DNA Framework-Engineered Assembly of Cyanine Dyes for Structural Identification of Nucleic Acids.

DNA nanostructures serve as precise templates for organizing organic dyes, enabling the creation of programmable artificial photonic systems with efficient light-harvesting and energy transfer capabilities. However, regulating the organization of organic dyes on DNA frameworks remains a great challenge. In this study, we investigated the factors influencing the self-assembly behavior of cyanine dye K21 on DNA frameworks. We observed that K21 exhibited diverse assembly modes, including monomers, H-aggregates, J-aggregates, and excimers, when combined with DNA frameworks. By manipulating conditions such as the ion concentration, dye concentration, and structure of DNA frameworks, we successfully achieved precise control over the assembly modes of K21. Leveraging K21's microenvironment-sensitive fluorescence properties on DNA nanostructures, we successfully discriminated between the chirality and topology structures of physiologically relevant G-quadruplexes. This study provides valuable insights into the factors influencing the dynamic assembly behavior of organic dyes on DNA framework nanostructures, offering new perspectives for constructing functional supramolecular aggregates and identifying DNA secondary structures.

A DNA origami device spatially controls CD95 signalling to induce immune tolerance in rheumatoid arthritis.

DNA origami is capable of spatially organizing molecules into sophisticated geometric patterns with nanometric precision. Here we describe a reconfigurable, two-dimensional DNA origami with geometrically patterned CD95 ligands that regulates immune cell signalling to alleviate rheumatoid arthritis. In response to pH changes, the device reversibly transforms from a closed to an open configuration, displaying a hexagonal pattern of CD95 ligands with ~10 nm intermolecular spacing, precisely mirroring the spatial arrangement of CD95 receptor clusters on the surface of immune cells. In a collagen-induced arthritis mouse model, DNA origami elicits robust and selective activation of CD95 death-inducing signalling in activated immune cells located in inflamed synovial tissues. Such localized immune tolerance ameliorates joint damage with no noticeable side effects. This device allows for the precise spatial control of cellular signalling, expanding our understanding of ligand-receptor interactions and is a promising platform for the development of pharmacological interventions targeting these interactions.

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.

Robotic Actuation-Mediated Quantitative Mechanogenetics for Noninvasive and On-Demand Cancer Therapy.

Cell mechanotransduction signals are important targets for physical therapy. However, current physiotherapy heavily relies on ultrasound, which is generated by high-power equipment or amplified by auxiliary drugs, potentially causing undesired side effects. To address current limitations, a robotic actuation-mediated therapy is developed that utilizes gentle mechanical loads to activate mechanosensitive ion channels. The resulting calcium influx precisely regulated the expression of recombinant tumor suppressor protein and death-associated protein kinase, leading to programmed apoptosis of cancer cell line through caspase-dependent pathway. In stark contrast to traditional gene therapy, the complete elimination of early- and middle-stage tumors (volume ≤ 100 mm3) and significant growth inhibition of late-stage tumor (500 mm3) are realized in tumor-bearing mice by transfecting mechanogenetic circuits and treating daily with quantitative robotic actuation in a form of 5 min treatment over the course of 14 days. Thus, this massage-derived therapy represents a quantitative strategy for cancer treatment.

An artificial protein modulator reprogramming neuronal protein functions.

Reversible protein phosphorylation, regulated by protein phosphatases, fine-tunes target protein function and plays a vital role in biological processes. Dysregulation of this process leads to aberrant post-translational modifications (PTMs) and contributes to disease development. Despite the widespread use of artificial catalysts as enzyme mimetics, their direct modulation of proteins remains largely unexplored. To address this gap and enable the reversal of aberrant PTMs for disease therapy, we present the development of artificial protein modulators (APROMs). Through atomic-level engineering of heterogeneous catalysts with asymmetric catalytic centers, these modulators bear structural similarities to protein phosphatases and exhibit remarkable ability to destabilize the bridging µ3-hydroxide. This activation of catalytic centers enables spontaneous hydrolysis of phospho-substrates, providing precise control over PTMs. Notably, APROMs, with protein phosphatase-like characteristics, catalytically reprogram the biological function of α-synuclein by directly hydrolyzing hyperphosphorylated α-synuclein. Consequently, synaptic function is reinforced in Parkinson's disease. Our findings offer a promising avenue for reprogramming protein function through de novo PTMs strategy.

Near-Field Terahertz Morphological Reconstruction Nanoscopy for Subsurface Imaging of Protein Layers.

