DNA Framework-Programmed Nanoscale Enzyme Assemblies.

Multienzyme assemblies mediated by multivalent interaction play a crucial role in cellular processes. However, the three-dimensional (3D) programming of an enzyme complex with defined enzyme activity in vitro remains unexplored, primarily owing to limitations in precisely controlling the spatial topological configuration. Herein, we introduce a nanoscale 3D enzyme assembly using a tetrahedral DNA framework (TDF), enabling the replication of spatial topological configuration and maintenance of an identical edge-to-edge distance akin to natural enzymes. Our results demonstrate that 3D nanoscale enzyme assemblies in both two-enzyme systems (glucose oxidase (GOx)/horseradish peroxidase (HRP)) and three-enzyme systems (amylglucosidase (AGO)/GOx/HRP) lead to enhanced cascade catalytic activity compared to the low-dimensional structure, resulting in ∼5.9- and ∼7.7-fold enhancements over homogeneous diffusional mixtures of free enzymes, respectively. Furthermore, we demonstrate the enzyme assemblies for the detection of the metabolism biomarkers creatinine and creatine, achieving a low limit of detection, high sensitivity, and broad detection range.

Concept and Development of Algebraic Topological Framework Nucleic Acids.

Nucleic acids are considered as promising materials for developing exquisite nanostructures from one to three dimensions. The advances of DNA nanotechnology facilitate ingenious design of DNA nanostructures with diverse shapes and sizes. Especially, the algebraic topological framework nucleic acids (ATFNAs) are functional DNA nanostructures that engineer guest molecules (e. g., nucleic acids, proteins, small molecules, and nanoparticles) stoichiometrically and spatially. The intrinsic precise properties and tailorable functionalities of ATFNAs hold great promise for biological applications, such as cell recognition and immunotherapy. This Perspective highlights the concept and development of precisely assembled ATFNAs, and outlines the new frontiers and opportunities for exploiting the structural advantages of ATFNAs for biological applications.

Proximity labeling-assisted click conjugation for electrochemical analysis of specific subpopulations in circulating extracellular vesicles.

Sensitive and accurate analysis of specific subpopulations in circulating extracellular vesicles (EVs) can provide a wealth of information for cancer diagnosis and management. Thus, we propose herein a new electrochemical biosensing method based on a proximity labeling-assisted click conjugation strategy. The method's core design is use of antibody-guided proximity labeling to equip target EVs with a large amount of alkyne groups, so that azide-tagged silver nanoparticles can be accurately loaded onto target EV surfaces, via click conjugation, to generate significant electrochemical responses. Adopting CD44-positive EVs as the model, the electrochemical method was demonstrated by analyzing target EVs across a wide linear range (103-109 particles/mL) with acceptable sensitivity and specificity. Satisfactory utility in clinical blood samples, and versatility with human epidermal growth factor receptor-2-positive EVs as alternative targets, were also shown. This method may thus provide a novel approach to specific subgroup analyses of circulating EVs, and is expected to offer reliable guidance for cancer diagnoses and management strategies.

High-throughput DNA synthesis for data storage.

With the explosion of digital world, the dramatically increasing data volume is expected to reach 175 ZB (1 ZB = 1012 GB) in 2025. Storing such huge global data would consume tons of resources. Fortunately, it has been found that the deoxyribonucleic acid (DNA) molecule is the most compact and durable information storage medium in the world so far. Its high coding density and long-term preservation properties make itself one of the best data storage carriers for the future. High-throughput DNA synthesis is a key technology for "DNA data storage", which encodes binary data stream (0/1) into quaternary long DNA sequences consisting of four bases (A/G/C/T). In this review, the workflow of DNA data storage and the basic methods of artificial DNA synthesis technology are outlined first. Then, the technical characteristics of different synthesis methods and the state-of-the-art of representative commercial companies, with a primary focus on silicon chip microarray-based synthesis and novel enzymatic DNA synthesis are presented. Finally, the recent status of DNA storage and new opportunities for future development in the field of high-throughput, large-scale DNA synthesis technology are summarized.

Twisted DNA Origami-Based Chiral Monolayers for Spin Filtering.

