Herein, we present an autocatalytic DNA circuit powered by a single polymerase. The autocatalysis was achieved via two displacement layers, where the output of one layer served as the trigger of the other. Each input was designed to release two outputs through polymerase-driven strand displacement. Such a design enabled rapid accumulation of output molecules and characteristic exponential signal amplification via a single enzyme.

Abstract Aspergillus fumigatus (AF) is the predominant pathogen implicated in invasive aspergillosis (IA) in humans; therefore, prompt and accurate detection is critical for the effective prevention and management of IA. This study developed a rapid detection system targeting the AF-specific anxC4 gene by integrating enzymatic recombinase amplification (ERA) with CRISPR/Cas12a. The reaction proceeds at a stable temperature of 37 °C, with amplification and detection systems separately positioned in the tube lid and bottom, respectively, effectively minimizing aerosol contamination typically associated with product transfers. To enhance sensitivity, the One-Pot method was optimized. Consequently, the fluorescence detection limit reached 1 fg/µL, and the sensitivity of the test strip reached 10 fg/µL, with no cross-reactivity observed against other fungi. Detection of AF was completed within 60 min, and results were visually displayed through fluorescence signals and nucleic acid test strips. Clinical practicality was further evaluated using aspergillosis samples, which demonstrated satisfactory performance. Pure culture results confirmed that out of 62 sputum samples, 32 were positive and 30 negative. Evaluation of 62 clinical samples using the One-Pot ERA-CRISPR/Cas12a system demonstrated sensitivity and specificity rates of 93.75% and 93.33%, respectively, via fluorescence detection, and 90.63% sensitivity and 96.67% specificity using lateral flow strips.

Opportunistic infections after organ transplantation require frequent monitoring over years. However, current diagnostic tests are inaccessible to patients in resource-limited settings. Here, we present a microfluidic-enhanced nucleic acid test for on-site reporting (MONITOR), a fully integrated, sample-in-answer-out platform for point-of-care (POC) detection of opportunistic infections. MONITOR employs a gene-specific DNAzyme that, upon integration with recombinase polymerase amplification, enables CRISPR-like trans-cleavage signal amplification with single-molecule sensitivity. It further incorporates a rapid, extraction-free protocol for processing the urine sample, lyophilized assay reagents for cold-chain-free storage and transportation, and a 3D-printed microfluidic cartridge for self-contained, contamination-free fluid handling. A portable real-time fluorescence reader and smartphone application are further developed to enable on-site quantification. Clinical validation showed complete concordance with qPCR across the tested cohorts and a field trial identified high-risk patients with systemic BK polyomavirus infection. MONITOR fulfills ideal criteria for POC testing, offering a practical solution for decentralized, quantitative viral monitoring in low-resource settings. Opportunistic infections are among the leading causes of graft loss for organ transplant recipients. Here authors introduce a protein-free CRISPR-like assay platform called MONITOR, which, when combined with pre-amplification, achieves single-molecule sensitivity, enabling sample-in-answer-out quantification of post-transplant infections in resource-limited settings.

This review provides a comprehensive overview of design principles and clinical applications of chemical tools for recognizing, discriminating, and enriching single nucleotide variants.

Molecular interactions involving nucleic acids constitute a fundamental paradigm in biological systems, governing processes ranging from gene expression to cellular signaling. Quantitative characterization of the thermodynamic and kinetic parameters of these interactions is critical not only for deciphering molecular mechanisms but also for rational design in biomedical engineering and nanomaterials science. This review systematically surveys six major categories of quantitative methods used to study nucleic acid interactions: spectroscopic methods, separation-based methods, calorimetric methods, surface-based binding assays, single-molecule methods, and DNA nanotechnology-based methods. Each category is discussed with respect to its principal advantages and inherent limitations. While conventional methods such as electrophoretic mobility shift assays (EMSA), isothermal titration calorimetry (ITC), and spectroscopic titrations have provided foundational insights, they often exhibit constraints in sensitivity, throughput, or applicability under physiologically relevant conditions. Recent advances in DNA nanotechnology, leveraging its inherent programmability and structural precision, have enabled the development of novel quantitative platforms. These include DNA origami-based single-molecule methods and homogeneous assays that support accurate and native thermodynamic profiling, significantly enhancing sensitivity and adaptability in physiologically relevant contexts. This review systematically surveys established methodologies and critically evaluates emerging DNA nanotechnology-driven strategies, highlighting their potential to advance the quantitative analysis of nucleic acid interactions.

