functions. Nevertheless, the manipulation of the assembly of exogenous molecules into artificial architectures in response to intracellular-specific signals remains a grand challenge. Herein, we achieve the precise self-assembly of deoxyribonucleic acid (DNA) network inside cancer cells, specifically responding to telomerase, and realize effective mitochondrial interference and the consequent regulation of cellular behaviors. Two functional DNA modules were designed: a mitochondria-targeting branched DNA and a telomerase-responsive linear DNA. Upon uptake by cancer cells, the telomerase primer in linear DNA responded to telomerase, and a strand displacement reaction was triggered by the reverse transcription of telomerase, thus releasing a linker DNA from the linear DNA. The linker DNA afterward hybridized with the branched DNA to form a DNA network on mitochondria. The DNA network interfered with the function of mitochondria, realizing the apoptosis of cancer cells. This system was further administered in a nude mouse tumor model, showing remarkable suppression of tumor growth. We envision that the telomerase-mediated intracellular self-assembly of the DNA network provides a promising route for cancer therapy.

Cascade dynamic assembly/disassembly of DNA nanoframework enabling the controlled delivery of CRISPR-Cas9 system.

CRISPR-Cas9 has been explored as a therapeutic agent for down-regulating target genes; the controlled delivery of Cas9 ribonucleoprotein (RNP) is essential for therapeutic efficacy and remains a challenge. Here, we report cascade dynamic assembly/disassembly of DNA nanoframework (NF) that enables the controlled delivery of Cas9 RNP. NF was prepared with acrylamide-modified DNA that initiated cascade hybridization chain reaction (HCR). Through an HCR, single-guide RNA was incorporated to NF; simultaneously, the internal space of NF was expanded, facilitating the loading of Cas9 protein. NF was designed with hydrophilic acylamino and hydrophobic isopropyl, allowing dynamic swelling and aggregation. The responsive release of Cas9 RNP was realized by introducing disulfide bond-containing N,N-bis(acryloyl)cystamine that was specifically in response to glutathione of cancer cells, triggering the complete disassembly of NF. In vitro and in vivo investigations demonstrated the high gene editing efficiency in cancer cells, the hypotoxicity in normal cells, and notable antitumor efficacy in a breast cancer mouse model.

A DNA-based hydrogel for exosome separation and biomedical applications.

Exosomes (EXOs) have been proven as biomarkers for disease diagnosis and agents for therapeutics. Great challenge remains in the separation of EXOs with high-purity and low-damage from complex biological media, which is critical for the downstream applications. Herein, we report a DNA-based hydrogel to realize the specific and nondestructive separation of EXOs from complex biological media. The separated EXOs were directly utilized in the detection of human breast cancer in clinical samples, as well as applied in the therapeutics of myocardial infarction in rat models. The materials chemistry basis of this strategy involved the synthesis of ultralong DNA chains via an enzymatic amplification, and the formation of DNA hydrogels through complementary base-pairing. These ultralong DNA chains that contained polyvalent aptamers were able to recognize and bind with the receptors on EXOs, and the specific and efficient binding ensured the selective separation of EXOs from media into the further formed networked DNA hydrogel. Based on this DNA hydrogel, rationally designed optical modules were introduced for the detection of exosomal pathogenic microRNA, which achieved the classification of breast cancer patients versus healthy donors with 100% precision. Furthermore, the DNA hydrogel that contained mesenchymal stem cell-derived EXOs was proved with significant therapeutic efficacy in repairing infarcted myocardium of rat models. We envision that this DNA hydrogel-based bioseparation system is promising as a powerful biotechnology, which will promote the development of extracellular vesicles in nanobiomedicine.

Lipid-, Inorganic-, Polymer-, and DNA-Based Nanocarriers for Delivery of the CRISPR/Cas9 system.