Protein layers formed on solid surfaces have important applications in various fields. High-resolution characterization of the morphological structures of protein forms in the process of developing protein layers has significant implications for the control of the layer's quality as well as for the evaluation of the layer's performance. However, it remains challenging to precisely characterize all possible morphological structures of protein in various forms, including individuals, networks, and layers involved in the formation of protein layers with currently available methods. Here, we report a terahertz (THz) morphological reconstruction nanoscopy (THz-MRN), which can reveal the nanoscale three-dimensional structural information on a protein sample from its THz near-field image by exploiting an extended finite dipole model for a thin sample. THz-MRN allows for both surface imaging and subsurface imaging with a vertical resolution of ∼0.5 nm, enabling the characterization of various forms of proteins at the single-molecule level. We demonstrate the imaging and morphological reconstruction of single immunoglobulin G (IgG) molecules, their networks, a monolayer, and a heterogeneous double layer comprising an IgG monolayer and a horseradish peroxidase-conjugated anti-IgG layer. The established THz-MRN presents a useful approach for the label-free and nondestructive study of the formation of protein layers.

Enhanced Detection of Novel Low-Frequency Gene Fusions via High-Yield Ligation and Multiplexed Enrichment Sequencing.

Panel-based methods are commonly employed for the analysis of novel gene fusions in precision diagnostics and new drug development in cancer. However, these methods are constrained by limitations in ligation yield and the enrichment of novel gene fusions with low variant allele frequencies. In this study, we conducted a pioneering investigation into the stability of double-stranded adapter DNA, resulting in improved ligation yield and enhanced conversion efficiency. Additionally, we implemented blocker displacement amplification, achieving a remarkable 7-fold enrichment of novel gene fusions. Leveraging the pre-enrichment achieved with this approach, we successfully applied it to Nanopore sequencing, enabling ultra-fast analysis of novel gene fusions within one hour with high sensitivity. This method offers a robust and remarkably sensitive mean of analyzing novel gene fusions, promising the discovery of pivotal biomarkers that can significantly improve cancer diagnostics and the development of new therapeutic strategies.

Upconversion dual-photosensitizer-expressing bacteria for near-infrared monochromatically excitable synergistic phototherapy.

Synergistic phototherapy stands for superior treatment prospects than a single phototherapeutic modality. However, the combined photosensitizers often suffer from incompatible excitation mode, limited irradiation penetration depth, and lack of specificity. We describe the development of upconversion dual-photosensitizer-expressing bacteria (UDPB) for near-infrared monochromatically excitable combination phototherapy. UDPB are prepared by integrating genetic engineering and surface modification, in which bacteria are encoded to simultaneously express photothermal melanin and phototoxic KillerRed protein and the surface primary amino groups are derived to free thiols for biorthogonal conjugation of upconversion nanoparticles. UDPB exhibit a near-infrared monochromatic irradiation-mediated dual-activation characteristic as the photothermal conversion of melanin can be initiated directly, while the photodynamic effect of KillerRed can be stimulated indirectly by upconverted visible light emission. UDPB also show living features to colonize hypoxic lesion sites and inhibit pathogens via bacterial community competition. In two murine models of solid tumor and skin wound infection, UDPB separately induce robust antitumor response and a rapid wound healing effect.

Programming crystallization kinetics of self-assembled DNA crystals with 5-methylcytosine modification.

Self-assembled DNA crystals offer a precise chemical platform at the ångström-scale for DNA nanotechnology, holding enormous potential in material separation, catalysis, and DNA data storage. However, accurately controlling the crystallization kinetics of such DNA crystals remains challenging. Herein, we found that atomic-level 5-methylcytosine (5mC) modification can regulate the crystallization kinetics of DNA crystal by tuning the hybridization rates of DNA motifs. We discovered that by manipulating the axial and combination of 5mC modification on the sticky ends of DNA tensegrity triangle motifs, we can obtain a series of DNA crystals with controllable morphological features. Through DNA-PAINT and FRET-labeled DNA strand displacement experiments, we elucidate that atomic-level 5mC modification enhances the affinity constant of DNA hybridization at both the single-molecule and macroscopic scales. This enhancement can be harnessed for kinetic-driven control of the preferential growth direction of DNA crystals. The 5mC modification strategy can overcome the limitations of DNA sequence design imposed by limited nucleobase numbers in various DNA hybridization reactions. This strategy provides a new avenue for the manipulation of DNA crystal structure, valuable for the advancement of DNA and biomacromolecular crystallography.

