Plasmonic nanomaterials bearing targeting ligands are of great interest for surface-enhanced Raman scattering (SERS)-based bioimaging applications. However, the practical utility of SERS-based imaging strategies has been hindered by the lack of a straightforward method to synthesize highly sensitive SERS-active nanostructures with high yield and efficiency. In this work, leveraging DNA origami principles, we report the first-in-class design of a SERS-based plasmonically coupled nanoprobe for targeted cancer imaging (SPECTRA). The nanoprobe harnesses a cancer cell targeting DNA aptamer sequence and vibrational tag with stretching frequency in the cell-silent Raman window. Through the integration of aptamer sequence specific for DU145 cells, we show the unique capabilities of SPECTRA for targeted imaging of DU145 cells. Our results demonstrate that the scalability, cost-effectiveness, and reproducibility of this method of fabrication of SERS nanoprobes can serve as a versatile platform for creating nanoprobes with broad applications in the fields of cancer biology and biomedical imaging.

Monotherapy, especially the use of antibodies targeting vascular endothelial growth factor (VEGF), has shown limitations in treating choroidal neovascularization (CNV) since reactive oxygen species (ROS) also exacerbate CNV formation. Herein, we developed a combination therapy based on a DNA origami platform targeting multiple components of ocular neovascularization. Our study demonstrated that ocular neovascularization was markedly suppressed by intravitreal injection of a rectangular DNA origami sheet modified with VEGF aptamers (Ap) conjugated to an anti-VEGF antibody (aV) via matrix metalloproteinase (MMP)-cleavable peptide linkers in a mouse model of CNV. Typically, the DNA origami-based therapeutic platform selectively accumulates in neovascularization lesions owing to the dual-targeting ability of the aV and Ap, followed by the cleavage of the peptide linker by MMPs to release the antibody. Together, the released antibody and Ap inhibited VEGF activity. Moreover, the residual bare DNA origami could effectively scavenge ROS, reducing oxidative stress at CNV sites and thus maximizing the synergistic effects of inhibiting neovascularization.

The engineering of enzymatic activity generally involves alteration of the protein primary sequences, which introduce structural changes that give rise to functional improvements. Mechanical forces have been used to interrogate protein biophysics, leading to deep mechanistic insights in single-molecule studies. Here, we use simple DNA springs to apply small pulling forces to perturb the active site of a thermostable alcohol dehydrogenase. Methods were developed to enable the study of different spring lengths and spring orientations under bulk catalysis conditions. Tension applied across the active site expanded the binding pocket volume and shifted the preference of the enzyme for longer chain-length substrates, which could be tuned by altering the spring length and the resultant applied force. The substrate specificity changes did not occur when the DNA spring was either severed or rotated by ∼90°. These findings demonstrate an alternative approach in protein engineering, where active site architectures can be dynamically and reversibly remodeled using applied mechanical forces.

DNA condensates, formed by liquid-liquid phase separation (LLPS), emerge as promising soft matter assemblies for creating artificial cells. The advantages of DNA condensates are their molecular permeability through the surface due to their membrane-less structure and their fluidic property. However, they face challenges in the design of their surface, e.g., unintended fusion and less regulation of permeable molecules. Addressing them, we report surface modification of DNA condensates with DNA origami nanoparticles, employing a Pickering-emulsion strategy. We successfully constructed core-shell structures with DNA origami coatings on DNA condensates and further enhanced the condensate stability toward fusion via connecting DNA origamis by responding to DNA input strands. The \'armoring\' prevented the fusion of DNA condensates, enabling the formation of multicellular-like structures of DNA condensates. Moreover, the permeability was altered through the state change from coating to armoring the DNA condensates. The armored DNA condensates have significant potential for constructing artificial cells, offering increased surface stability and selective permeability for small molecules while maintaining compartmentalized space and multicellular organization.

The detection of a single-enzyme catalytic reaction by surfaced-enhanced Raman scattering (SERS) is presented by utilizing DNA origami-based plasmonic antennas. A single horseradish peroxidase (HRP) was accommodated on a DNA origami nanofork plasmonic antenna (DONA) containing gold nanoparticles, enabling the tracing of single-molecule SERS signals during the peroxide reduction reaction. This allows monitoring of the structure of a single enzymatic catalytic center and products under suitable liquid conditions. Herein, we demonstrate the chemical changes of HRP and the appearance of tetramethylbenzidine (TMB), which works as a hydrogen donor before and after the catalytic reaction. The results show that the iron in HRP adopts Fe4+ and low spin states with the introduction of H2O2, indicating compound-I formation. Density functional theory (DFT) calculations were performed for later catalytic steps to rationalize the experimental Raman/SERS spectra. The presented data provide several possibilities for tracking single biomolecules in situ during a chemical reaction and further developing plasmon-enhanced biocatalysis.