The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (CRISPR/Cas9) system has been widely explored for the precise manipulation of target DNA and has enabled efficient genomic editing in cells. Recently, CRISPR/Cas9 has shown promising potential in biomedical applications, including disease treatment, transcriptional regulation and genome-wide screening. Despite these exciting achievements, efficient and controlled delivery of the CRISPR/Cas9 system has remained a critical obstacle to its further application. Herein, we elaborate on the three delivery forms of the CRISPR/Cas9 system, and discuss the composition, advantages and limitations of these forms. Then we provide a comprehensive overview of the carriers of the system, and focus on the nonviral nanocarriers in chemical methods that facilitate efficient and controlled delivery of the CRISPR/Cas9 system. Finally, we discuss the challenges and prospects of the delivery methods of the CRISPR/Cas9 system in depth, and propose strategies to address the intracellular and extracellular barriers to delivery in clinical applications.

DNA-guided self-assembly in living cells.

Self-assembly processes exist widely in life systems and play essential roles in maintaining life activities. It is promising to explore the molecular fundamentals and mechanisms of life systems through artificially constructing self-assembly systems in living cells. As an excellent self-assembly construction material, deoxyribonucleic acid (DNA) has been widely used to achieve the precise construction of self-assembly systems in living cells. This review focuses on the recent progress of DNA-guided intracellular self-assembly. First, the methods of intracellular DNA self-assembly based on the conformational transition of DNA are summarized, including complementary base pairing, the formation of G-quadruplex/i-motif, and the specific recognition of DNA aptamer. Next, The applications of DNA-guided intracellular self-assembly on the detection of intracellular biomolecules and the regulation of cell behaviors are introduced, and the molecular design of DNA in the self-assembly systems is discussed in detail. Ultimately, the challenges and opportunities of DNA-guided intracellular self-assembly are commented.

DNA Nanomaterial-Based Optical Probes for Exosomal miRNA Detection.

Micro ribonucleic acids (miRNAs) in exosomes have been proven as reliable biomarkers to detect disease progression. In recent years, deoxyribonucleic acid (DNA)-based nanomaterials show great potential in the field of diagnosis due to the programmable sequence, various molecule recognition and predictable assembly/disassembly of DNA. In this review, we focus on the molecular design and detection mechanism of DNA nanomaterials, and the developed DNA nanomaterial-based optical probes for exosomal miRNA detection are summarized and discussed. The rationally-designed DNA sequences endows these probes with low background signal and high sensitivity in exosomal miRNA detection, and the detection mechanisms based on different DNA nanomaterials are detailly introduced. At the end, the challenges and future opportunities of DNA nanomaterial-based optical probes in exosomal miRNA detection are discussed. We envision that DNA nanomaterial-based optical probes will be promising in precise biomedicine.

Assembly of Rolling Circle Amplification-Produced Ultralong Single-Stranded DNA to Construct Biofunctional DNA Materials.

The Review by Yang, Yao and colleagues (DOI: 10.1002/chem.202202673) describes recent developments in biofunctional DNA hydrogels and DNA nanocomplexes based on rolling circle amplification (RCA) and introduces assembly strategies and functionalization methods of the ultralong single-strand DNA produced by RCA to construct biofunctional materials.

Dynamic assembly of DNA-ceria nanocomplex in living cells generates artificial peroxisome.

Intracellular accumulation of reactive oxygen species (ROS) leads to oxidative stress, which is closely associated with many diseases. Introducing artificial organelles to ROS-imbalanced cells is a promising solution, but this route requires nanoscale particles for efficient cell uptake and micro-scale particles for long-term cell retention, which meets a dilemma. Herein, we report a deoxyribonucleic acid (DNA)-ceria nanocomplex-based dynamic assembly system to realize the intracellular in-situ construction of artificial peroxisomes (AP). The DNA-ceria nanocomplex is synthesized from branched DNA with i-motif structure that responds to the acidic lysosomal environment, triggering transformation from the nanoscale into bulk-scale AP. The initial nanoscale of the nanocomplex facilitates cellular uptake, and the bulk-scale of AP supports cellular retention. AP exhibits enzyme-like catalysis activities, serving as ROS eliminator, scavenging ROS by decomposing H2O2 into O2 and H2O. In living cells, AP efficiently regulates intracellular ROS level and resists GSH consumption, preventing cells from redox dyshomeostasis. With the protection of AP, cytoskeleton integrity, mitochondrial membrane potential, calcium concentration and ATPase activity are maintained under oxidative stress, and thus the energy of cell migration is preserved. As a result, AP inhibits cell apoptosis, reducing cell mortality through ROS elimination.

