Programming DNA Nanoassemblies into Polyvalent Lysosomal Degraders for Potent Degradation of Pathogenic Membrane Proteins.

Lysosome-targeting chimera (LYTAC) shows great promise for protein-based therapeutics by targeted degradation of disease-associated membrane or extracellular proteins, yet its efficiency is constrained by the limited binding affinity between LYTAC reagents and designated proteins. Here, we established a programmable and multivalent LYTAC system by tandem assembly of DNA into a high-affinity protein degrader, a heterodimer aptamer nanostructure targeting both pathogenic membrane protein and lysosome-targeting receptor (insulin-like growth factor 2 receptor, IGF2R) with adjustable spatial distribution or organization pattern. The DNA-based multivalent LYTACs showed enhanced efficacy in removing immune-checkpoint protein programmable death-ligand 1 (PD-L1) and vascular endothelial growth factor receptor 2 (VEGFR2) in tumor cell membrane that respectively motivated a significant increase in T cell activity and a potent effect on cancer cell growth inhibition. With high programmability and versatility, this multivalent LYTAC system holds considerable promise for realizing protein therapeutics with enhanced activity.

A Methyl-Engineered DNAzyme for Endogenous Alkyltransferase Monitoring and Self-Sufficient Gene Regulation.

The on-demand gene regulation is crucial for extensively exploring specific gene functions and developing personalized gene therapeutics, which shows great promise in precision medicines. Although some nucleic acid-based gene regulatory tools (antisense oligonucleotides and small interfering RNAs) are devised for achieving on-demand activation, the introduction of chemical modifications may cause undesired side effects, thereby impairing the gene regulatory efficacy. Herein, a methyl-engineered DNAzyme (MeDz) is developed for the visualization of endogenous alkyltransferase (AGT) and the simultaneous self-sufficiently on-demand gene regulation. The catalytic activity of DNAzyme can be efficiently blocked by O6-methylguanine (O6MeG) modification and specifically restored via the AGT-mediated DNA-repairing pathway. This simply designed MeDz is demonstrated to reveal AGT of varying expression levels in different cells, opening the possibility to explore the AGT-related biological processes. Moreover, the AGT-guided MeDz exhibits cell-selective regulation on the human early growth response-1 (EGR-1) gene, with efficient gene repression in breast cancer cells and low effectiveness in normal cells. The proposed MeDz offers an attractive strategy for on-demand gene regulation, displaying great potential in biomedical applications.

On-Site Multiply Stimulated Self-Confinement of an Integrated DNA Cascade Circuit for Highly Reliable Intracellular Imaging of miRNA and In Situ Interrogation of the Relevant Regulatory Pathway.

Artificial DNA circuits represent a versatile yet promising toolbox for in situ monitoring and concomitant regulation of diverse biological events within live cells. Nonetheless, their performance is significantly impeded by the diffusion-dominated slow reaction kinetics and the uncontrollable off-target activation. Herein, a self-localized cascade (SLC) circuit is reported for the robust and efficient microRNA (miRNA) analysis in living cells. The SLC circuit consists of the cell-specific localization module and the analyte-specific signal amplification module. By integrating the reaction probes of these two modules, the complexity of the system is reduced to realize the responsive co-localization of circuitry probes and the simultaneous cascade signal amplification. Taking advantage of the specifically activated, self-localized, and cascade design, the SLC circuit successfully achieves the robust miRNA-21 (miR-21) imaging and the accurate cells differentiation. Moreover, the reverse regulation mechanism is successfully explored between messenger RNA (mRNA) and miRNA through the engineered SLC circuit and further elucidates the underlying signaling pathways between them. Therefore, the SLC circuit provides a powerful tool for the sensitive detection of intracellular biomolecules and the study of the corresponding cell regulatory mechanisms.

Programming Fast DNA Amplifier Circuits with Versatile Toehold Exchange Pathway.

