Urinary tract infections (UTI) are among the most prevalent infectious diseases, demanding rapid diagnosis to provide timely therapeutic interventions. Urinary molecules can indicate the responses of the host cells to pathogenic infections, which may serve as markers for UTI diagnosis. Sensitive and multiplex detection of urinary markers in one-pot has remained challenging. Herein, we developed an allosteric DNAzyme-based biosensor, denoted as SMART (sensitive and multiplex detection of ATP and miRNAs for UTI diagnosis), to enable rapid UTI diagnosis. Multiple sensing modules were integrated into a unimolecular DNA strand of perfect stoichiometry and excellent thermodynamic stability that collectively led to enhanced detection capability. The binding of targets to the detection module induces conformational reconfiguration of SMART to activate sequence-specific catalytic cleavage against fluorescent RNA reporters. SMART demonstrated remarkable sensitivity (ATP: ∼pM; miRNAs: ∼fM) and multiplexing capability (∼4 markers) to realize extraction-free, preamplification-free, and rapid (∼2.5 hr) detection of UTI markers. SMART-based UTI diagnosis model yielded a detection accuracy of 95.5% in a cohort of 164 patients. SMART may serve as a technical platform for detecting various markers that could be applied to the diagnosis of UTI and many other diseases in the future.

Circulating nucleic acids are emerging disease markers whose clinical applications are hindered by the lack of rapid, sensitive, convenient, and cost-effective detection assays. Inspired by the natural replication of virus, here, we developed a biomimetic one-step, one-pot, isothermal detection assay named RAPID to realize rapid and sensitive detection of nucleic acids with minimal reliance on instruments. The core element of RAPID is an autocatalytic molecular sensor that exploits the viral replication endonuclease of duck circovirus (i.e., DCV) to transform rolling circle amplification (RCA) from linear to exponential. DCV cleaves target-induced, RCA-generated amplicons into target analogs to prime secondary RCA reactions to catalytically propagate the amplification sensor. RAPID enables rapid (∼10 min), ultrasensitive (attomolar sensitivity), and direct (RNA extraction-free) detection of microRNAs and viral RNAs that is compatible with smartphone-based fluorescence detection devices. RAPID exhibited pronounced clinical translational capability by quantitatively profiling a panel of six miRNAs to achieve accurate discrimination of prostate cancer from benign prostatic hyperplasia that is exceptionally important but challenging in clinics. Furthermore, RAPID demonstrated rapid detection of influenza A viral infections of high accuracy in point-of-care settings. Simple nucleic acid detection assays like RAPID could largely promote the development and application of liquid biopsy molecular diagnostics.

Objective·To achieve the rapid detection of Staphylococcus aureus using nanopore Cas9-targeted sequencing (nCATS) technology, and simultaneously perform staphylococcal protein A (spa) typing and staphylococcal cassette chromosome mec (SCCmec) typing.Methods·The spa gene and SCCmec gene elements were selected as two regions of interest (ROIs) for targeted sequencing. Four types of CRISPR RNAs (crRNAs) were designed to form Cas9 ribonucleoproteins (RNPs) to cleave sequences flanking the two ROIs. For each crRNA, a 42 bp synthetic target DNA was designed. Appropriate crRNAs were screened, the cleavage system was optimized, and both the cleavage reaction time and Cas9 RNP synthesis temperature were determined based on the results of Cas9 RNP cleavage efficiency testing. Genomic DNA was extracted from a methicillin-resistant Staphylococcus aureus (MRSA) strain, and the ends flanking the cleaved ROIs were dephosphorylated and dA-tailed. Sequencing adapters were ligated, and sequencing was performed using a nanopore platform. The quality scores of the sequencing data were analyzed, and the obtained nucleic acid sequences were compared with those in the mecA, spa and SCCmec databases. Based on the comparison results, the presence of Staphylococcus aureus, MRSA or not, and spa and SCCmec types were determined.Results·Two sets of crRNAs were designed. Based on grayscale analysis of electrophoresis results, the set with higher cleavage efficiency was selected for further experiments. Optimization showed that a 1∶1 ratio of Cas9 RNP to target DNA, a 15 min cleavage reaction time, and a Cas9 RNP synthesis temperature of 25 ℃ yielded a cleavage efficiency of 87.41%. nCATS sequencing quality scores ranged between 15 (Q15) and 20 (Q20), indicating an approximate sequencing accuracy of 99%. Sequence comparisons with the mecA, spa and SCCmec databases revealed that the strain's spa type was t2 and its SCCmec type was Ⅱ. These results were consistent with those obtained by PCR amplification sequencing and multiplex PCR.Conclusion·The nCATS technique enables rapid detection of Staphylococcus aureus, while simultaneously providing spa and SCCmec typing information.