DNA Supramolecular Assembly on Micro/Nanointerfaces for Bioanalysis.

Facing increasing demand for precision medicine, materials chemistry systems for bioanalysis with accurate molecular design, controllable structure, and adjustable biological activity are required. As a genetic biomacromolecule, deoxyribonucleic acid (DNA) is created via precise, efficient, and mild processes in life systems and can in turn precisely regulate life activities. From the perspective of materials chemistry, DNA possesses the characteristics of sequence programmability and can be endowed with customized functions by the rational design of sequences. In recent years, DNA has been considered to be a potential biomaterial for analysis and has been applied in the fields of bioseparation, biosensing, and detection imaging. To further improve the precision of bioanalysis, the supramolecular assembly of DNA on micro/nanointerfaces is an effective strategy to concentrate functional DNA modules, and thus the functions of DNA molecules for bioanalysis can be enriched and enhanced. Moreover, the new modes of DNA supramolecular assembly on micro/nanointerfaces enable the integration of DNA with the introduced components, breaking the restriction of limited functions of DNA materials and achieving more precise regulation and manipulation in bioanalysis. In this Account, we summarize our recent work on DNA supramolecular assembly on micro/nanointerfaces for bioanalysis from two main aspects. In the first part, we describe DNA supramolecular assembly on the interfaces of microscale living cells. The synthesis strategy of DNA is based on rolling-circle amplification (RCA), which generates ultralong DNA strands according to circular DNA templates. The templates can be designed with complementary sequences of functional modules such as aptamers, which allow DNA to specifically bind with cellular interfaces and achieve efficient cell separation. In the second part, we describe DNA supramolecular assembly on the interfaces of nanoscale particles. DNA sequences are designed with functional modules such as targeting, drug loading, and gene expression and then are assembled on interfaces of particles including upconversion nanoparticles (UCNPs), gold nanoparticles (AuNPs), and magnetic nanoparticle (MNPs). The integration of DNA with these functional particles achieves cell manipulation, targeted tumor imaging, and cellular regulation. The processes of interfacial assembly are well controlled, and the functions of the obtained bioanalytical materials can be flexibly regulated. We envision that the work on DNA supramolecular assembly on micro/nanointerfaces will be a typical paradigm for the construction of more bioanalytical materials, which we hope will facilitate the development of precision medicine.

Multimodules integrated functional DNA nanomaterials for intelligent drug delivery.

Deoxyribonucleic acid (DNA) has been an emerging building block to construct functional biomaterials. Due to their programmable sequences and rich responsiveness, DNA has attracted rising attention in the construction of intelligent nanomaterials with predicable nanostructure and adjustable functions, which has shown great potential in drug delivery. On the one hand, the DNA sequences with molecule recognition, responsiveness, and therapeutic efficacy can be easily integrated to the framework of DNA nanomaterials by sequence designing; on the other hand, the rich chemical groups on DNA molecules provide binding points for other functional units. In this review, we divided the functionalization modules in the construction of DNA nanomaterials into three types, including targeting modules, responsive modules, and therapeutic modules. Based on these modules, five DNA kinds of representative nanomaterials applied in drug delivery were introduced, including DNA nanogel, DNA origami, DNA framework, DNA nanoflower, and DNA hybrid nanosphere. Finally, we discussed the challenges in the transition of DNA materials to clinical applications. We expect that this review can help readers to obtain a deeper understanding of DNA materials, and further promote the development of these intelligent materials to real world's application. This article is categorized under: Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

T Lymphocyte-Captured DNA Network for Localized Immunotherapy.