Renal ischemia-reperfusion injury (IRI) is a major contributing factor to the development of acute kidney injury (AKI) and has resulted in considerable morbidity and mortality. Persistent inflammatory responses and excessive reactive oxygen species (ROS) in the kidney following IRI can severely delay tissue repair, making it challenging to effectively promote IRI regeneration. Herein, we report an approach to enhance immunotherapy using interleukin-10 (IL-10) to promote IRI regeneration by loading IL-10 onto rectangular DNA origami nanostructures (rDON). rDON can significantly enhance the renal accumulation and retention time of IL-10, enabling it to effectively polarize type 1 macrophages into type 2 macrophages, thereby significantly reducing proinflammatory factors and increasing anti-inflammatory factors. In addition, DNA origami helps mitigate the harmful effects of ROS during renal IRI. The administration of IL-10-loaded DNA origami effectively improves kidney function, resulting in a notable reduction in blood urea nitrogen, serum uric acid, and serum creatinine levels. Our study demonstrates that the integration of anti-inflammatory cytokines within DNA origami holds promise as a strategic approach for cytokine immunotherapy in patients with AKI and other renal disorders.

Biological nanopores crucially control the import and export of biomolecules across lipid membranes in cells. They have found widespread use in biophysics and biotechnology, where their typically narrow, fixed diameters enable selective transport of ions and small molecules, as well as DNA and peptides for sequencing applications. Yet, due to their small channel sizes, they preclude the passage of large macromolecules, e.g., therapeutics. Here, the unique combined properties of DNA origami nanotechnology, machine-inspired design, and synthetic biology are harnessed, to present a structurally reconfigurable DNA origami MechanoPore (MP) that features a lumen that is tuneable in size through molecular triggers. Controllable switching of MPs between 3 stable states is confirmed by 3D-DNA-PAINT super-resolution imaging and through dye-influx assays, after reconstitution of the large MPs in the membrane of liposomes via an inverted-emulsion cDICE technique. Confocal imaging of transmembrane transport shows size-selective behavior with adjustable thresholds. Importantly, the conformational changes are fully reversible, attesting to the robust mechanical switching that overcomes pressure from the surrounding lipid molecules. These MPs advance nanopore technology, offering functional nanostructures that can be tuned on-demand - thereby impacting fields as diverse as drug delivery, biomolecule sorting, and sensing, as well as bottom-up synthetic biology.

Staphylococcus aureus (SA) poses a serious risk to human and animal health, necessitating a low-cost and high-performance analytical platform for point-of-care diagnostics. Cellulose paper-based field-effect transistors (FETs) with RNA-cleaving DNAzymes (RCDs) can fulfill the low-cost requirements, however, its high hydrophilicity and lipophilicity hinder biochemical modification and result in low sensitivity, poor mechanical stability and poor fouling performance. Herein, we proposed a controllable self-cleaning FET to simplify biochemical modification and improve mechanical stability and antifouling performance. Then, we constructed an RCD-based DNA nanotree to significantly enhance the sensitivity for SA detection. For controllable self-cleaning FET, 1 H,1 H,2 H,2 H-perfluorodecyltrimethoxysilane based-polymeric nanoparticles were synthesized to decorate cellulose paper and whole carbon nanofilm wires. O2 plasma was applied to regulate to reduce fluorocarbon chain density, and then control the hydrophobic-oleophobic property in sensitive areas. Because negatively charged DNA affected the sensitivity of semiconducting FETs, three Y-shaped branches with low-cost were designed and applied to synthesize an RCD-based DNA-Nanotree based on similar DNA-origami technology, which further improved the sensitivity. The trunk of DNA-Nanotree was composed of RCD, and the canopy was self-assembled using multiple Y-shaped branches. The controllable self-cleaning FET biosensor was applied for SA detection without cultivation, which had a wide linear range from 1 to 105 CFU/mL and could detect a low value of 1 CFU/mL.

Circular dichroism (CD) spectroscopy has been extensively utilized for detecting and distinguishing the chirality of diverse substances and structures. However, CD spectroscopy is inherently weak and conventionally associated with chiral sensing, thus constraining its range of applications. Here, we report a DNA-origami-empowered metasurface sensing platform through the collaborative effect of metasurfaces and DNA origami, enabling achiral/slightly chiral sensing with high sensitivity via the enhanced ΔCD. An anapole metasurface, boasting over 60 times the average optical chirality enhancement, was elaborately designed to synergize with reconfigurable DNA origami. We experimentally demonstrated the detection of achiral/slightly chiral DNA linker strands via the enhanced ΔCD of the proposed platform, whose sensitivity was a 10-fold enhancement compared with the platform without metasurfaces. Our work presents a high-sensitivity platform for achiral/slightly chiral sensing through chiral spectroscopy, expanding the capabilities of chiral spectroscopy and inspiring the integration of multifunctional artificial nanostructures across diverse domains.

DNA nanostructures have been utilized to study biological mechanical processes and construct artificial nanosystems. Many application scenarios necessitate nanodevices able to robustly generate large single molecular forces. However, most existing dynamic DNA nanostructures are triggered by probabilistic hybridization reactions between spatially separated DNA strands, which only non-deterministically generate relatively small compression forces (≈0.4 piconewtons (pN)). Here, an intercalator-triggered dynamic DNA origami nanostructure is developed, where large amounts of local binding reactions between intercalators and the nanostructure collectively lead to the robust generation of relatively large compression forces (≈11.2 pN). Biomolecular loads with different stiffnesses, 3, 4, and 6-helix DNA bundles are efficiently bent by the compression forces. This work provides a robust and powerful force-generation tool for building highly chemo-mechanically coupled molecular machines in synthetic nanosystems.