A 3D DNA spatial chip (DSC) based on an icosahedral DNA origami framework is introduced to construct customized circular single-stranded DNA (c-ssDNA) for data storage. Within the confined space of the DSC, thirty addressable location sequences extending from the framework edges are available for designing circular paths and directing the assembly of a series of information oligonucleotides for efficient ligation. This strategy is verified by constructing c-ssDNAs from up to 15 fragments to encode two poems (800 and 860 nucleotides). Using orthogonal location sites, both poems are simultaneously assembled within a single DSC and read out together. Rolling circle amplification (RCA) and nanopore sequencing enable complete retrieval of all the above data files. The DSCs with distinct fluorescent labels and capture sequences are further functionalized on their outer surfaces, allowing magnetic bead-based retrieval and rapid identification of specific datasets from a mixture. Moreover, the DSCs maintain data integrity after storage under various conditions. These findings demonstrate that the 3D DNA spatial chip provides an efficient approach for assembling long c-ssDNA for data storage, addressing limitations by reducing redundancy, enhancing stability, and enabling multiplexed storage and retrieval.

The rising global burden of cancer underscores the urgent demand for minimally invasive precision diagnostic methods. Extracellular vesicles (EVs) are emerging cancer liquid biopsy biomarkers carrying promising molecular markers such as membrane proteins. However, conventional approaches to EV membrane protein profiling remain limited by low multiplexing capability, high sample consumption, and complex operational workflows. Herein, we report a DNA encoded multi-round profiling of extracellular vesicle membrane proteins for cancer diagnostics (DETECT) strategy that enables detection of EV membrane proteins with high sensitivity and scalability. This method leverages engineered aptamer probes to facilitate the capture and multi-round in situ detection of 9 EV surface proteins. DETECT integrates aptamer recognition with hybridization chain reaction (HCR) for signal amplification, followed by enzymatic cleavage for complete signal erasure, thereby enabling cyclic detection of multiple protein targets on the same EVs population. Clinical validation with EVs isolated from 48 serum samples of three cancer (gastric, breast, and prostate) demonstrated DETECT's capability to uncover cancer-specific membrane protein fingerprints, which achieved 100 % accuracy in differentiating cancers from noncancers and 83.3 % classification accuracy in differentiating three cancer types. DETECT represents a feasible, robust, and scalable technical platform for profiling EV surface proteins that shall hold great application potential in cancer diagnostics and beyond.

MicroRNAs (miRNAs) play important regulatory roles in biology. Direct sequencing of miRNAs in full-length can reveal comprehensive information on their sequences, abundance, and modifications, which, however, has yet to be achieved due to their extremely short length (∼22 nt). Herein, we developed Direct-miR-seq, a nanopore-based direct RNA sequencing (DRS) method that elongates miRNAs at both the 5' and 3' ends by ligating with custom nucleic acid adaptors to ensure full-length sequencing of miRNAs with high yield and accuracy. Compared to standard DRS, Direct-miR-seq enabled sequencing of the whole sequence of miRNAs, achieved a 26-fold sequencing yield, and exhibited reduced bias across miRNA species along with low sequencing error rates. We applied Direct-miR-seq to native RNA populations from cells and human serum to demonstrate its capability to selectively capture miRNAs of known sequences in complex RNA environments for revealing quantitative information in abundance and m6A modification at single-molecule and single-base resolution of ∼100 miRNA species in a single sequencing event. We envision that Direct-miR-seq may be translated toward a variety of biological and medical applications by sequencing miRNAs and other small RNAs.