DNA monolayers with inherent chirality play a pivotal role across various domains including biosensors, DNA chips, and bioelectronics. Nonetheless, conventional DNA chiral monolayers, typically constructed from single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), often lack structural orderliness and design flexibility at the interface. Structural DNA nanotechnology has emerged as a promising solution to tackle these challenges. In this study, we present a strategy for crafting highly adaptable twisted DNA origami-based chiral monolayers. These structures exhibit distinct interfacial assembly characteristics and effectively mitigate the structural disorder of dsDNA monolayers, which is constrained by a limited persistence length of ∼50 nm of dsDNA. We highlight the spin-filtering capabilities of seven representative DNA origami-based chiral monolayers, demonstrating a maximal one-order-of-magnitude increase in spin-filtering efficiency per unit area compared with conventional dsDNA chiral monolayers. Intriguingly, our findings reveal that the higher-order tertiary chiral structure of twisted DNA origami further enhances the spin-filtering efficiency. This work paves the way for the rational design of DNA chiral monolayers.

Directing the Encapsulation of Single Cells with DNA Framework Nucleator-Based Hydrogel Growth.

Encapsulating individual mammalian cells with biomimetic materials holds potential in ex vivo cell culture and engineering. However, current methodologies often present tradeoffs between homogeneity, stability, and cell compatibility. Here, inspired by bacteria that use proteins stably anchored on their outer membranes to nucleate biofilm growth, we develop a single-cell encapsulation strategy by using a DNA framework structure as a nucleator (DFN) to initiate the growth of DNA hydrogels under cell-friendly conditions. We find that among the tested structures, the tetrahedral DFN can evenly and stably reside on cell membranes, effectively initiating hybridization chain reactions which generate homogeneously dense yet flexible single-cell encapsulation for diverse cell lines. The encapsulation persists for up to 72 hours in a serum-containing cell culture environment, representing a ~70-fold improvement compared to encapsulations mediated by single-stranded DNA nucleators. The metabolism and proliferation of the encapsulated cells are suppressed, but can be restored to the original efficiencies upon release, suggesting the superior cell compatibility of the encapsulation. We also find that compared to naked cells, the encapsulated cells exhibit a lower autophagy level after undergoing mechanical stress, suggesting the protective effect of the DNA encapsulation. This method may provide a new tool for ex vivo cell engineering.

Functionalized tetrahedral DNA frameworks for the capture of circulating tumor cells.

Identification and characterization of circulating tumor cells (CTCs) from blood samples of patients with cancer can help monitor parameters such as disease stage, disease progression and therapeutic efficiency. However, the sensitivity and specificity of current multivalent approaches used for CTC capture is limited by the lack of control over the ligands' position. In this Protocol Update, we describe DNA-tetrahedral frameworks anchored with aptamers that can be configured with user-defined spatial arrangements and stoichiometries. The modified tetrahedral DNA frameworks, termed 'n-simplexes', can be used as probes to specifically target receptor-ligand interactions on the cell membrane. Here, we describe the synthesis and use of n-simplexes that target the epithelial cell adhesion molecule expressed on the surface of CTCs. The characterization of the n-simplexes includes measuring the binding affinity to the membrane receptors as a result of the spatial arrangement and stoichiometry of the aptamers. We further detail the capture of CTCs from patient blood samples. The procedure for the preparation and characterization of n-simplexes requires 11.5 h, CTC capture from clinical samples and data processing requires ~5 h per six samples and the downstream analysis of captured cells typically requires 5.5 h. The protocol is suitable for users with basic expertise in molecular biology and handling of clinical samples.

Construction of Double-enzyme Complexes with DNA Framework Nanorulers for Improving Enzyme Cascade Catalytic Efficiency.

Efficient biocatalytic cascade reactions play a crucial role in guiding intricate, specific and selective intracellular transformation processes. However, the catalytic activity of the enzyme cascade reaction in bulk solution was greatly impacted by the spatial morphology and inter-enzyme distance. The programmability and addressability nature of framework nucleic acid (FNA) allows to be used as scaffold for immobilization and to direct the spatial arrangement of enzyme cascade molecules. Here, we used tetrahedral DNA framework (TDF) as nanorulers to assemble two enzymes for constructing a double-enzyme complex, which significantly enhance the catalytic efficiency of sarcosine oxidase (SOx)/horseradish peroxidase (HRP) cascade system. We synthesized four types of TDF nanorulers capable of programming the lateral distance between enzymes from 5.67 nm to 12.33 nm. Enzymes were chemical modified by ssDNA while preserving most catalytic activity. Polyacrylamide gel electrophoresis (PAGE), transmission electron microscopy (TEM) and atomic force microscopy (AFM) were used to verify the formation of double-enzyme complex. Four types of double-enzyme complexes with different enzyme distance were constructed, in which TDF26 (SOx+HRP) exhibited the highest relative enzyme cascade catalytic activity, ~3.11-fold of free-state enzyme. Importantly, all the double-enzyme complexes demonstrate a substantial improvement in enzyme cascade catalytic activity compared to free enzymes.