DNA amplifier circuits establish powerful tools to dynamically control molecular assembly for computation, sensing, and biological applications. However, the slow reaction speed remains a major barrier to their practical utility. Here, diverse fast DNA amplifier circuits termed toehold exchange polymerization (TEP) and toehold exchange catalysis (TEC) using toehold exchange-mediated assembly as a fundamental mechanism are built. Both TEP and TEC with a duplex and a hairpin can respond within minutes to diverse nucleic acid inputs with high fidelity. In addition, the circuits can amplify live-cell signals for fluorescence imaging target RNA dynamics and discriminating different cell lines. Compared with existing DNA circuits that involve time scales of hours for transducing small signals, TEP and TEC exhibit much faster dynamics, simpler design, and comparable sensitivity. These features make TEP and TEC promising platforms to develop programmable nucleic acid tools and devices and to create fast sensing and processing systems, amenable to wide practical applications.

Enpp1 mutations promote upregulation of hedgehog signaling in heterotopic ossification with aging.

INTRODUCTION: Heterotopic ossification of the tendon and ligament (HOTL) is a chronic progressive disease that is usually accompanied by thickening and ossification of ligaments and high osteogenic activity of the surrounding ligament tissue. However, the molecular mechanism of maintaining the cellular phenotype of HOTL remains unclear. MATERIALS AND METHODS: We first constructed a model of HOTL, Enpp1flox/flox/EIIa-Cre mice, a novel genetic mouse system. Imaging, histological, and cell-level analyses were performed to investigate the progressive ossification of the posterior longitudinal ligament, Achilles tendons, and degeneration joints caused by Enpp1 deficiency. RESULTS: The results indicate that Enpp1 deficiency led to markedly progressive heterotopic ossification (HO), especially spine, and Achilles tendons, and was associated with progressive degeneration of the knees. The bone mass was decreased in the long bone. Furthermore, fibroblasts from Enpp1flox/flox/EIIa-Cre mice had greater osteogenic differentiation potential following induction by osteogenesis, accompanied by enhanced hedgehog (Hh) signaling. In addition, fibroblast cells show senescence, and aggravation of the senescence phenotype by further osteogenic induction. CONCLUSION: Our study indicated that with increasing age, mutations in Enpp1 promote ectopic ossification of spinal ligaments and endochondral ossification in tendons and further aggravate knee degeneration by upregulating hedgehog signaling.

Hook-Like DNAzyme-Activated Autocatalytic Biosensor for the Universal Detection of Pathogenic Bacteria.

DNAzyme-based assays have found extensive utility in pathogenic bacteria detection but often suffer from limited sensitivity and specificity. The integration of a signal amplification strategy could address this challenge, while the existing combination methods require extensive modification to accommodate various DNAzymes, limiting the wide-spectrum bacteria detection. We introduced a novel hook-like DNAzyme-activated autocatalytic nucleic acid circuit for universal pathogenic bacteria detection. The hook-like connector DNA was employed to seamlessly integrate the recognition element DNAzyme with the isothermal enzyme-free autocatalytic hybridization chain reaction and catalytic hairpin assembly for robust exponential signal amplification. This innovative autocatalytic circuit substantially amplifies the output signals from the DNAzyme recognition module, effectively overcoming DNAzyme's inherent sensitivity constraints in pathogen identification. The biosensor exhibits a strong linear response within a range of 1.5 × 103 to 3.7 × 107 CFU/mL, achieving a detection limit of 1.3 × 103 CFU/mL. Noted that the sensor's adaptability as a universal detection platform is established by simply modifying the hook-like connector module, enabling the detection of various pathogenic bacteria of considerable public health importance reported by the World Health Organization, including Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Salmonella typhimurium. Additionally, the specificity of DNAzyme in bacterial detection is markedly improved due to the signal amplification process of the autocatalytic circuit. This hook-like DNAzyme-activated autocatalytic platform presents a versatile, sensitive, and specific approach for pathogenic bacteria detection, promising to significantly expand the applications of DNAzyme in bacteria detection.

Development of a mouse model of chronic ventral spinal cord compression: Neurobehavioral, radiological, and pathological changes.

