The limited repertoire of experimentally validated RNA-targeting nucleases has constrained both mechanistic studies and the efficient discovery of novel enzymes for RNA biotechnology. This challenge is particularly pronounced for prokaryotic Argonaute (Ago) proteins, where the scarcity of confirmed RNA-targeting members and a lack of clarity regarding RNA specificity determinants hinder systematic exploration. Although machine learning offers a potential solution, its application is often impeded by the scarcity of labeled training data in this field. To address these limitations, we developed the self-iterative hierarchical ensemble model (SIM), which integrates hierarchical ensemble learning with a self-training strategy. This approach bypasses the dependency on large-scale experimental datasets, allowing SIM to iteratively expand its predictive capability from minimal initial labeled data. When applied to prokaryotic Agos, SIM identified six high-confidence RNA-targeting candidates, five of which were experimentally validated (83% success rate). Notably, SIM identified three uncharacterized Agos harboring a novel N-terminal domain, defining a previously unrecognized subclass. Biochemical and in vivo validations of Haloferax profundi Ago (HpAgo) confirmed its RNA cleavage activity and a distinctive RNA modification-sensing capability. We leveraged this latter finding to develop a rapid, cost-effective method for quantifying modified RNAs. Our study not only expands the repertoire of RNA-targeting tools but also establishes SIM as a generalizable framework for protein function prediction under data-scarce conditions. This work has broad implications for both RNA biotechnology and the application of machine learning in data-limited fields.

Bacteria have evolved a myriad of host-defense systems that protect against invasive mobile genetic elements. The DdmDE module, which consists of a DNA-guided, DNA-targeting prokaryotic Argonaute (pAgo) DdmE and a helicase-nuclease enzyme DdmD, represents a model anti-plasmid system. Here, we demonstrate that a double-stranded DNA (dsDNA)-destabilizing force generates transient bubbles that provoke promiscuous binding by DNA-guided DdmE. The resulting nucleoprotein complex discriminates on-targets from off-targets primarily through differences in dissociation rates. Remarkably, DNA-bound DdmE recruits a DdmD dimer to drive dsDNA shortening against a resisting force. Further analysis attributes this shortening to target-centered, bidirectional dsDNA unwinding and single-stranded DNA (ssDNA) extrusion by DdmD, in which monomeric DdmD engages both unwound strands. During this process, free DdmD rapidly associates with the two extruded strands and catalyzes ssDNA digestion with a sequence preference for a 5' guanine. Our findings provide a dynamic perspective on how pAgos cooperate with accessory factors to achieve plasmid clearance.

N6-Methyladenosine (m6A) is the most prevalent RNA chemical modification in epitranscriptomics and is closely associated with disease development. Current m6A detection techniques predominantly rely on specific antibodies to capture modified transcripts, followed by detection through sequencing or PCR. However, these methods are complex to operate and often suffer from limited resolution, as well as low sensitivity. Here, we developed a unique site-specific m6A detection strategy denoted as STEM-M6A-PCR (Specific Terminal-Mediated m6A PCR). This approach employs a well-designed capture primer and the specific properties of Bst DNA polymerase to trigger the generation of a specific end that forms a complete hairpin structure, enabling exponential amplification for highly sensitive detection. STEM-M6A-PCR achieves a detection limit of 100 aM for target m6A-RNA with a total RNA input as low as 250 fg. Notably, this method is extraordinarily capable of site-specific resolution for closely adjacent m6A modifications through the rational design of capture primers. As an antibody-independent approach, STEM-M6A-PCR offers a powerful tool for the precise identification and quantification of m6A modifications in biological studies.

CRISPR-based molecular diagnostics have emerged as powerful and programmable platforms that enable sensitive and specific detection for disease management and epidemiological surveillance. Advances in CRISPR engineering and assay design are driving the emergence of next-generation detection platforms that are highly sensitive, rapid, and amenable to field deployment. These engineering breakthroughs have the potential to reshape point-of-care tests (POCT) and transform how emerging and persistent health threats are monitored in decentralized and resource-limited settings. Herein, we systematically review the recent advancements in CRISPR engineering strategies aimed at improving detection sensitivity and specificity, eliminating the dependence on preamplification, and enabling robust POC deployment. The discussed strategies encompass both the rational engineering of CRISPR ribonucleoproteins (RNPs) and the optimization of downstream signaling modules for molecular diagnostic applications. We further highlight key challenges and future perspectives that may inspire impactful research directions and accelerate the advancement of CRISPR engineering strategies toward robust, field-deployable POCT platforms.