A Programmable DNAzyme for the Sensitive Detection of Nucleic Acids.

Nucleic acids in biofluids are emerging biomarkers for the molecular diagnostics of diseases, but their clinical use has been hindered by the lack of sensitive detection assays. Herein, we report the development of a sensitive nucleic acid detection assay named SPOT (sensitive loop-initiated DNAzyme biosensor for nucleic acid detection) by rationally designing a catalytic DNAzyme of endonuclease capability into a unified one-stranded allosteric biosensor. SPOT is activated once a nucleic acid target of a specific sequence binds to its allosteric module to enable continuous cleavage of molecular reporters. SPOT provides a highly robust platform for sensitive, convenient and cost-effective detection of low-abundance nucleic acids. For clinical validation, we demonstrated that SPOT could detect serum miRNAs for the diagnostics of breast cancer, gastric cancer and prostate cancer. Furthermore, SPOT exhibits potent detection performance over SARS-CoV-2 RNA from clinical swabs with high sensitivity and specificity. Finally, SPOT is compatible with point-of-care testing modalities such as lateral flow assays. Hence, we envision that SPOT may serve as a robust assay for the sensitive detection of a variety of nucleic acid targets enabling molecular diagnostics in clinics.

Data Storage Using DNA.

The exponential growth of global data has outpaced the storage capacities of current technologies, necessitating innovative storage strategies. DNA, as a natural medium for preserving genetic information, has emerged as a highly promising candidate for next-generation storage medium. Storing data in DNA offers several advantages, including ultrahigh physical density and exceptional durability. Facilitated by significant advancements in various technologies, such as DNA synthesis, DNA sequencing, and DNA nanotechnology, remarkable progress has been made in the field of DNA data storage over the past decade. However, several challenges still need to be addressed to realize practical applications of DNA data storage. In this review, the processes and strategies of in vitro DNA data storage are first introduced, highlighting recent advancements. Next, a brief overview of in vivo DNA data storage is provided, with a focus on the various writing strategies developed to date. At last, the challenges encountered in each step of DNA data storage are summarized and promising techniques are discussed that hold great promise in overcoming these obstacles.

Reconstruction of Vesicle Assemblies with DNA Nanorulers for Resolving Heterogeneity of Vesicles in Live Cells.

Nanoscale vesicles such as synaptic vesicles play a pivotal role in efficient interneuronal communications in vivo. However, the coexistence of single vesicle and vesicle clusters in living cells increases the heterogeneity of vesicle populations, which largely complicates the quantitative analysis of the vesicles. The high spatiotemporal monitoring of vesicle assemblies is currently incompletely resolved. Here, this work uses synthetic vesicles and DNA nanorulers to reconstruct in vitro the vesicle assemblies that mimic vesicle clusters in living cells. DNA nanorulers program the lateral distance of vesicle assemblies from 3 to 10 nm. This work uses the carbon fiber nanoelectrode (CFNE) to amperometric monitor artificial vesicle assemblies with sub-10 nm interspaces, and obtain a larger proportion of complex events. This work resolves the heterogeneity of individual vesicle release kinetics in PC12 cells with the temporal resolution down to ≈0.1 ms. This work further analyzes the aggregation state of intracellular vesicles and the exocytosis of living cells with electrochemical vesicle cytometry. The results indicate that the exocytosis of vesicle clusters is critically dependent on the size of clusters. This technology has the potential as a tool to shed light on the heterogeneity analysis of vesicle populations.

Programmable Atom-Like Nanoparticle Reporters for High-Precision Urinalysis of In Situ Membrane Proteins.

