DNA-FLASH─a DNAzyme Walker-Based Nanosensor for Digital Biosensing at the Point of Care.

Spatially Confined DNA Nanodevices for Sensing and In Situ Imaging of Biomolecules

Leveraging the predictability of Watson–Crick base pairing and the programmability of nucleic-acid structures, DNA nanodevices can be rationally designed into diverse architectures that incorporate functional motifs such as aptamers and DNAzymes. These multifunctional constructs enable the integration of target recognition, signal transduction, and amplification within a single framework, facilitating versatile biomolecular sensing. However, translating conventional DNA nanodevices into practical applications within complex biological samples remains challenging due to their high susceptibility to nuclease degradation, which compromises structural integrity and leads to signal leakage. Additionally, the low abundance of target analytes and the crowded nature of biological fluids significantly reduce molecular collision frequency and slow interfacial reaction kinetics, thereby resulting in poor sensitivity and a high detection limit. Therefore, developing next-generation DNA nanodevices with enhanced structural stability and improved reaction efficiency is crucial for their practical use in real biological environments. Confinement refers to the spatial restriction of molecules, reactants, or reaction interfaces within nanometer-sized domains, where their physicochemical behaviors differ markedly from those in bulk solutions. Under confined conditions, reactive components can be shielded from external interference, thus enhancing molecular stability. Moreover, confined environments can locally enrich reactants and reduce the configurational entropy of reaction components, greatly improving the thermodynamic and kinetic efficiency of molecular interactions. Thus, integrating confinement effects into DNA nanodevices offers a promising strategy for the synergistically enhancing both structural robustness and sensing performance in complex biological samples. In this Account, we describe our recent efforts to design and develop spatially confined DNA nanodevices aimed at improving molecular sensing in real biological samples. By engineering the molecular interaction interface of DNA nanodevices through confinement strategies, we have developed four representative classes of such materials, that are cavity-confined, surface-confined, topological structure-confined, and network-confined DNA nanodevices. We systematically explore their advanced applications in biomarker sensing within biological samples and discuss the underlying mechanisms responsible for the enhanced structural stability and the analytical performance, including sensitivity, limit of detection, and reaction kinetics. The experimental progress summarized in this Account suggests that confinement strategy could serve as a versatile design principle for engineering DNA nanodevice interfaces, thereby advancing practical biosensing applications in complex biological environments.

A dual-recognition fluorescence chip utilizing a substrate-borne DNA walker for precision profiling of small extracellular vesicles.

A fluorescence chip based on a membrane-anchored, bipedal DNAzyme walker assembled on small extracellular vesicles (sEVs) surfaces via catalytic hairpin assembly (CHA) opens a new paradigm for designing efficient DNA nanomachines on membrane-enveloped targets.

Programmable 2D-to-3D structural transformation of DNA nanostructures triggered by single-stranded RNA.

Controllable structural transformation of DNA nanostructures offers substantial potential for molecular devices. Toehold-mediated strand displacement, a common strategy for driving such rearrangements, generally requires a dedicated toehold sequence to initiate the reaction, making the regulation of conformational changes in perfectly matched DNA systems challenging. To circumvent this limitation, we herein efficiently assembled a two-dimensional (2D) DNA triangle using only two short DNA strands. Inspired by the structural diversity of RNA, we found that strand displacement induced by the corresponding RNA sequence can alter the assembly pathway of the triangle without a toehold, leading to the formation of a three-dimensional (3D) RNA-DNA hybrid quadrilateral. All nanostructures and their transformation pathways were confirmed by cryogenic-electron microscopy (cryo-EM) and mass spectrometry (MS) analyses. Further, to investigate whether our designed transformable DNA nanostructure can serve as a target-specific signal reporting system, we developed a nucleic acid detection strategy based on isothermal amplification coupled with single-stranded RNA displacement-induced transformation of the DNA nanostructure (SDT), which successfully detected human papillomavirus (HPV) in clinical samples. Our experimental results demonstrate that RNA strand displacement provides new insights into constructing transformable DNA nanostructures and offers a novel design strategy for dynamic DNA nanostructures in molecular diagnostics.

Programmable two-dimensional CoOOH nanosheets with a hybridization chain reaction amplification system for label-free fluorescent detection of miRNA-205 in colorectal cancer

Construction of multivalent aptamer-based fluorogenic DNA nanostructures for label-free fluorescent biosensors.

Fluorescent light-up aptamers (FLAPs) are attractive signaling tools due to their label-free fluorescence and programmability. However, DNA-based FLAP sensors are often constrained by low fluorogenic activation efficiency, limited intrinsic target responsiveness, and insufficient signal amplification. In this study, we integrated a DNA-based FLAP (DAP) with a three-branched DNA (TBD) scaffold to construct a multivalent FLAP nanostructure (DAP-TBD) that exhibited enhanced fluorogenic activation toward Auramine O (AO). Compared with monovalent DAP, the trivalent DAP-TBD showed a 3.3-fold increase in fluorogenic activation efficiency, indicating a clear multivalent effect. The system adopts a modular architecture in which DAP functions as a replaceable FLAP model. Using a target-triggered catalytic DNA assembly (CDA) circuit composed of three rationally designed hairpin DNAs, multiple DAP-TBD nanostructures are assembled isothermally, enabling target-triggered signal generation and amplification without the need for labeled probes. The CDA-based DAP-TBD system selectively detects target single-stranded DNA with a detection limit of 235 pM. Furthermore, coupling the DAP-TBD system with an exonuclease-assisted hairpin and an aptazyme enabled label-free detection of microRNA and adenosine triphosphate, demonstrating the versatility of the system as a signal sensor that can interface with diverse upstream transducer modules. The system requires no fluorophore or quencher labeling, relies on only three unmodified DNA strands, and offers programmability, modularity, and cost-effectiveness. Overall, this study presents a multivalent DNA-based FLAP nanostructure and provides a versatile strategy for developing label-free fluorescent biosensors.

Encoded DNA Nanoswitch Array for Multiplexed MicroRNA Profiling via Nanopore Readout

DNA nanoswitches are programmable molecular devices that convert target recognition events into highly specific and controllable conformational changes. Nevertheless, their application in multiplexed analysis of low abundance miRNAs remains limited, and methodological breakthroughs are urgently needed for analysis in complex clinical samples. Here, we have seamlessly integrated rolling circle amplification (RCA), cloverleaf structured DNA nanoswitch arrays, and glass nanopore detection technology to construct a high throughput analysis platform, which is expected to be applied in the diagnostic research of acute myocardial infarction (AMI). The design leverages RCA to convert target miRNAs into stable DNA reporters followed by enzymatic cleavage, which enables sensitive molecular detection and effectively alleviates nanopore clogging. The cloverleaf design enhances signal amplitude by 70.77% over conventional dumbbell structures. In clinical plasma samples, the platform accurately differentiated AMI patients from healthy controls by profiling a panel of five potential AMI characteristic miRNAs. A reference free ternary encoding (RFTC) strategy further enables a 12 carrier DNA nanoswitch array capable of simultaneously detecting up to 60 targets. By combining label free DNA nanoswitch arrays with glass nanopore sensing, this work establishes a robust methodological framework for multiplex nucleic acid analysis. The platform also exhibits promising potential for analyzing circulating miRNAs and screening acute myocardial infarction related biomarkers, offering a facile modular strategy for flexible detection of diverse molecular targets in future clinical translational research.