ABSTRACT Cell‐specific detection of aberrant mRNA in blood is essential for diagnosing and treating hematological malignancies. However, current sensors are unable to function in unprocessed whole blood due to limitations in chemical stability and cell‐targeting capability. Here, we engineer a cell‐resolved ultrastable sensor for hematology (CRUSH) via spherical‐nucleic‐acid (SNA) technology, which enables live‐cell detection of leukemia fusion transcripts in unprocessed whole blood. CRUSH employs stoichiometrically controlled thiol protector to achieve a defect‐free thiol monolayer on AuNPs. This design feature endows CRUSH with a record‐breaking stability, withstanding 0.1 M dithiothreitol, a 10,000‐fold improvement over conventional SNAs. We also demonstrate that the phagocytic bias of myeloid cells over lymphoid cells drives selective internalization of CRUSH in myeloid lineages in whole blood. Leveraging cellular selectivity and engineered stability, CRUSH offers a mixed‐and‐read diagnostic test, where lyophilized sensors are directly mixed with whole blood samples, followed by standard flow cytometry analysis. This one‐step test detects BCR‐ABL1 fusions in living myeloid cells with high specificity and robustness, enabling accurate discrimination of multilineage acute lymphoblastic leukemia within 1 h. Our study bridges biosensing innovation with urgent diagnostic needs, offering a rapid, specific, and robust tool for accurate diagnosis and treatment of hematologic malignancies.

The translation of nucleic acid testing to point-of-care settings is hindered by the reliance on target amplification, which introduces complexity and contamination risks. Herein, we report a target-amplification-free assay for the direct detection of Staphylococcus aureus 16S rRNA, utilizing a cascaded DNAzyme and Nicking endonuclease reaction (DNECR). This system integrates a panel of target-specific multicomponent DNAzyme (MNAzyme) for primary recognition and signal amplification with a sterically blocked bipedal DNA walker (BDW) for cascade signal amplification. Upon target binding, the activated MNAzyme cleaves the blocker to initiate the BDW, which then traverses a spherical nucleic acid track via nicking endonuclease activity, generating amplified fluorescent signals. This cascaded design achieved a detection limit of 102 CFU/mL for cultured S. aureus with high specificity. Clinical validation using 12 patient sputum samples demonstrated 100% diagnostic sensitivity and specificity, confirming the potential of DNECR as a robust, amplification-free platform for rapid pathogen detection at the point of care.

Complementarity between nucleic acids via Watson-Crick base pairing formulates the basic principle for designing hybridization probes but often suffers low sequence selectivity against single nucleotide mutations. Herein, we report mismatch-exchange as a new design principle that allows the highly sensitive and robust discrimination of single nucleotide polymorphisms (SNPs) by simply manipulating the number and position of mismatches in both probes and the reaction products. Leveraging mismatches to drive the strand-exchange and finetuning the specificity, mismatch-exchange is particularly advantageous for analyzing complex nucleic acid targets containing multiple nearby SNPs. Both selective tolerance to synonymous SNPs and OR-gate-based detection of clustered drug-resistant SNPs were demonstrated. Once deployed to nucleic acid testing in clinical settings, mismatch-exchange enabled the discrimination of multiple lamivudine-resistant hepatitis B virus mutants in a clinical cohort containing 65 clinical plasma samples.