The expression of disease-specific membrane proteins (MPs) is a crucial indicator for evaluating the onset and progression of diseases. Urinalysis of in situ MPs has the potential for point-of-care disease diagnostics, yet remains challenging due to the lack of molecular reporter to transform the expression information of in situ MPs into the measurable urine composition. Herein, a series of tetrahedral DNA frameworks (TDFs) are employed as the cores of programmable atom-like nanoparticles (PANs) to direct the self-assembly of PAN reporters with defined ligand valence and spatial distribution. With the rational spatial organization of ligands, the interaction between PAN reporters and MPs exhibits superior stability on cell-membrane interface under renal tubule-mimic fluid microenvironment, thus enabling high-fidelity conversion of MPs expression level into binding events and reverse assessment of in situ MP levels via measurement of the renal clearance efficiency of PAN reporters. Such PAN reporter-mediated signal transformation mechanism empowers urinalysis of the onset of acute kidney injury at least 6 h earlier than the existing methods with an area under the curve of 100%. This strategy has the potential for urinalysis of a variety of in situ membrane proteins.

Programmable DNA Hydrogel Assisting Microcrystal Formulations for Sustained Locoregional Drug Delivery in Surgical Residual Tumor Lesions and Lymph Node Metastasis.

Surgical residual tumor lesions (R1 resection of surgical procedures (e.g., liver cancer infiltrating the diaphragm, surgical residual breast cancer, postoperative residual ovarian cancer) or boundary residual after ablation) and lymph node metastasis that cannot be surgically resected (retroperitoneal lymph nodes) significantly affect postoperative survival of tumor patients. This clinical conundrum poses three challenges for local drug delivery systems: stable and continuous delivery, good biocompatibility, and the ability to package new targeted drugs that can synergize with other treatments. Here, a drug-laden hydrogel generated from pure DNA strands and highly programmable in adjusting its mesh size is reported. Meanwhile, the DNA hydrogel can assist the microcrystallization of novel radiosensitizing drugs, ataxia telangiectasia and rad3-related protein (ATR) inhibitor (Elimusertib), further facilitating its long-term release. When applied to the tumor site, the hydrogel system demonstrates significant antitumor activity, minimized systemic toxicity, and has a modulatory effect on the tumor-immune cell interface. This drug-loaded DNA-hydrogel platform represents a novel modality for adjuvant therapy in patients with surgical residual tumor lesions and lymph node metastasis.

DNA-based programmable gate arrays for general-purpose DNA computing.

The past decades have witnessed the evolution of electronic and photonic integrated circuits, from application specific to programmable1,2. Although liquid-phase DNA circuitry holds the potential for massive parallelism in the encoding and execution of algorithms3,4, the development of general-purpose DNA integrated circuits (DICs) has yet to be explored. Here we demonstrate a DIC system by integration of multilayer DNA-based programmable gate arrays (DPGAs). We find that the use of generic single-stranded oligonucleotides as a uniform transmission signal can reliably integrate large-scale DICs with minimal leakage and high fidelity for general-purpose computing. Reconfiguration of a single DPGA with 24 addressable dual-rail gates can be programmed with wiring instructions to implement over 100 billion distinct circuits. Furthermore, to control the intrinsically random collision of molecules, we designed DNA origami registers to provide the directionality for asynchronous execution of cascaded DPGAs. We exemplify this by a quadratic equation-solving DIC assembled with three layers of cascade DPGAs comprising 30 logic gates with around 500 DNA strands. We further show that integration of a DPGA with an analog-to-digital converter can classify disease-related microRNAs. The ability to integrate large-scale DPGA networks without apparent signal attenuation marks a key step towards general-purpose DNA computing.

Meta-DNA Strand Displacement for Sub-Micron-Scale Autonomous Reconfiguration.

Dynamic molecular interactions in chemical reaction networks lead to complex behaviors in living systems. Whereas recent advances in programming DNA molecular reactions have reached a high level of complexity at molecular and nanometer scales, achieving programmable autonomous behavior at submicron or even larger scales remains challenging. Here, we present a mechanism of meta-DNA strand displacement reactions (M-SDRs) that is mediated solely by meta-toehold (M-toehold) using versatile submicron building blocks of meta-DNA (M-DNA). M-SDR emulates the toehold binding and branch migration processes of conventional strand displacement. Importantly, the kinetics of M-SDR can be modulated over a range of five orders of magnitude reaching a maximum rate of about 1.62 × 105 M-1 s-1. Further, we demonstrate the use of M-SDR to program autonomous reconfiguration in information transmission and logical computation systems. We envision that M-SDR serves as a versatile mechanism for emulating autonomous behavior approaching the cellular level.