The introduction of peptide bonds into oligonucleotides as a backbone has led to the development of powerful peptide nucleic acids. However, the integration of amino acid residues (R groups) as nucleobase surrogates in oligonucleotides remains largely unexplored, presenting opportunities for creating chimeras with unique properties. In this study, we describe the design and synthesis of oligonucleotides (ONs: molecular beacons, aptamers, and ASOs) functionalized with leucine-mimicking elements (LEs), specifically isobutyl, neopentyl, and trimethylsilyl ethyl. By design and synthesis of corresponding phosphoramidites, oligonucleotide chimeras containing mono-, di-, and poly-LEs were constructed by solid phase synthesis, including the PTK7 protein-targeted aptamer Sgc8c, STAT3 (signal transducer and activator of transcription 3), and HER2 mRNA-targeted antisense oligonucleotide (ASO). Through fluorescence assays, we analyzed the stability of poly-LE-induced helical structures and investigated the assembly of molecular beacons via hydrophobic interactions. The introduction of poly-LE helices into aptamers and the incorporation of a dileucine-like moiety into antisense oligonucleotides demonstrated the functional versatility of these chimeras in improved internalization properties while adding hydrophobic elements. Notably, poly-LE incorporation significantly improved the target binding affinity and in vivo tumor-targeting efficiency of the aptamer. This work presents a novel approach to oligonucleotide engineering, offering new insights into protein-like functions in DNA and expanding the scope for molecular design in biological and therapeutic applications.

Diagnosis of influenza A viral infection is crucial for preventing disease transmission and providing effective clinical treatments. There is an increasing need for convenient detection methods to enable simple yet precise identification of viral infections. Herein, a nucleic acid probe-enabled lateral flow assay (NALFA) is developed to realize visual detection and identification of influenza A viral infections (H1N1 and H3N2) of high sensitivity and specificity. Viral RNA targets are recognized by a padlock probe, which is circularized to induce rolling circle amplification (RCA). RCA products are enzymatically cleaved into short amplicons to complex with capture DNA probes for gold colloidal-induced visual lateral flow assay. NALFA achieved attomolar (aM) sensitivity for both standard viral RNAs, along with high specificity. While applying clinical samples (16 H1N1 patients, 12 healthy controls), NALFA exhibited high detection accuracy to successfully discriminate infected samples from noninfected samples. NALFA represents a potent and convenient nucleic acid detection assay that shall find its applications in fields of viral detection and beyond.

Cell membrane vesicles (CMVs) have been extensively used as delivery vehicles for a variety of cargos, which are generally prepared via membrane extrusion. Extruded CMVs are not necessarily to have the outer membrane facing outward due to the randomness of membrane wrapping. Nanoparticles have been used to serve as cores to direct the membrane orientation of CMVs; nevertheless, there is a lack of methods to efficiently sort coreless CMVs of desired orientations. Herein, we utilized a group of functional DNA probes to reveal the random distribution of membrane orientations of coreless CMVs after extrusion, producing either right-side-out vesicles (RSVs) or inside-out vesicles (ISVs). More importantly, DNA probes that protrude out from the outer membrane can serve as handles for efficiently sorting out RSVs from ISVs to produce vesicles with a dominant right-side-out orientation. We investigated three methods to enrich RSVs, including strand displacement reaction, photo cleavage (PC), and enzymatic cleavage. Among them, PC exhibits the highest enrichment efficiency (∼93%) and RSVs purity (∼85.4%), which therefore is recommended for future applications. This work revealed the mixed orientations of coreless CMVs and provided a technical platform to efficiently enrich CMVs of wanted membrane orientations that shall be useful toward a vast array of biomedical applications.

Extracellular vesicles (EVs) have emerged as valuable sources for liquid biopsy in disease diagnostics, given their protein and nucleic acid cargoes (e.g., miRNA, mRNA, glycoRNA) can serve as critical biomarkers. DNA nanotechnology, leveraging its inherent programmability, high specificity, and powerful signal amplification capability, offers a transformative approach for the bioanalysis of EVs. This review summarizes recent advances in DNA nanotechnology-based analytical methodologies for detecting EV-associated proteins and nucleic acids. We detail the underlying principles, applications, and performance of key strategies, including aptamer-based recognition, enzyme-free catalytic amplification circuits (e.g., HCR, CHA), enzyme catalytic amplification techniques (e.g., RCA, CRISPR-Cas systems), and DNA nanostructures-assisted amplification. The integration of these DNA tools into multiplexed detection platforms is also discussed. Finally, current challenges and future perspectives concerning clinical translation of EV detection are presented.