Objectives: The main objective of this study was to establish a mouse model of spinal ligament ossification to simulate the chronic spinal cord compression observed in patients with ossification of the posterior longitudinal ligament (OPLL). The study also aimed to examine the mice's neurobiological, radiological, and pathological changes. Methods: In the previous study, a genetically modified mouse strain was created using Crispr-Cas9 technology, namely, Enpp1 flox/flox /EIIa-Cre (C57/B6 background), to establish the OPLL model. Wild-type (WT) mice without compression were used as controls. Functional deficits were evaluated through motor score assessment, inclined plate testing, and gait analysis. The extent of compression was determined using CT imaging. Hematoxylin and eosin staining, luxol fast blue staining, TUNEL assay, immunofluorescence staining, qPCR, and Western blotting were performed to evaluate levels of apoptosis, inflammation, vascularization, and demyelination in the study. Results: The results demonstrated a gradual deterioration of compression in the Enpp1 flox/flox /EIIa-Cre mice group as they aged. The progression rate was more rapid between 12 and 20 weeks, followed by a gradual stabilization between 20 and 28 weeks. The scores for spinal cord function and strength, assessed using the Basso Mouse Scale and inclined plate test, showed a significant decline. Gait analysis revealed a noticeable reduction in fore and hind stride lengths, stride width, and toe spread. Chronic spinal cord compression resulted in neuronal damage and activated astrocytes and microglia in the gray matter and anterior horn. Progressive posterior cervical compression impeded blood supply, leading to inflammation and Fas-mediated neuronal apoptosis. The activation of Bcl2 and Caspase 3 was associated with the development of progressive neurological deficits (p < 0.05). Conclusions: The study presents a validated model of chronic spinal cord compression, enabling researchers to explore clinically relevant therapeutic approaches for OPLL.

Endogenous Glutathione-Activated Nucleic Acid Molecular Circuitry for Cell-Specific MicroRNA Imaging.

Sensitive and reliable microRNA imaging in living cells has significant implications for clinical diagnosis and monitoring. Catalytic DNA circuits have emerged as potent tools for tracking these intracellular biomarkers and probing the corresponding biochemical processes. However, their utility is hindered by the low resistance to external interference, leading to undesired off-site activation and consequent signal leakage. Therefore, achieving the endogenous control of the DNA circuit's activation is preferable to the reliable target analysis in living cells. In this study, we attempted to address this challenge by engineering a simple yet effective endogenous glutathione (GSH)-regulated hybridization chain reaction (HCR) circuit for acquiring high-contrast miRNA imaging. Initially, the HCR hairpin reactants were blocked by the engineered disulfide-integrated DNA duplex, thus effectively passivating their sensing function. And the precaged HCR hairpin was liberated by the cell-specific GSH molecule, thus initiating the HCR system for selectively amplified detection of microRNA-21 (miR-21). This approach prevented unwanted signal leakage before exposure into target cells, thus ensuring robust miR-21 imaging with high accuracy and reliability in specific tumor cells. Moreover, the endogenously responsive HCR circuit established a link between the small regulatory factors and miRNA, thereby enhancing the signal gain. In summary, the endogenously activatable DNA circuit represents a versatile toolbox for robust bioanalysis and exploration of potential signaling pathways in living cells.

A Methylation-Gated DNAzyme Circuit for Spatially Controlled Imaging of MicroRNA in Cells and Animals.

Epigenetic modification plays an indispensable role in regulating routine molecular signaling pathways, yet it is rarely used to modulate molecular self-assembly networks. Herein, we constructed a bioorthogonal demethylase-stimulated DNA circuitry (DSC) system for high-fidelity imaging of microRNA (miRNA) in live cells and mice by eliminating undesired off-site signal leakage. The simple and robust DSC system is composed of a primary cell-specific circuitry regulation (CR) module and an ultimate signal-transducing amplifier (SA) module. After the modularly designed DSC system was delivered into target live cells, the DNAzyme of the CR module was site-specifically activated by endogenous demethylase to produce fuel strands for the subsequent miRNA-targeting SA module. Through the on-site and multiply guaranteed molecular recognitions, the lucid yet efficient DSC system realized the reliably amplified in vivo miRNA sensing and enabled the in-depth exploration of the demethylase-involved signal pathway with miRNA in live cells. Our bioorthogonally on-site-activated DSC system represents a universal and versatile biomolecular sensing platform via various demethylase regulations and shows more prospects for more different personalized theragnostics.