Multidrug-resistant (MDR) bacterial infections and persistent oxidative stress exert a prolonged, hostile inflammatory pressure in chronic skin wounds, posing a formidable clinical challenge. Current antibiotic-based modalities often fall short in inflammation resolution and cutaneous regeneration. Here, we present the design of a reactive oxygen species (ROS)-balancing MXene/MoSe2 (MX/Mo), with MoSe2 synthesized on MXene surfaces via a solvothermal method. In infections, MX/Mo under ultrasound activation promotes efficient electron-hole separation to trigger a localized ROS storm. This disturbs the bacterial respiratory chain and irreversibly blocks adenosine triphosphate synthesis, causing lethal energy deprivation and MDR bacterial eradication. During healing, MX/Mo without ultrasound exhibits significant multienzyme-like activity to autonomously scavenge residual ROS, alleviating oxidative injury. In in vivo studies, MX/Mo exhibits superior antibacterial and wound healing efficacy by rapidly suppressing severe Methicillin-resistant Staphylococcus aureus infections and creating a favorable microenvironment with balanced ROS levels, thereby triggering angiogenesis and promoting collagen deposition for tissue regeneration. We anticipate that MX/Mo addresses skin wounds with multifactorial pathologies by integrating antibacterial activity and oxidative stress regulation, which offers new possibilities for treating a broad range of bacterial infection-driven disorders.

Purinergic signaling dysregulation (e.g., excessive extracellular ATP, exATP) plays a critical role in the pathology of inflammatory disorders, but current efforts in drug development for blocking purinergic receptors are unsatisfactory. Here, inspired by natural metabolite sensing/signaling system, we develop a DNA origami-based ATP-sensing nanodevice (ND) for fine-tuning purinergic signaling and immune homeostasis. This ND composes a tubular DNA origami equipped with ATP sensors and the catalytic subunits (ENPP1-CD73 pairs), which can sense high levels of exATP and then expose the catalytic subunits for metabolizing exATP to adenosine, thereby driving an immune switch from exATP-mediated proinflammatory signals to adenosine-mediated immunosuppressive signals. Further surface displaying of ND on the monocytes (ND@Monos) enables its active inflamed site-targeting to restore immunometabolic hemostasis and reduce inflammation in diverse models in vivo. This study highlights that design of metabolite-sensing NDs is a promising strategy for controlling the homeostasis of cell metabolism and the immune response. Purinergic signalling dysregulation can drive inflammatory disorders, but effective targeted therapy is still lacking. Here, authors develop a smart DNA origami nanodevice that can precisely sense and metabolize high levels of extracellular ATP, thereby fine-tuning purinergic signalling and immune homeostasis.

Developing sustainable, high-performance fabrics from biomass waste is vital to mitigate the environmental impact of conventional fabric industries. Mycelium, an emerging biomanufacturing platform that directly upcycles low-value plant biomass waste into fabric-like materials, shows great promise. However, its application in wearable systems remains constrained by poor durability and limited breathable water-resistance. Here, we report a molecular engineering strategy, via isophorone diisocyanate (IPDI) mediated crosslinking, to transform raw mycelium network into a high-performance biofabric (MEM-biofabric). Benefiting from this molecular design, the MEM-biofabric exhibits significantly enhanced mechanical properties, with its tensile strength (10.1 MPa) and tear resistance (6.0 N mm(-1)) increasing by 1.77 and similar to 7-fold, respectively, along with enhanced softness and flexibility. Moreover, the material achieves superior breathable water-resistance, combining remarkable hydrophobicity (contact angle (CA): 152.8 +/- 1.3 degrees) with high water vapor transmission rate (WVTR) (2.8 +/- 0.17 kg m(-2) day(-1)). More interestingly, the material also demonstrates strong resistance to biodegradation (maintaining structural integrity after 50 days of soil burial) and outstanding biocompatibility (cell viability >95%). This work establishes a scalable and eco-friendly molecular design principle for natural biofabric, paving the way toward next-generation bio-based wearable materials that integrate functionality, comfort, and sustainability.

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.