RNA-based fluorescent light-up aptamers (FLAPs) have been progressively developed as imaging probes because of their high signal-to-noise ratio. However, it remains a challenge to use these light-up aptamers due to their poor folding and stability. Leveraging DNA nanotechnology, we investigated whether a DNA origami template could improve folding and further enhance the functionality of FLAPs, namely, the corresponding fluorescence intensities. We utilized aptamer Broccoli and its cognate fluorogen DFHBI-1T as a model. When singular aptamer Broccoli was scaffolded on DNA origami, DNA brick-based nanostructures, DNA double helices, and even on structures as simple as a DNA hairpin stem, our results showed that the fluorescence intensities could be significantly enhanced. These findings show a positive correlation between the fluorogen activity of light-up aptamers and the DNA stem length, potentially mediated by the improved structural stability of the DNA stem, as determined by their simulated thermodynamic properties. Our studies provide a new method to design and enhance the fluorescence behavior of FLAPs, especially structures with a G-quadruplex-based fluorogen recognition region.

The combination of DNA nanotechnology and Nano Gold (NG) plasmon has opened exciting possibilities for a new generation of functional plasmonic systems that exhibit tailored optical properties and find utility in various applications. In this review, the booming development of dynamic gold nanostructures are summarized, which are formed by DNA self-assembly using DNA-modified NG, DNA frameworks, and various driving forces. The utilization of bottom-up strategies enables precise control over the assembly of reversible and dynamic aggregations, nano-switcher structures, and robotic nanomachines capable of undergoing on-demand, reversible structural changes that profoundly impact their properties. Benefiting from the vast design possibilities, complete addressability, and sub-10 nm resolution, DNA duplexes, tiles, single-stranded tiles and origami structures serve as excellent platforms for constructing diverse 3D reconfigurable plasmonic nanostructures with tailored optical properties. Leveraging the responsive nature of DNA interactions, the fabrication of dynamic assemblies of NG becomes readily achievable, and environmental stimulation can be harnessed as a driving force for the nanomotors. It is envisioned that intelligent DNA-assembled NG nanodevices will assume increasingly important roles in the realms of biological, biomedical, and nanomechanical studies, opening a new avenue toward exploration and innovation. This review summarizes dynamic gold nanostructures composed with DNA-modified nano gold, DNA frameworks, and various driving forces. The reversible and dynamic aggregations, nano-switcher structures, and robotic nanomachines are precisely controlled on demand. The advantages and disadvantages of the driving forces/actuator stimuli, such as temperature, light, pH, ion concentration, and oligonucleotides are discussed in detail.image

Bioactive hydrogel materials have great potential for applications in bone tissue engineering. However, fabrication of functional hydrogels that mimic the natural bone extracellular matrix (ECM) remains a challenge, because they need to provide mechanical support and embody physiological cues for angiogenesis and osteogenesis. Inspired by the features of ECM, we constructed a dual-component composite hydrogel comprising interpenetrating polymer networks of gelatin methacryloyl (GelMA) and deoxyribonucleic acid (DNA). Within the composite hydrogel, the GelMA network serves as the backbone for mechanical and biological stability, whereas the DNA network realizes dynamic capabilities (e.g., stress relaxation), thereby promoting cell proliferation and osteogenic differentiation. Furthermore, functional aptamers (Apt19S and AptV) are readily attached to the DNA network to recruit bone marrow mesenchymal stem cells (BMSCs) and achieve sustained release of loaded vascular endothelial growth factor towards angiogenesis. Our results showed that the composite hydrogel could facilitate the adhesion of BMSCs, promote osteogenic differentiation by activating focal adhesion kinase (FAK)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/β-Catenin signaling pathway, and eventually enhance vascularized bone regeneration. This study shows that the multifunctional composite hydrogel of GelMA and DNA can successfully simulate the biological functions of natural bone ECM and has great potential for repairing bone defects.

Cell membranes are widely recognized for their ability to effectively block the entry of harmful substances and impede the absorption of useful substances, such as drugs, by cells. Biomolecular drugs suffer from reduced stability and are vulnerable to degradation by various biological enzymes, thereby reducing their capacity to effectively target their intended site of action. This chapter investigates the impact of DNA nanostructure geometry as a drug delivery platform on cytokinesis. It discusses various design methods of DNA nanostructures, such as DNA origami, single-stranded DNA tiles, and dynamic DNA structures. The chapter provides an overview of the types and mechanisms of endocytosis, including phagocytosis, macropinocytosis, and protein-mediated internalization pathways. It summarizes the mechanisms of how different morphologies of DNA nanostructures enter the cell and their resulting cellular effects. The utilization of dynamic DNA structures has significantly increased in the biomedical field, as well as in fundamental research, due to their various applications.