Construction of Multiply Guaranteed DNA Sensors for Biological Sensing and Bioimaging Applications.

Nucleic acids exhibit exceptional functionalities for both molecular recognition and catalysis, along with the capability of predictable assembly through strand displacement reactions. The inherent programmability and addressability of DNA probes enable their precise, on-demand assembly and accurate execution of hybridization, significantly enhancing target detection capabilities. Decades of research in DNA nanotechnology have led to advances in the structural design of functional DNA probes, resulting in increasingly sensitive and robust DNA sensors. Moreover, increasing attention has been devoted to enhancing the accuracy and sensitivity of DNA-based biosensors by integrating multiple sensing procedures. In this review, we summarize various strategies aimed at enhancing the accuracy of DNA sensors. These strategies involve multiple guarantee procedures, utilizing dual signal output mechanisms, and implementing sequential regulation methods. Our goal is to provide new insights into the development of more accurate DNA sensors, ultimately facilitating their widespread application in clinical diagnostics and assessment.

A causal examination of the correlation between hormonal and reproductive factors and low back pain.

Background: The relationship between hormonal fluctuations in the reproductive system and the occurrence of low back pain (LBP) has been widely observed. However, the causal impact of specific variables that may be indicative of hormonal and reproductive factors, such as age at menopause (ANM), age at menarche (AAM), length of menstrual cycle (LMC), age at first birth (AFB), age at last live birth (ALB) and age first had sexual intercourse (AFS) on low back pain remains unclear. Methods: This study employed Bidirectional Mendelian randomization (MR) using publicly available summary statistics from Genome Wide Association Studies (GWAS) and FinnGen Consortium to investigate the causal links between hormonal and reproductive factors on LBP. Various MR methodologies, including inverse-variance weighted (IVW), MR-Egger regression, and weighted median, were utilized. Sensitivity analysis was conducted to ensure the robustness and validity of the findings. Subsequently, Multivariate Mendelian randomization (MVMR) was employed to assess the direct causal impact of reproductive and hormone factors on the risk of LBP. Results: After implementing the Bonferroni correction and conducting rigorous quality control, the results from MR indicated a noteworthy association between a decreased risk of LBP and AAM (OR=0.784, 95% CI: 0.689-0.891; p=3.53E-04), AFB (OR=0.558, 95% CI: 0.436-0.715; p=8.97E-06), ALB (OR=0.396, 95% CI: 0.226-0.692; p=0.002), and AFS (OR=0.602, 95% CI: 0.518-0.700; p=3.47E-10). Moreover, in the reverse MR analysis, we observed no significant causal effects of LBP on ANM, AAM, LMC and AFS. MVMR analysis demonstrated the continued significance of the causal effect of AFB on LBP after adjusting for BMI. Conclusion: Our study explored the causal relationship between ANM, AAM, LMC, AFB, AFS, ALB and the prevalence of LBP. We found that early menarche, early age at first birth, early age at last live birth and early age first had sexual intercourse may decrease the risk of LBP. These insights enhance our understanding of LBP risk factors, offering valuable guidance for screening, prevention, and treatment strategies for at-risk women.

Scaling up of a Self-Confined Catalytic Hybridization Circuit for Robust microRNA Imaging.

The precise regulation of cellular behaviors within a confined, crowded intracellular environment is highly amenable in diagnostics and therapeutics. While synthetic circuitry system through a concatenated chemical reaction network has rarely been reported to mimic dynamic self-assembly system. Herein, a catalytic self-defined circuit (CSC) for the hierarchically concatenated assembly of DNA domino nanostructures is engineered. By incorporating pre-sealed symmetrical fragments into the preying hairpin reactants, the CSC system allows the hierarchical DNA self-assembly via a microRNA (miRNA)-powered self-sorting catalytic hybridization reaction. With minimal strand complexity, this self-sustainable CSC system streamlined the circuit component and achieved localization-intensified cascaded signal amplification. Profiting from the self-adaptively concatenated hybridization reaction, a reliable and robust method has been achieved for discriminating carcinoma tissues from the corresponding para-carcinoma tissues. The CSC-sustained self-assembly strategy provides a comprehensive and smart toolbox for organizing various hierarchical DNA nanostructures, which may facilitate more insights for clinical diagnosis and therapeutic assessment.