Eradicating the bacteria on the surface of titanium‐based (Ti) implants and optimizing the local immune environment is urgently needed to minimize the clinical implantation failures. Here, an ultrasound (US)‐responsive Ti implant aiming at killing bacteria is prepared by building strontium titanate (SrTiO 3 ) nanoarrays on Ti substrate through a multi‐step hydrothermal method and the subsequent decoration of Au nanoparticles (Au NPs). As an n‐type semiconductor, SrTiO 3 can combine with Au NPs to form a Mott–Schottky heterostructure. The results reveal that the built‐in electric field of the M–S heterojunction composed of Au NPs and SrTiO 3 significantly promotes the separation of electrons and holes while reducing the bandgap. Under US irradiation, Au@SrTiO 3 generates a large amount of reactive oxygen species (ROS) to kill bacteria, meanwhile eliminating bacterial biofilms that adhere to the implant surface. Besides, Au NPs accelerate bone remodeling by modulating macrophage polarization toward inflammation‐suppressive orientation. Also, SrTiO 3 and Au NPs synergistically directed the osteogenic differentiation. The implantation in vivo shows that continuously optimizing the osteogenic microenvironment can obviously accelerate the osseointegration of Ti implant. Transcriptomic analysis reveals the in vivo pro‐osteointegration mechanism of heterostructured Au@SrTiO 3 nanoarrays on Ti implant.

Once bone is damaged, a rapid surge of reactive oxygen species (ROS) and anaerobic glycolysis will create an oxidative stress and acidic microenvironment, thus inhibiting osteogenic factor expression and disrupting bone regeneration. To address this, we innovatively developed an alkaline hydrogel-coated poly(ether ether ketone) (PEEK) porous scaffold with VitaFlux functionality in this study. This decorated PEEK scaffold distinct from those hydrogel-perfused scaffolds with obstructed pores still kept its initial 3D-printed pore structure to allow the ingrowth of bone tissues. The results showed that the modified scaffold could not only exhibit significant efficacy in lowering oxidative stress, neutralizing acidic microenvironment, and activating TGF-β1 expression in serum through its inherent alkalinity but also recruit stem cells to the bone defect site and upregulate osteogenic factor expression via the TGF-β1/Smad signaling pathway. This study proved the obvious advantage of the modified scaffold in enhancing bone regeneration by the synergy of immune microenvironment remodeling and osteogenic factor expression enhancement, thus providing a facile method to modify bone scaffold which can facilitate a desirable bone repair process through self-activating the transfer of growth factors between the material and tissues.

Aberrant CpG island methylation serves as a pivotal biomarker for cancer diagnosis, with accuracy substantially enhanced by analyzing multiple loci. Current techniques, such as bisulfite conversion or restriction enzyme-based methods, often fall short in delivering efficient multiplexed genomic methylation analysis using standard PCR platforms. Herein, we introduce an innovative bisulfite-free, multiplex assay-multiple specific terminal mediated methylation PCR (multi-STEM MePCR). This assay integrates a methylation-dependent restriction endonuclease (MDRE) with a novel multiplex PCR, leveraging innovative stem-loop structured assays for simultaneous detection of multiple CpG sites. As a proof-of-concept, the multi-STEM MePCR platform simultaneously achieved quantification of three methylation model sites down to ten copies per tube, accompanied by a broader linear dynamic range, and attained a sensitivity of 0.1% against a background of 10 000 unmethylated gene copies. Crucially, by markedly minimizing cross-reactivity and reducing competition among targets, this technique adeptly distinguishes between sites exhibiting significant variations in methylation abundance. Additionally, this method effectively detects digestion products of various sizes, demonstrating clinical precision comparable to bisulfite sequencing, yet with simpler operation, less time, and lower cost. This multi-STEM PCR technology pioneers an advanced strategy for multiplexed methylation analysis, which is essential for epigenetic research and clinical DNA methylation diagnostics.

N6-Methyladenosine (m6A) ranks among the most prevalent modifications in RNA, which serves as a biomarker for diseases, such as lung cancer. Herein, we developed a CRISPR/Cas13a-Csm6 tandem assay (termed CRISPRm6A assay) allowing for preamplification-free, sensitive, and rapid detection of RNA m6A modifications. The coupling of Cas13a-Csm6 tandem with MazF endoribonuclease enables the assay to identify m6A RNA with single-base resolution. Compared to the CRISPRm6A assay using Cas13a alone, the tandem CRISPRm6A assay yielded an improved sensitivity for RNA detection by ∼22 times, thus enabling preamplification-free detection of RNA m6A. Particularly, the proposed assay enabled quantification of m6A abundance down to 0.5% at the picomole level in lncRNA MALAT1 and demonstrated a 100% correlation in diagnosing nonsmall cell lung cancer. In summary, the CRISPRm6A assay supports two key applications in biological samples: (1) precise determination of m6A sites and (2) quantitative measurement of m6A fractions. Therefore, the CRISPR tandem method presents a promising tool for RNA epigenetics-based diagnostics.

