Simulation-Guided Rational Design of DNA Walker-Based Theranostic Platform.

Biomolecule-functionalized nanoparticles represent a type of promising biomaterials in biomedical applications owing to their excellent biocompatibility and versatility. DNA-based reactions on nanoparticles have enabled emerging applications including intelligent biosensors, drug delivery, and biomimetic devices. Among the reactions, strand hybridization is the critical step to control the sensitivity and specificity of biosensing, and the efficiency of drug delivery. However, a comprehensive understanding of DNA hybridization on nanoparticles is still lacking, which may differ from the process in homogeneous solutions. To address this limitation, coarse-grained model-based molecular dynamic simulation is harnessed to disclose the critical factors involved in intermolecular hybridization. Based on simulation guidance, DNA walker-based smart theranostic platform (DWTP) based on "on-particle" hybridization is developed, showing excellent consistency with simulation. DWTP is successfully applied for highly sensitive miRNA 21 detection and tumor-specific miRNA 21 imaging, driven by tumor-endogenous APE 1 enzyme. It enables the precise release of antisense oligonucleotide triggered by tumor-endogenous dual-switch miRNA 21 and APE 1, facilitating effective gene silencing therapy with high biosafety. The simulation of "on-particle" DNA hybridization has improved the corresponding biosensing performance and the release efficiency of therapeutic agents, representing a conceptually new approach for DNA-based device design.

A cascade signal-amplified fluorescent biosensor combining APE1 enzyme cleavage-assisted target cycling with rolling circle amplification.

A cascade signal-amplified fluorescent biosensor was developed for miRNA-21 detection by combining APE1 enzyme-assisted target recycling and rolling circle amplification strategy. A key feature of this biosensor is its dual-trigger mechanism, utilizing both tumor-endogenous miRNA-21 and the APE1 enzyme in the initial amplification step, followed by a second rolling circle amplification reaction. This dual signal amplification cascade significantly enhanced sensitivity, achieving a detection limit of 3.33 pM. Furthermore, this biosensor exhibited excellent specificity and resistance to interference, allowing it to effectively distinguish and detect the target miRNA-21 in the presence of multiple interfering miRNAs. Moreover, the biosensor maintained its robust detection capabilities in a 10% serum environment, demonstrating its potential for clinical disease diagnosis applications.

Increased serum levels of sCD40L were associated with rapidly progressive interstitial lung disease in idiopathic inflammatory myopathies.

OBJECTIVES: Whether coagulopathy exists in development of idiopathic inflammatory myopathies associated rapidly progressive interstitial lung disease (IIMs-RPILD) is unclear. In this study, we aimed to investigate soluble CD40 ligand and D-dimer levels in RPILD patients. METHODS: Patients with IIMs-ILD were enrolled and classified as RPILD and stable-ILD group. Clinical data, laboratory examinations including coagulation-associated parameters and the myositis antibodies status, chest high-resolution computed tomography (HRCT) findings and treatment regimens were collected and serum levels of sCD40L were detected by ELISA. Univariable and adjusted multivariable cox regression were performed to identify risk factors for 6-month mortality, and further to select predictors for establishing predictive model for RPILD. RESULTS: Eighty patients with IIMs-ILD were enrolled and 34 of them were diagnosed as RPILD while 46 as stable-ILD. Multivariable cox regression showed that albumin<32.4 g/L and sCD40L<1658.55 pg/ml were independent risk factors of short-term mortality in RPILD. A SMAD model consisting of serum sCD40L>1054 pg/ml, anti-MDA5 positivity, albumin<32.4 g/L and D-dimer>0.865 mg/L were generated. The odds for RPILD with SMAD score of 0, 1, 2, 3 and 4 were 0, 26.9%, 66.7%, 91.7% and 100%. The 6-month survival stratified by mild (SMAD score 0), moderate (SMAD score 1 and 2) and severe group (SMAD score 3 and 4) were 100%, 79.5% and 20%, respectively. CONCLUSIONS: We established a predictive model for IIMs-RPILD, which provided a clue that coagulopathy might exist in IIMs-RPILD and could help to better treat patients with RPILD. This model awaits further validations.

Anti-Ro52 antibodies are associated with the prognosis of adult idiopathic inflammatory myopathy-associated interstitial lung disease.