Multiply Guaranteed Catalytic DNA Circuit for Cancer-Cell-Selective Imaging of miRNA and Robust Evaluation of Drug Resistance.

Catalytic DNA circuits are desirable for sensitive bioimaging in living cells; yet, it remains a challenge to monitor these intricate signal communications because of the uncontrolled circuitry leakage and insufficient cell selectivity. Herein, a simple yet powerful DNA-repairing enzyme (APE1) activation strategy is introduced to achieve the site-specific exposure of a catalytic DNA circuit for realizing the selectively amplified imaging of intracellular microRNA and robust evaluation of the APE1-involved drug resistance. Specifically, the circuitry reactants are firmly blocked by the enzyme recognition/cleavage site to prevent undesirable off-site circuitry leakage. The caged DNA circuit has no target-sensing activity until its circuitry components are activated via the enzyme-mediated structural reconstitution and finally transduces the amplified fluorescence signal within the miRNA stimulation. The designed DNA circuit demonstrates an enhanced signal-to-background ratio of miRNA assay as compared with the conventional DNA circuit and enables the cancer-cell-selective imaging of miRNA. In addition, it shows robust sensing performance in visualizing the APE1-mediated chemoresistance in living cells, which is anticipated to achieve in-depth clinical diagnosis and chemotherapy research.

Genetically proxied PCSK9 inhibition is associated with reduced psoriatic arthritis risk.

BACKGROUND: Lipid pathways play a crucial role in psoriatic arthritis development, and some lipid-lowering drugs are believed to have therapeutic benefits due to their anti-inflammatory properties. Traditional observational studies face issues with confounding factors, complicating the interpretation of causality. This study seeks to determine the genetic link between these medications and the risk of psoriatic arthritis. METHODS: This drug target study utilized the Mendelian randomization strategy. We harnessed high-quality data from population-level genome-wide association studies sourced from the UK Biobank and FinnGen databases. The inverse variance-weighted method, complemented by robust pleiotropy methods, was employed. We examined the causal relationships between three lipid-lowering agents and psoriatic arthritis to unveil the underlying mechanisms. RESULTS: A significant association was observed between genetically represented proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition and a decreased risk of psoriatic arthritis (odds ratio [OR]: 0.51; 95% CI 0.14-0.88; P < 0.01). This association was further corroborated in an independent dataset (OR 0.60; 95% CI 0.25-0.94; P = 0.03). Sensitivity analyses affirmed the absence of statistical evidence for pleiotropic or genetic confounding biases. However, no substantial associations were identified for either 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors or Niemann-Pick C1-like 1 inhibitors. CONCLUSIONS: This Mendelian randomization analysis underscores the pivotal role of PCSK9 in the etiology of psoriatic arthritis. Inhibition of PCSK9 is associated with reduced psoriatic arthritis risk, highlighting the potential therapeutic benefits of existing PCSK9 inhibitors.

Transarterial Chemoembolization Plus Tyrosinkinase Inhibitors and PD-1 Inhibitors for Spontaneously Ruptured Hepatocellular Carcinoma.