Detection methods with single-nucleotide specificity are essential tools for nucleic acid analysis in diverse clinical and biological settings. However, both hybridization-based and enzyme-based methods are only effective for discriminating single-nucleotide mutations at certain positions, making it difficult to detect nucleic acid targets having multiple nearby mutations. Herein, we describe the design of cooperative recognition probes (CRPs) that integrate both hybridization and ligation-based recognition mechanisms and thus are highly effective for discriminating mutations throughout all positions. The cooperative nature of CRPs further enables AND-gate-based detection of multiple nearby mutations with high fidelity and specificity. Moreover, CRPs generate circular or linear ligation products that can be readily amplified by rolling circle amplification or polymerase chain reaction, making our strategy readily adaptable to diverse biological and clinical settings. Leveraging CRPs, we demonstrate the detection of nucleic acid targets that are difficult to be discriminated using conventional strategies, such as the highly specific discrimination of microRNA from its family members and isoforms, and the high-fidelity identification of drug-resistant single-nucleotide variants in the presence of nearby synchronous mutations in lung cancer samples.

Marine biofouling is an important factor that affects the service life of marine equipment. In this work, phenol source capsaicin-mimicking N-(4-hydroxy-3-methoxy-benzyl) acrylamide, two amine sources, 3-aminopropyltriethoxysilane (APTES) and furfurylamine (FFA), together with paraformaldehyde were used to prepare two benzoxazine coatings, P(HA) and P(HF). By reaction with iodomethane, the quaternary ammonium group was introduced into the N position of the oxazine ring. Quaternarily ammonified benzoxazine coatings P(HAI) and P(HFI) show exciting antibacterial performance compared to that of the untreated benzoxazine coating. The P(HFI) coating exhibited 93.8%, 96.4%, 99.9%, and 99.9% killing rates for Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis, respectively. The quaternary ammonization treatment also precipitated further enhanced anti-alga activity and excellent real-sea antifouling performances. However, the introduction of the ionic groups hindered the thermal polymerization of benzoxazine, leading to more defects on the coating surface, resulting in corrosion resistance that was poorer than that of HA and HF as detected from electrochemical corrosion and neutral salt spray experiments. The corrosion current density of the P(HA) and P(HF) coatings is 2 orders greater than that of the P(HAI) and P(HFI) coatings. Our results shed light on developing new quaternary ammonization treatment routes for polymeric coatings for the desired functions.

Circulating cell-free DNA (cfDNA) has emerged as a promising noninvasive diagnostic tool for liquid biopsy, with abnormal DNA methylation serving as a key biomarker for cancer screening and early diagnosis. However, the low abundance and high fragmentation of cfDNA present significant challenges to the sensitivity and specificity of the current methylation detection methods. We aim to develop a highly sensitive methylation detection method for fragmented cfDNA that does not require bisulfite conversion. In this study, we combined double restriction enzyme digestion with specific terminal-mediated polymerase chain reaction (STEM-PCR) to establish a cfDNA methylation detection method, namely, the dRE-STEM method. The dRE-STEM method could detect methylated cfDNA on the PCR platform without the need for cumbersome bisulfite conversion. A case-control study was conducted to validate the diagnostic value of colorectal cancer (CRC)-related specific methylated locates using the dRE-STEM method. The dRE-STEM method successfully detected methylation ratios as low as 3% using just 4 ng of cfDNA input, and the best diagnostic model achieved 80.2% (95% CI, 70.6-87.4%) sensitivity and 80.9% (95% CI, 71.9-87.7%) specificity for CRC, significantly outperforming conventional protein markers. The dRE-STEM method offers a promising approach for accurate methylation quantification in low-abundance, highly fragmented cfDNA, making it particularly suitable for routine clinical practice.