Edge Length-Programmed Single-Stranded RNA Origami for Predictive Innate Immune Activation and Therapy.

Ligands targeting nucleic acid-sensing receptors activate the innate immune system and play a critical role in antiviral and antitumoral therapy. However, ligand design for in situ stability, targeted delivery, and predictive immunogenicity is largely hampered by the sophisticated mechanism of the nucleic acid-sensing process. Here, we utilize single-stranded RNA (ssRNA) origami with precise structural designability as nucleic acid sensor-based ligands to achieve improved biostability, organelle-level targeting, and predictive immunogenicity. The natural ssRNAs self-fold into compact nanoparticles with defined shapes and morphologies and exhibit resistance against RNase digestion in vitro and prolonged retention in macrophage endolysosomes. We find that programming the edge length of ssRNA origami can precisely regulate the degree of macrophage activation via a toll-like receptor-dependent pathway. Further, we demonstrate that the ssRNA origami-based ligand elicits an anti-tumoral immune response of macrophages and neutrophils in the tumor microenvironment and retards tumor growth in the mouse pancreatic tumor model. Our ssRNA origami strategy utilizes structured RNA ligands to achieve predictive immune activation, providing a new solution for nucleic acid sensor-based ligand design and biomedical applications.

A Membrane Tension-Responsive Mechanosensitive DNA Nanomachine.

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

Framework-Hotspot Enhanced Trans Cleavage of CRISPR-Cas12a for Clinical Samples Detection.

The trans-cleavage property of CRISPR-Cas12a system makes it an excellent tool for disease diagnosis. Nevertheless, most methods based on CRISPR-Cas system still require pre-amplification of the target to achieve the desired detection sensitivity. Here we generate Framework-Hotspot reporters (FHRs) with different local densities to investigate their effect on trans-cleavage activity of Cas12a. We find that the cleavage efficiency increases and the cleavage rate accelerates with increasing reporter density. We further construct a modular sensing platform with CRISPR-Cas12a-based target recognition and FHR-based signal transduction. Encouragingly, this modular platform enables sensitive (100 fM) and rapid (<15 min) detection of pathogen nucleic acids without pre-amplification, as well as detection of tumor protein markers in clinical samples. The design provides a facile strategy for enhanced trans cleavage of Cas12a, which accelerates and broadens its applications in biosensing.

A new paradigm in transplant immunology: At the crossroad of synthetic biology and biomaterials.

Solid organ transplant (SOT) recipients require meticulously tailored immunosuppressive regimens to minimize graft loss and mortality. Traditional approaches focus on inhibiting effector T cells, while the intricate and dynamic immune responses mediated by other components remain unsolved. Emerging advances in synthetic biology and material science have provided novel treatment modalities with increased diversity and precision to the transplantation community. This review investigates the active interface between these two fields, highlights how living and non-living structures can be engineered and integrated for immunomodulation, and discusses their potential application in addressing the challenges in SOT clinical practice.

DNA-framework-based multidimensional molecular classifiers for cancer diagnosis.

A molecular classification of diseases that accurately reflects clinical behaviour lays the foundation of precision medicine. The development of in silico classifiers coupled with molecular implementation based on DNA reactions marks a key advance in more powerful molecular classification, but it nevertheless remains a challenge to process multiple molecular datatypes. Here we introduce a DNA-encoded molecular classifier that can physically implement the computational classification of multidimensional molecular clinical data. To produce unified electrochemical sensing signals across heterogeneous molecular binding events, we exploit DNA-framework-based programmable atom-like nanoparticles with n valence to develop valence-encoded signal reporters that enable linearity in translating virtually any biomolecular binding events to signal gains. Multidimensional molecular information in computational classification is thus precisely assigned weights for bioanalysis. We demonstrate the implementation of a molecular classifier based on programmable atom-like nanoparticles to perform biomarker panel screening and analyse a panel of six biomarkers across three-dimensional datatypes for a near-deterministic molecular taxonomy of prostate cancer patients.