Programming Intracellular Clustering of Spiky Nanoparticles via Liposome Encapsulation.

The intracellular clustering of anisotropic nanoparticles is crucial to the improvement of the localized surface plasmon resonance (LSPR) for phototherapy applications. Herein, we programmed the intracellular clustering process of spiky nanoparticles (SNPs) by encapsulating them into an anionic liposome via a frame-guided self-assembly approach. The liposome-encapsulated SNPs (lipo-SNPs) exhibited distinct and enhanced lysosome-triggered aggregation behavior while maintaining excellent monodispersity, even in acidic or protein-rich environments. We explored the enhancement of the photothermal therapy performance for SNPs as a proof of concept. The photothermal conversion efficiency of lipo-SNPs clusters significantly increased 15 times compared to that of single lipo-SNPs. Upon accumulation in lysosomes with a 2.4-fold increase in clustering, lipo-SNPs resulted in an increase in cell-killing efficiency to 45% from 12% at 24 µg/mL. These findings indicated that liposome encapsulation provides a promising approach to programing nanoparticle clustering at the target site, which facilitates advances in the development of smart nanomedicine with programmable enhancement in LSPR.

An intelligent DNA nanodevice for precision thrombolysis.

Thrombosis is a leading global cause of death, in part due to the low efficacy of thrombolytic therapy. Here, we describe a method for precise delivery and accurate dosing of tissue plasminogen activator (tPA) using an intelligent DNA nanodevice. We use DNA origami to integrate DNA nanosheets with predesigned tPA binding sites and thrombin-responsive DNA fasteners. The fastener is an interlocking DNA triplex structure that acts as a thrombin recognizer, threshold controller and opening switch. When loaded with tPA and intravenously administrated in vivo, these DNA nanodevices rapidly target the site of thrombosis, track the circulating microemboli and expose the active tPA only when the concentration of thrombin exceeds a threshold. We demonstrate their improved therapeutic efficacy in ischaemic stroke and pulmonary embolism models, supporting the potential of these nanodevices to provide accurate tPA dosing for the treatment of different thromboses.

Spacer-Programmed Two-Dimensional DNA Origami Assembly.

Two-dimensional (2D) DNA origami assembly represents a powerful approach to the programmable design and construction of advanced 2D materials. Within the context of hybridization-mediated 2D DNA origami assembly, DNA spacers play a pivotal role as essential connectors between sticky-end regions and DNA origami units. Here, we demonstrated that programming the spacer length, which determines the binding radius of DNA origami units, could effectively tune sticky-end hybridization reactions to produce distinct 2D DNA origami arrays. Using DNA-PAINT super-resolution imaging, we unveiled the significant impact of spacer length on the hybridization efficiency of sticky ends for assembling square DNA origami (SDO) units. We also found that the assembly efficiency and pattern diversity of 2D DNA origami assemblies were critically dependent on the spacer length. Remarkably, we realized a near-unity yield of ∼98% for the assembly of SDO trimers and tetramers via this spacer-programmed strategy. At last, we revealed that spacer lengths and thermodynamic fluctuations of SDO are positively correlated, using molecular dynamics simulations. Our study thus paves the way for the precision assembly of DNA nanostructures toward higher complexity.

Extended Enrichment for Ultrasensitive Detection of Low-Frequency Mutations by Long Blocker Displacement Amplification.

Detecting low-frequency DNA mutations hotspots cluster is critical for cancer diagnosis but remains challenging. Quantitative PCR (qPCR) is constrained by sensitivity, and allele-specific PCR is restricted by throughput. Here we develop a long blocker displacement amplification (LBDA) coupled with qPCR for ultrasensitive and multiplexed variants detection. By designing long blocker oligos to perfectly match wildtype sequences while mispairing with mutants, long blockers enable 14-44 nt enrichment regions which is 2-fold longer than normal BDA in the experiments. For wild template with a specific nucleotide, LBDA can detect different mutation types down to 0.5 % variant allele frequency (VAF) in one reaction, with median enrichment fold of 1,000 on 21 mutant DNA templates compared to the wild type. We applied LBDA-qPCR to detect KRAS and NRAS mutation hotspots, utilizing a single plex assay capable of covering 81 mutations and tested in synthetic templates and colorectal cancer tissue samples. Moreover, the mutation types were verified through Sanger sequencing, demonstrating concordance with results obtained from next generation sequencing. Overall, LBDA-qPCR provides a simple yet ultrasensitive approach for multiplexed detection of low VAF mutations hotspots, presenting a powerful tool for cancer diagnosis and monitoring.