Biomarkers are crucial physiological and pathological indicators in the host. Over the years, numerous detection methods have been developed for biomarkers, given their significant potential in various biological and biomedical applications. Among these, the detection system based on functionalized DNA origami has emerged as a promising approach due to its precise control over sensing modules, enabling sensitive, specific, and programmable biomarker detection. We summarize the advancements in biomarker detection using functionalized DNA origami, focusing on strategies for DNA origami functionalization, mechanisms of biomarker recognition, and applications in disease diagnosis and monitoring. These applications are organized into sections based on the type of biomarkers - nucleic acids, proteins, small molecules, and ions - and concludes with a discussion on the advantages and challenges associated with using functionalized DNA origami systems for biomarker detection.

Tumor-derived small extracellular vesicle (sEV) microRNAs (miRNAs) are emerging biomarkers for cancer diagnostics. Conventional sEV miRNA detection methods necessitate the lysis of sEVs, rendering them laborious and time-consuming and potentially leading to damage or loss of miRNAs. Membrane fusion-based in situ detection of sEV miRNAs involves the preparation of probe-loaded vesicles (e.g., liposomes or cellular vesicles), which are typically sophisticated and require specialist equipment. Membrane perforation methods employ chemical treatments that can induce severe miRNA degradation or leaks. Inspired by previous studies that loaded nucleic acids into EVs or cells using hydrophobic tethers for therapeutic applications, herein, we repurposed this strategy by conjugating a hydrophobic tether onto molecular beacons to aid their transportation into sEVs, allowing for in situ detection of miRNAs in a fusion-free and multiplexing manner. This method enables simultaneous detection of multiple miRNA species within serum-derived sEVs for the diagnosis of prostate cancer, breast cancer, and gastric cancer with an accuracy of 83.3%, 81.8%, and 100%, respectively, in a cohort of 66 individuals, indicating that it holds a high application potential in clinical diagnostics.

How to realize fM-level detection of viral DNA without target amplification is a challenge. Traditionally, preamplification of low-abundant DNA targets is a prerequisite, which not only increases the risk of infectious material leakage, enlarges the chance of false positive detection, but also prolongs the overall detection time. Herein, we developed a combinatory CRISPR-Cas12a detection system from three aspects to enhance its limit of detection (LOD) to the femtomolar level which is three orders of magnitude lower than a conventional protocol. Specifically, the detection sensitivity of CRISPR-Cas12a system was enhanced by utilizing multiplex crRNAs for simultaneous recognition of multiple distinct sites of the same viral DNA target, by employing a novel molecular reporter with G-triplex structure that exhibits a significantly enhanced cleaving tendency by Cas12a, by exploring the optimal molecular coexistence reaction environment for maintaining enzyme activity which was the key for sensing. It is worth noting that we have for the first time discovered an environment where sensitive G-triplex reporter forms at a low K+ concentration without damaging CRISPR-Cas12a activity. Using this system, we demonstrated ultrasensitive detection of plasmids containing monkeypox (Mpox) viral DNA sequences in a mimicking physiological scenario. More importantly, we realized sensitive and specific detection and classification of human papillomavirus (HPV) subtypes from clinical samples. Moreover, this design successfully circumvented intricate procedures and the signal readout was not contingent upon the use of unconventional equipment, suggesting its great promise of expanding into a portable, field-deployable test kit for rapid, sensitive, and specific detection of various pathogens.

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.

Structural DNA nanotechnologyis capable of fabricatingdesignernanoscale artificial architectures. Developing simple and yet versatileassembly methods to construct large DNA structures of defined spatialfeatures and dynamic capabilities has remained challenging. Herein,we designed a molecular assembly system where DNA tiles can assembleinto tubes and then into large one-dimensional DNA bundles followinga hierarchical pathway. A cohesive link was incorporated into thetile to induce intertube binding for the formation of DNA bundles.DNA bundles with length of dozens of micrometers and width of hundredsof nanometers were produced, whose assembly was revealed to be collectivelydetermined by cationic strength and linker designs (binding strength,spacer length, linker position, etc.). Furthermore, multicomponentDNA bundles with programmable spatial features and compositions wererealized by using various distinct tile designs. Lastly, we implementeddynamic capability into large DNA bundles to realize reversible reconfigurationsamong tile, tube, and bundles following specific molecular stimulations.We envision this assembly strategy can enrich the toolbox of DNA nanotechnologyfor rational design of large-size DNA materials of defined featuresand properties that may be applied to a variety of fields in materialsscience, synthetic biology, biomedical science, and beyond.