PURPOSE: To compare the efficacy and safety of transcatheter arterial chemoembolization (TACE) in combination with tyrosinkinase inhibitors (TKI) and PD-1 inhibitors, versus TACE monotherapy for the treatment of ruptured hepatocellular carcinoma (HCC). MATERIALS AND METHODS: This study included 104 patients with ruptured HCC receiving either combination therapy or TACE monotherapy at two centers between June 2015 and June 2022. Propensity score matching (PSM) analysis was used at a 1:2 ratio to reduce bias between the two groups. The primary outcome measures were overall survival (OS) and progression-free survival (PFS), and the secondary outcome measures were the occurrence of adverse events (AEs, Common Terminology Criteria for AEs, version 5.0.) and the peritoneal metastasis rate. RESULTS: A total of 69 patients were enrolled after PSM, including 23 patients in the combination group and 46 patients in the monotherapy group. The combination group exhibited a significantly longer median OS (553 days, 95% confidence interval [CI] 222.6-883.9) compared to the monotherapy group (105 days, 95% CI 81.2-128.7; P < 0.001). Similarly, the combination group showed a better median PFS (356 days, 95% CI 299.5-412.4) compared to the monotherapy group (97 days, 95% CI 75.9-118.1; P < 0.001). Moreover, there was no significant difference in the peritoneal metastasis rate (combination group: 8.6% vs. monotherapy group: 26.1%, P = 0.119). Grade 3 AEs occurred at a rate of 21.7% and 13% in combination and monotherapy groups, respectively. No Grade 4/5 AEs were observed in either group. CONCLUSIONS: Our study demonstrated that the combination of TACE with TKI and PD-1 inhibitors significantly enhances OS and PFS compared to TACE monotherapy in ruptured HCC patients. Furthermore, this combined approach exhibited an acceptable safety profile.

A Programmable DNAzyme for the Sensitive Detection of Nucleic Acids.

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.

Enzyme-Free Dynamic DNA Reaction Networks for On-Demand Bioanalysis and Bioimaging.

ConspectusThe pursuit of in-depth studying the nature and law of life activity has been dominating current research fields, ranging from fundamental biological studies to applications that concern synthetic biology, bioanalysis, and clinical diagnosis. Motivated by this intention, the spatiotemporally controlled and in situ analysis of living cells has been a prospective branch by virtue of high-sensitivity imaging of key biomolecules, such as biomarkers. The past decades have attested that deoxyribonucleic acid (DNA), with biocompatibility, programmability, and customizable features, is a competitive biomaterial for constructing high-performance molecular sensing tools. To conquer the complexity of the wide extracellular-intracellular distribution of biomarkers, it is a meaningful breakthrough to explore high-efficiently amplified DNA circuits, which excel at operating complex yet captivating dynamic reaction networks for various bioapplications. In parallel, the multidimensional performance improvements of nucleic acid circuits, including the availability, detection sensitivity, and reliability, are critical parameters for realizing accurate imaging and cell regulation in bioanalysis.In this Account, we summarize our recent work on enzyme-free dynamic DNA reaction networks for bioanalysis from three main aspects: DNA circuitry functional extension of molecular recognition for epigenetic analysis and regulation, DNA circuitry amplification ability improvement for sensitive biomarker detection, and site-specific activation of DNA circuitry systems for reliable and accurate cell imaging. In the first part, we have designed an epigenetically responsive deoxyribozyme (DNAzyme) circuitry system for intracellular imaging and gene regulation, which enriches the possible analyzed species by chemically modifying conventional DNAzyme. For example, an exquisite N6-methyladenine (m6A)-caged DNAzyme was built for achieving the precise FTO (fat mass and obesity-associated protein)-directed gene regulation. In addition, varieties of DNAzyme-based nanoplatforms with self-sufficient cofactor suppliers were assembled, which subdued the speed-limiting hardness of DNAzyme cofactors in live-cell applications. In the second part, we have developed a series of hierarchically assembled DNA circuitry systems to improve the signal transduction ability of traditional DNA circuits. First, the amplification ability of the DNAzyme circuit has been significantly enhanced via several heterogeneously or homogeneously concatenated circuitry models. Furthermore, a feedback reaction pathway was integrated into these concatenated circuits, thus dramatically increasing the amplification efficiency. Second, considering the complex cellular environment, we have simplified the redundancy of multicomponents or reaction procedures of traditional cascaded circuits, relying on the minimal component complexity and merely one modular catalytic reaction, which guaranteed high cell-delivering uniformity while fostering reaction kinetics and analysis reliability. In the third part, we have constructed in-cell-selective endogenous-stimulated DNA circuitry systems via the multiply guaranteed molecular recognitions, which could not only eliminate the signal leakage, but could also retain its on-site and multiplex signal amplification. Based on the site-specific activation strategy, more circuitry availability in cellular scenarios has been acquired for reliable and precise biological sensing and regulation. These enzyme-free dynamic DNA reaction networks demonstrate the purpose-to-concreteness engineering for tailored multimolecule recognition and multiple signal amplification, achieving high-gain signal transduction and high-reliability targeted imaging in bioanalysis. We envision that the enzyme-free dynamic DNA reaction network can contribute to more bioanalytical layouts, which will facilitate the progression of clinical diagnosis and prognosis.