Assessment of bacterial viability is vital for food and clinical biosafety, as it reveals pathogen survival and metabolic state, helping predict their potential to persist and cause illness. Herein, we presented a visible light-driven photoelectrochemical assay (termed DzPEC) designed for signal-on detection of live pathogenic bacteria. The DzPEC assay was fabricated using a DNAzyme-functionalized Z-scheme g-C3N4/V2C bio-heterojunction as a photoresposive material and SiO2 as a photoquencher. The DzPEC assay, which utilizes DNAzyme to target the metabolic endoprotein RNase H2 that is secreted into the extracellular matrix, allows for bacterial viability assessment. Leveraging the Z-scheme heterojunction with high photoactive performance, the DzPEC assay for Salmonella enterica (S. enterica), used as a bacteria model, exhibited a detection limit of 141 CFU/mL and the ability to detect live bacterial abundnace as low as 0.1 %. The DzPEC assay demonstrated a strong correlation with RT-qPCR in the detection of S. enterica contamination in complex food and clinical matrices. These findings highlighted the potential of the DzPEC assay in bacterial viability phenotyping and live bacteria-associated biosafety monitoring.

Accurate identification and quantification of 5-hydroxymethylcytosine (5hmC) can help elucidate its function in gene expression and disease pathogenesis. Current 5hmC analysis methods still present challenges, especially for clinical applications, such as having a risk of false-positive results and a lack of sufficient sensitivity. Herein, a 5hmC quantification method for fragment-specific DNA sequences with extreme specificity, high sensitivity, and clinical applicability was established using a quantitative real-time PCR (qPCR)-based workflow through the combination of enzymatic digestion and biological deamination strategy (EDD-5hmC assay). The EDD-5hmC approach enriched glycosylated 5hmC via enzyme digestion and then APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like)-mediated deamination to efficiently differentiate between various cytosine(C) modification states, resulting in 5hmC quantification with extreme specificity such that nonspecific amplification is reduced over eight million-fold. Moreover, the nondestructive biological treatment process of the EDD-5hmC assay exhibits high sensitivity, yielding the limit of detection of 30 aM. For the first time, we measured 5hmC levels in colorectal cancer tissues and matched paracancerous tissues to evaluate the ability to differentiate colorectal cancer, with the area under the receiver operating characteristic curve of up to 82.8% for the single gene of Septin9 and 83.6% for the combinations of Septin9 and Syndecan-2 (SDC2), demonstrating the EDD-5hmC assay is a promising method with clinical applicability for accurately quantifying the 5hmC level.

In-field molecular diagnostics of plant pathogens are critical for crop disease management and precision agriculture, but tools are still lacking. Herein, we present a bioluminescent molecular diagnostic assay capable of detecting viable pathogens directly in minimally processed plant samples, enabling rapid and precise in-field crop disease diagnosis. The assay, called bioluminescent craspase diagnostics (BioCrastics), leverages newly discovered RNA-activated protease of CRISPR (Craspase) with enzymatic luminescence to generate a cascaded amplification, thus bypasses nucleic acid purification and amplification while achieving sub-nanogram sensitivity for fungal pathogens. Using wheat stripe rust as a proof of concept, we demonstrate direct pathogen detection in crude leaf homogenates within 40 min, early identification of infections 6 days prior to symptom emergence. Notably, the assay, via targeting pathogenic RNAs, specifically quantifies viable fungi, overcoming false positives from dead pathogens-a limitation of PCR-based methods that impairs disease risk assessment. Featuring simplified sample processing, portable detection, and species-specific accuracy, BioCrastics establishes a field-deployable tool that bridges the gap between laboratory-level precision and on-farm diagnostic needs for crop disease management.

Foodborne pathogen issue threats human health and remains to be a worldwide challenge. Therefore, rapid, sensitive and point-of-care testing technologies are crucial for food safety and human health. Nanomaterial-based nucleic acid assays hold the promise for meeting the requirements of foodborne pathogen detection. Nucleic acid amplification can generate abundant copies of target genes and high specificty for sequence identification, while nanotechnology allows to transfer the gene information to be readable signals for constructing various biosensors. This review presents nanotechnological nucleic acid biosensors for pathogen detection by the category of nucleic acid amplification techniques and nanomaterials including carbon nanomaterials, metal nanomaterials, magnetic nanomaterials and quantum dots, which provides a cutting-edge solution for monitoring food safety. These approaches achieve sensitive detection of foodborne pathogens, and enable qualitative and quantitative analysis in the complex food matrixes, and hold potential for on-site detection. Challenges and potential solutions in this field are discussed, which is expected to inspire innovation on nanomaterial and biotechnical strategies to improve food safety.