We report a LEGO-inspired multipiece chip (LIM-Chip), a modular microfluidic system capable of streamlining multiple nucleic acid amplification and detection steps through on-demand chip assembly via universal 3D-printed side release buckle connectors. The clinical applicability of the LIM-Chip is demonstrated by mediating a DNAzyme-based isothermal nucleic acid amplification assay for the sensitive detection of HPV16 DNA in clinical cervical samples.

Background:Membrane Spanning 4-Domains A3 (MS4A3) has been confirmed to possess significant tumor-suppressive potential in various malignancies. However, its expression characteristics and clinical prognostic value in colon cancer (CC) still lack systematic and in-depth investigation. This study aimed to systematically investigate the expression pattern, prognostic value, immune microenvironment association, and biological function of MS4A3 in CC through integrated bioinformatics analyses and experimental validation. Methods:This study utilized The Cancer Genome Atlas-Colon Adenocarcinoma (TCGA-COAD) cohort to screen for genes significantly associated with CC and combined multiple independent Gene Expression Omnibus (GEO) datasets to validate the expression patterns and prognostic significance of MS4A3. Key biological pathways were identified through gene set enrichment analysis (GSEA), and tumor immune infiltration characteristics were evaluated using the CIBERSORT algorithm. Additionally, the expression of MS4A3 and its impacts on cellular functions were validated at the cellular level through quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, Cell Counting Kit-8 (CCK-8), EdU, Transwell, and TUNEL assays. Results:Analysis of public datasets revealed that MS4A3 is significantly downregulated in CC tissues, and its low expression is an independent risk factor for shortened overall survival (OS). GSEA indicated that MS4A3 downregulation is closely associated with the aberrant activation of the pentose phosphate pathway. Immune infiltration analysis showed that low MS4A3 expression is closely linked to the enrichment of M2 macrophages and neutrophils, as well as the upregulation of multiple immune checkpoint genes. In vitro experiments further confirmed that MS4A3 was lowly expressed in CC cell lines. Its overexpression significantly inhibited CC cell viability, proliferation, migration, and invasion, while simultaneously promoting cell apoptosis. Conclusions:MS4A3 expression is significantly decreased in CC tissues and is significantly correlated with poor prognosis, suggesting that this gene may serve as a potential prognostic biomarker.

Nucleic acid strand displacement reactions (SDRs) are fundamental building blocks of dynamic DNA nanotechnology. A detailed understanding of their kinetics is crucial for designing efficient sequences and regulating reaction networks with applications in biosensing, synthetic biology, biocomputing, and medical diagnostics. Since the development of toehold-mediated strand displacement, researchers have devised many strategies to adjust reaction kinetics. These efforts have expanded the available tools in DNA nanotechnology. This review summarizes the basic principles and recent advances in activation strategies, emphasizing the role of strand proximity as a central driving force. Proximity-based approaches include toehold docking, associative toeholds, remote toeholds, and allosteric designs, as well as strategies that operate without explicit toehold motifs. These methods enable flexible and scalable construction of DNA reaction networks. We further discuss how combining different activation and kinetic control approaches gives rise to dynamic networks with complex and dissipative behaviors, providing new directions for DNA-based nanotechnology.

Programming microorganism adhesions to engineer multicellular microbial communities holds promise for synthetic biology and medicine. Current chemical and genetic engineering approaches often lack specificity or require engineered bacteria, making the design of responsive interactions challenging. Here, we demonstrate the use of functional DNA as programmable surface receptors to regulate the patterns and behaviors of microbial communities. Using metabolic labeling and hydrophobic insertion, we modified various microorganisms with DNA, including Gram-positive and Gram-negative bacteria, and dormant spores. By incorporating distinct sequences, we achieved precise spatial control of bi- and tricomponent microbial assemblies, forming diverse morphologies like core-shell and selective clusters. Stimuli-responsive clustering was successfully realized using aptamers, strand displacement, and reverse-Hoogsteen base pairing, with oligonucleotides or small molecules as exogenous cues. This work extends the use of functional DNA to control microbial interactions, enabling living communities with dynamic biofunctions, such as biofilm formation, antibiotic sensitivity, and quorum sensing, in response to biological triggers.