DNA Origami-Enabled Gene Localization of Repetitive Sequences.

Repetitive sequences, which make up over 50% of human DNA, have diverse applications in disease diagnosis, forensic identification, paternity testing, and population genetic analysis due to their crucial functions for gene regulation. However, representative detection technologies such as sequencing and fluorescence imaging suffer from time-consuming protocols, high cost, and inaccuracy of the position and order of repetitive sequences. Here, we develop a precise and cost-effective strategy that combines the high resolution of atomic force microscopy with the shape customizability of DNA origami for repetitive sequence-specific gene localization. "Tri-block" DNA structures were specifically designed to connect repetitive sequences to DNA origami tags, thereby revealing precise genetic information in terms of position and sequence for high-resolution and high-precision visualization of repetitive sequences. More importantly, we achieved the results of simultaneous detection of different DNA repetitive sequences on the gene template with a resolution of ∼6.5 nm (19 nt). This strategy is characterized by high efficiency, high precision, low operational complexity, and low labor/time costs, providing a powerful complement to sequencing technologies for gene localization of repetitive sequences.

DNA as a universal chemical substrate for computing and data storage.

DNA computing and DNA data storage are emerging fields that are unlocking new possibilities in information technology and diagnostics. These approaches use DNA molecules as a computing substrate or a storage medium, offering nanoscale compactness and operation in unconventional media (including aqueous solutions, water-in-oil microemulsions and self-assembled membranized compartments) for applications beyond traditional silicon-based computing systems. To build a functional DNA computer that can process and store molecular information necessitates the continued development of strategies for computing and data storage, as well as bridging the gap between these fields. In this Review, we explore how DNA can be leveraged in the context of DNA computing with a focus on neural networks and compartmentalized DNA circuits. We also discuss emerging approaches to the storage of data in DNA and associated topics such as the writing, reading, retrieval and post-synthesis editing of DNA-encoded data. Finally, we provide insights into how DNA computing can be integrated with DNA data storage and explore the use of DNA for near-memory computing for future information technology and health analysis applications.

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.

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.

Programmed Remodeling of the Tumor Milieu to Enhance NK Cell Immunotherapy Combined with Chemotherapy for Pancreatic Cancer.

Natural killer (NK) cell-based adoptive immunotherapy has demonstrated encouraging therapeutic effects in clinical trials for hematological cancers. However, the effectiveness of treatment for solid tumors remains a challenge due to insufficient recruitment and infiltration of NK cells into tumor tissues. Herein, a programmed nanoremodeler (DAS@P/H/pp) is designed to remodel dense physical stromal barriers and for dysregulation of the chemokine of the tumor environment to enhance the recruitment and infiltration of NK cells in tumors. The DAS@P/H/pp is triggered by the acidic tumor environment, resulting in charge reversal and subsequent hyaluronidase (HAase) release. HAase effectively degrades the extracellular matrix, promoting the delivery of immunoregulatory molecules and chemotherapy drugs into deep tumor tissues. In mouse models of pancreatic cancer, this nanomediated strategy for the programmed remodeling of the tumor microenvironment significantly boosts the recruitment of NK92 cells and their tumor cell-killing capabilities under the supervision of multiplexed near-infrared-II fluorescence.

DNA Framework-Templated Fabrication of Ultrathin Electroactive Gold Nanosheets.

Generally, two-dimensional gold nanomaterials have unique properties and functions that offer exciting application prospects. However, the crystal phases of these materials tend to be limited to the thermodynamically stable crystal structure. Herein, we report a DNA framework-templated approach for the ambient aqueous synthesis of freestanding and microscale amorphous gold nanosheets with ultrathin sub-nanometer thickness. We observe that extended single-stranded DNA on DNA nanosheets can induce site-specific metallization and enable precise modification of the metalized nanostructures at predefined positions. More importantly, the as-prepared gold nanosheets can serve as an electrocatalyst for glucose oxidase-catalyzed aerobic oxidation, exhibiting enhanced electrocatalytic activity (~3-fold) relative to discrete gold nanoclusters owing to a larger electrochemical active area and wider band gap. The proposed DNA framework-templated metallization strategy is expected to be applicable in a broad range of fields, from catalysis to new energy materials.