The efficient isolation of immune cells with high purity and low cell damage is important for immunotherapy and remains highly challenging. We herein report a cell capture DNA network containing polyvalent multimodules for the specific isolation and in situ incubation of T lymphocytes (T-cells). Two ultralong DNA chains synthesized by an enzymatic amplification process were rationally designed to include functional multimodules as cell anchors and immune adjuvants. Mutually complementary sequences facilitated the formation of a DNA network and encapsulation of T-cells, as well as offering cutting sites of a restriction enzyme for the responsive release of T-cells and immune adjuvants. The purity of captured tumor-infiltrating T-cells reached 98%, and the viability of T-cells maintained ∼90%. The T-cells-containing DNA network was further administrated to a tumor lesion for localized immunotherapy. Our work provides a robust nanobiotechnology for efficient isolation of immune cells and other biological particles.

Rolling circle amplification (RCA)-based DNA hydrogel.

DNA hydrogels have unique properties, including sequence programmability, precise molecular recognition, stimuli-responsiveness, biocompatibility and biodegradability, that have enabled their use in diverse applications ranging from material science to biomedicine. Here, we describe a rolling circle amplification (RCA)-based synthesis of 3D DNA hydrogels with rationally programmed sequences and tunable physical, chemical and biological properties. RCA is a simple and highly efficient isothermal enzymatic amplification strategy to synthesize ultralong single-stranded DNA that benefits from mild reaction conditions, and stability and efficiency in complex biological environments. Other available methods for synthesis of DNA hydrogels include hybridization chain reactions, which need a large amount of hairpin strands to produce DNA chains, and PCR, which requires temperature cycling. In contrast, the RCA process is conducted at a constant temperature and requires a small amount of circular DNA template. In this protocol, the polymerase phi29 catalyzes the elongation and displacement of DNA chains to amplify DNA, which subsequently forms a 3D hydrogel network via various cross-linking strategies, including entanglement of DNA chains, multi-primed chain amplification, hybridization between DNA chains, and hybridization with functional moieties. We also describe how to use the protocol for isolation of bone marrow mesenchymal stem cells and cell delivery. The whole protocol takes ~2 d to complete, including hydrogel synthesis and applications in cell isolation and cell delivery.

Recent Progress of Extracellular Vesicle Engineering.

Extracellular vesicles (EVs) are nanoscale phospholipid bilayer membrane vesicles which contain varied active biomolecules. As natural carriers, EVs can deliver endogenous cargos to target tissues safely and effectively. However, the applications of natural released EVs are limited by their low yield and heterogeneity. Engineering EVs can endow them with more functions and better performances to address these issues. EVs can be modified and engineered to improve the yield, targeting efficiency, and content of beneficial cargos. Herein, the strategies of engineering EVs through genetic modification of EVs are introduced; the molecular modification of the EV membrane and the loading of nucleic acids are summarized; the building of EV mimetic nanovesicles are reviewed. Overall, we anticipate that readers will gain a better understanding of the progress of EV engineering, which will help to promote the development of the technologies and applications in this field.

Self-assembly of stem cell membrane-camouflaged nanocomplex for microRNA-mediated repair of myocardial infarction injury.