ABO-incompatible kidney transplantation is widely used to meet the escalating need for organs. Current recipient-centric desensitization protocols involving antibody depletion through plasmapheresis increase the risk of infections, perioperative bleeding events and costs. Here we present a donor-centric desensitization protocol, converting type-A kidneys into enzyme-converted O kidneys during hypothermic perfusion to remove the A antigen from the kidneys. An ex vivo model resulted in no antibody-mediated injury. Encouraged by this, an enzyme-converted O kidney was transplanted into a type-O brain-dead recipient with a high titre of anti-A antibody, and no hyperacute rejection was observed. The graft was well tolerated with no evidence of antibody-mediated rejection for 2 days. Antibody-mediated lesions and complement deposition were found starting 3 days post-transplant, coinciding with A-antigen regeneration, and later higher Banff scores, suggesting an immune-mediated response. Single-cell sequencing confirms the elevated expression of accommodation-related genes, suggesting the potential for longer-term tolerance. This study provides a donor-centric organ engineering strategy and has the potential to broaden the reach of ABO-incompatible kidney transplantation, improving the fairness of and access to organ allocation. An ex vivo model and pre-clinical study in a brain-dead recipient provide enzyme-converted O organs to avoid hyperacute rejection in ABO-incompatible kidney transplant patients.

Nucleic acids can form diverse non-canonical structures, such as G-quadruplexes (G4s) and i-motifs (iMs), which are critical in biological processes and disease pathways. This study presents an innovative probe design strategy based on groove size differences, leading to the development of BT-Cy-1, a supramolecular cyanine probe optimized by fine-tuning dimer "thickness". BT-Cy-1 demonstrated high sensitivity in detecting structural transitions and variations in G4s and iMs, even in complex environments with excess dsDNA. Applied to clinical blood samples, it revealed significant differences in RNA G4 and iM levels between liver cancer patients and healthy individuals, marking the first report of altered iM levels in clinical samples. This work highlights a novel approach for precise nucleic acid structural profiling, offering insights into their biological significance and potential in disease diagnostics.

Isothermal nucleic acid amplification techniques are promising alternatives to polymerase chain reaction (PCR) for amplifying and detecting nucleic acids under resource-limited conditions. While many isothermal amplification strategies, such as recombinase polymerase amplification (RPA), offer comparable sensitivity to PCR, they often lack the specificity and robustness for discriminating single nucleotide variants (SNVs), mainly due to the uncontrolled production of massive amplicons. Herein, we introduce a mismatch-guided DNA assembly (MGDA) approach capable of discriminating SNVs in the presence of high concentrations of wild-type (WT) interferences. We show that an optimal MGDA design can effectively suppress interfering signals from WT while maintaining high detection signals for the targeted SNV. A further introduction of a competitive sink probe allowed the detection of challenging SNVs, such as those containing G-T wobbles, with high sensitivity and specificity. Because it is highly specific and tolerant to massively produced interfering amplicons during isothermal nucleic acid amplification, we integrated MGDA with RPA for discriminating clinically relevant SNVs in point-of-care settings. We demonstrate that our RPA-MGDA is highly sensitive and specific, allowing the detection of as low as 1 aM SNVs with an allele frequency of 0.5%. We also evaluated the clinical potential of RPA-MGDA by analyzing epidermal growth factor receptor L858R mutations in tumor tissue samples collected from non-small-cell lung cancer patients (n = 44). A multiplexed RPA-MGDA assay was also developed for the simultaneous detection of pharmacogenetic mutations in buccal swab samples (n = 30).