Spatiotemporally Programmed Disassembly of Multifunctional Integrated DNAzyme Nanoplatfrom for Amplified Intracellular MicroRNA Imaging.

The sensing performance of DNAzymes in live cells is tremendously hampered by the inefficient and inhomogeneous delivery of DNAzyme probes and their incontrollable off-site activation, originating from their susceptibility to nuclease digestion. This requires the development of a more compact and robust DNAzyme-delivering system with site-specific DNAzyme activation property. Herein, a highly compact and robust Zn@DDz nanoplatform is constructed by integrating the unimolecular microRNA-responsive DNA-cleaving DNAzyme (DDz) probe with the requisite DNAzyme Zn2+ -ion cofactors, and the amplified intracellular imaging of microRNA via the spatiotemporally programmed disassembly of Zn@DDz nanoparticles is achieved. The multifunctional Zn@DDz nanoplatform is simply composed of a structurally blocked self-hydrolysis DDz probe and the inorganic Zn2+ -ion bridge, with high loading capacity, and can effectively deliver the initially catalytic inert DDz probe and Zn2+ into living cells with enhanced stabilities. Upon their entry into the acidic microenvironment of living cells, the self-sufficient Zn@DDz nanoparticle is disassembled to release DDz probe and simultaneously supply Zn2+ -ion cofactors. Then, endogenous microRNA-21 catalyzes the reconfiguration and activation of DDz for generating the amplified readout signal with multiply guaranteed imaging performance. Thus, this work paves an effective way for promoting DNAzyme-based biosensing systems in living cells, and shows great promise in clinical diagnosis.

Rational Engineering of a Multifunctional DNA Assembly for Enhanced Antibacterial Efficacy and Accelerated Wound Healing.

DNA-based assemblies hold immense prospects for antibacterial application, yet are constrained by their poor specificity and deficient antibacterial delivery. Herein, the fabrication of a versatile rolling circle amplification (RCA)-sustained DNA assembly is reported, encoding simultaneously with multivalent aptamers and tandem antibacterial agents, for target-specific and efficient antibacterial application. In the compact RCA-sustained antibacterial platform, the facilely organized multivalent aptamers guarantee the target bacteria-specific delivery of sufficient antibacterial agents which is assembled through DNA-stabilizing silver nanostructures. It is shown that the biocompatible DNA system could enhance bacteria elimination and simultaneously facilitate wound healing in vivo. By virtue of the programmable RCA assembly, the present RCA-sustained system provides a highly modular and scalable approach to design versatile multifunctional therapeutic systems.

Engineering of a Self-Regulatory Bidirectional DNA Assembly Circuit for Amplified MicroRNA Imaging.

The engineering of catalytic hybridization DNA circuits represents versatile ways to orchestrate a complex flux of molecular information at the nanoscale, with potential applications in DNA-encoded biosensing, drug discovery, and therapeutics. However, the diffusive escape of intermediates and unintentional binding interactions remain an unsolved challenge. Herein, we developed a compact, yet efficient, self-regulatory assembly circuit (SAC) for achieving robust microRNA (miRNA) imaging in live cells through DNA-templated guaranteed catalytic hybridization. By integrating the toehold strand with a preblocked palindromic fragment in the stem domain, the proposed miniature SAC system allows the reactant-to-template-controlled proximal hybridization, thus facilitating the bidirectional-sustained assembly and the localization-intensified signal amplification without undesired crosstalk. With condensed components and low reactant complexity, the SAC amplifier realized high-contrast intracellular miRNA imaging. We anticipate that this simple and template-controlled design can enrich the clinical diagnosis and prognosis toolbox.