Mesenchymal stem cells-derived exosomes have shown promising therapeutic effect on myocardial infarction (MI). The major hurdles remain for the use of exosomes primarily due to the low yields from cell cultures coupled with complicated purification processes. Herein we report the self-assembly of stem cell membrane-camouflaged exosome-mimicking nanocomplex that recapitulates exosome functions, achieving efficient microRNA (miRNA) delivery and miRNA-mediated myocardial repair. The nanocomplex is constructed via the self-assembly of mesenchymal stem cell membrane on miRNA loaded mesoporous silica nanoparticle surface, which enables high miRNA loading capacity and protects miRNA from degradation in body fluid. The nanocomplex can escape the clearance of immunologic system, and target to ischemic injured cardiomyocytes. miRNA is triggered to release and binds to target mRNA, which inhibits the translation of apoptosis-related proteins, and consequently promotes the proliferation of cardiomyocytes. In the MI mouse model, the administration of exosome-mimicking nanocomplex effectively leads to preservation of viable myocardium and augmentation of cardiac functions.

Double Rolling Circle Amplification Generates Physically Cross-Linked DNA Network for Stem Cell Fishing.

Stem cells have been widely studied in cell biology and utilized in cell-based therapies, and fishing stem cells from marrow is highly challenging due to the ultralow content. Herein, a physically cross-linked DNA network-based cell fishing strategy is reported, achieving efficient capture, 3D envelop, and enzyme-triggered release of bone marrow mesenchymal stem cells (BMSCs). DNA network is constructed via a double rolling circle amplification method and through the intertwining and self-assembly of two strands of ultralong DNA chains. DNA-chain-1 containing aptamer sequences ensures specific anchor with BMSCs from marrow. Hybridization between DNA-chain-1 and DNA-chain-2 enables the cross-link of cell-anchored DNA chains to form a 3D network, thus realizing cell envelop and separation. DNA network creates a favorable microenvironment for 3D cell culture, and remarkably the physically cross-linked DNA network shows no damage to cells. DNA network is digested by nuclease, realizing the deconstruction from DNA network to fragments, and achieving enzyme-triggered cell release; after release, the activity of cells is well maintained. The strategy provides a powerful and effective method for fishing stem cells from tens of thousands of nontarget cells.

Super-Soft and Super-Elastic DNA Robot with Magnetically Driven Navigational Locomotion for Cell Delivery in Confined Space.

Soft organisms such as earthworms can access confined, narrow spaces, inspiring scientists to fabricate soft robots for in vivo manipulation of cells or tissues and minimally invasive surgery. We report a super-soft and super-elastic magnetic DNA hydrogel-based soft robot (DNA robot), which presents a shape-adaptive property and enables magnetically driven navigational locomotion in confined and unstructured space. The DNA hydrogel is designed with a combinational dynamic and permanent crosslinking network through chain entanglement and DNA hybridization, resulting in shear-thinning and cyclic strain properties. DNA robot completes a series of complex magnetically driven navigational locomotion such as passing through narrow channels and pipes, entering grooves and itinerating in a maze by adapting and recovering its shape. DNA robot successfully works as a vehicle to deliver cells in confined space by virtue of the 3D porous networked structure and great biocompatibility.

Luminescent Ultralong Microfibers Prepared through Supramolecular Self-Assembly of Lanthanide Ions and Thymidine in Water.

Luminescent materials are of great interest in many fields, such as fluorescent sensing and medical imaging. Here, the construction of lanthanide-based luminescent ultralong microfibers through supramolecular self-assembly (SSA) is reported. Nucleosides (thymidine in particular), the building blocks of nucleic acids, were used as new ligands to mediate the formation of luminescent microfibers in water. The length of microfibers from thymidine-lanthanide ion (Eu and Tb) SSA was on the centimeter scale. Notably, the microfibers exhibited strong luminescence because the lanthanide ions had been chelated, sensitized and effectively protected by thymidine molecules in water. Only when the stoichiometric ratio of lanthanide ion to thymidine was 1:3 and the pH of the solution was 7, are luminescent microfibers formed. Other nucleosides, such as adenosine, cytidine, and guanosine, could not form microfibers with the lanthanide ions. This work opens a new avenue for constructing nucleoside-lanthanide SSA architectures, which hold great potential in biological and optical related applications.