A label-free, orthogonal dual-channel CRISPR-Cas platform is developed for the simultaneous detection of diverse pathogens. Cas12a is reported by a G4-PPIX complex, and Cas13 by a split Broccoli-DFHBI aptamer, which ensure exclusive channel specificity and eliminate optical cross-talk. Clinical tests on 50 samples show 100% concordance with PCR.

In this research, we developed a multi-functional probe (NBN), capable of concurrently responding to pH, viscosity and hypochlorous acid. NBN realized reversible pH detection through the interconversion of phenol hydroxyl and quinone, and realized the detection of HClO through the chlorination of benzothiazole moiety rather than the oxidation of olefine double bond. Notably, NBN was the first HClO probe to achieve ratiometric detection by fluorescence red-shift. NBN exhibited excellent responsiveness and could respond promptly to pH/HClO/viscosity. It was utilized to detect pH and HClO in actual water samples. Additionally, NBN was also applied to assess viscosity and HClO level in cells, as well as the thickening effect of various thickeners.

Single nucleotide polymorphism (SNP) discrimination plays an important role in precision medicine by providing insights into an individual's genetic information. The low abundance of the mutant target and the minimal impact caused by SNP on the whole nucleic acid sequence remain a challenge during discrimination. Herein, a cascade strand displacement reaction mediated label-free Cas12a sensing platform is established for SNP analysis. Split G-quadruplex (G4) motif is recruited as label-free signal output for Cas12a sensing system, and hence overcomes relying of fluorescence labeled substrates like conventional method. The established platform exhibits excellent single base difference discrimination ability attributing to the cascade strand displacement process, during which, single-base mismatch will affect the strand-exchange rate significantly. The limit of detection reaches 1 copy / test after integration with isothermal preamplification. The proposed method can discriminate as low as 0.1 % single base variation and perform robustly in biological matrixes. Human buccal swab samples are successfully genotyped with high accuracy.

DNA-mediated synthetic cell communication enabling non-natural signaling and regulatory pathways is highly attractive but relies on direct cell contacts. Here, we report a diffusion-based synthetic cell communication capable of regulating migration behaviors of epithelial cell adhesion molecule (EpCAM)-overexpressed cancer cells in response to apoptosis events at distal sites. This synthetic cell communication network is enabled by a multitasking DNA nanomachine that not only mediates signal production, transmission, and regulation of cell migration but also amplifies signaling ligands in situ in response to specific receiver cells to overcome the signal attenuation during diffusion. Leveraging this diffusion-based synthetic cell communication network, we demonstrate the inhibition of cancer cell migrations in response to distal apoptosis events induced by an anticancer drug. Our system enriches current DNA nanotechnological tools for manipulating cellular interactions and function. It also directs a possible intervening strategy to reduce the invasiveness of cancer cells.

Achieving strong yet reversible adhesion via minimalist molecular design remains a critical challenge for next-generation supramolecular materials. Here, a dual-end modular adhesion strategy is presented based on a small organic molecule incorporating carboxylic acid and triphenylphosphonium terminals linked by a flexible alkyl spacer. This design enables synergistic noncovalent interactions-including hydrogen bonding, dipole-dipole interactions, and electrostatic forces-to construct a thermally reconfigurable supramolecular network. Upon mild heating, the system transitions from ordered to amorphous states, facilitating dynamic cohesion and interfacial adaptability across both hydrophilic and hydrophobic substrates. The resulting adhesive achieves high lap-shear strength (up to 4.6 MPa on polyethylene terephthalate (PET)), rapid curing, and exceptional resistance to solvents, humidity, and low temperatures. Moreover, it enables fully reversible adhesion and closed-loop recyclability. Combined experimental characterizations and molecular simulations reveal how the interplay of molecular architecture and noncovalent synergy governs adhesion performance. This work provides a generalizable framework for the design of sustainable, programmable supramolecular adhesives.