OBJECTIVES: In the present study, we aimed to assess the prevalence and clinical significance of anti-Ro52 antibodies in a cohort of patients with idiopathic inflammatory myopathy-associated interstitial lung disease (IIM-ILD) with different myositis-specific autoantibodies (MSAs). METHODS: A cohort of 267 IIM-ILD patients, including 62 patients with PM, 126 patients with DM and 79 patients with clinically amyopathic DM (CADM) were retrospectively analysed in this study. Clinical and laboratory findings, pulmonary function tests (PFTs), HRCT patterns and treatment information were compared between patients with and without anti-Ro52 antibodies. The association between prognosis and anti-Ro52 antibodies was also evaluated based on different MSA subgroups. RESULTS: Anti-Ro52 antibodies were more frequent in patients with anti-MDA5 (62.1%, P < 0.01) and anti-Jo1 (64.9%, P < 0.01) antibodies than in those with other MSAs. The proportion of patients with anti-Jo1 antibodies was higher in the anti-Ro52 antibody-positive group than in the anti-Ro52 antibody-negative group. Patients with anti-Ro52 antibodies were more likely to exhibit the Gottron sign than the anti-Ro52 antibody-negative group (P < 0.001). Furthermore, it was a predictive factor for rapid progression interstitial lung disease (RP-ILD) (P = 0.001) and was also associated with a higher mortality rate (log-rank test, P = 0.001). Furthermore, RP-ILD was more frequently exhibited in anti-MDA5- and anti-Ro52-positive patients. Moreover, anti-Ro52 antibody positivity was closely associated with a higher mortality rate in anti-MDA5-ILD patients (log-rank test, P < 0.05). CONCLUSIONS: Anti-Ro52 antibodies were highly prevalent in patients with anti-MDA5 and anti-Jo1 antibodies. Within all patients with IIM-ILD, those with anti-Ro52 autoantibodies had a higher frequency of RP-ILD and a poorer prognosis, especially in the anti-MDA5 antibody subgroup.

Cytokeratin 19 fragment is associated with severity and poor prognosis of interstitial lung disease in anti-MDA5 antibody-positive dermatomyositis.

OBJECTIVES: In the present study, we aimed to assess the clinical significance of cytokeratin 19 fragment (CYFRA21-1) in patients with anti-melanoma differentiation-associated gene 5 (MDA5) antibody-positive DM-interstitial lung disease (MDA5-DM-ILD). METHODS: A total of 73 MDA5-DM-ILD patients were retrospectively analysed in this work. Their clinical characteristics, including clinical manifestations, laboratory findings, peripheral blood lymphocyte subsets and lung function, were compared between patients with acute/subacute interstitial pneumonia (A/SIP) and chronic interstitial pneumonia (CIP). The level of serum CYFRA21-1 was also compared between the above-mentioned two groups of patients, and its association with the clinical features and mortality of MDA5-DM-ILD was also evaluated. RESULTS: Of the 73 MDA5-DM-ILD patients, 26 patients exhibited the A/SIP pattern. The level of serum CYFRA21-1 was higher in MDA5-DM patients with A/SIP compared with the CIP group (P = 0.009). Lower oxygenation index (OI), CD3+CD4+ T cell counts and percentage of CD3+CD4+ cells were also observed in MDA5-DM patients with A/SIP compared with the CIP group. Higher serum CYFRA21-1, lower OI, and lower zone consolidation were associated with a higher risk of A/SIP in MDA5-DM-ILD. In addition, 38 decedents with MDA5-DM-ILD exhibited a greater level of CYFRA21-1 compared with 35 survivors (P < 0.001). Furthermore, it was a prognostic factor and also associated with a higher mortality rate (log-rank test, P < 0.001). CONCLUSIONS: CYFRA21-1 could be a useful serum indicator associated with occurrence of A/SIP in MDA5-DM-ILD. Moreover, it was associated with a poor survival in MDA5-DM-ILD patients.

The circular RNA circ_GRHPR promotes NSCLC cell proliferation and invasion via interactions with the RNA-binding protein PCBP2.

As the most common malignancy, lung cancer is characterised by high rates of occurrence and mortality. Although circular RNAs (circRNAs) are known to act as important regulators in cancer, their role in lung cancer remains poorly understood. In this study, circ_GRHPR expression was found to be significantly upregulated in the serum of five patients with non-small cell lung cancer (NSCLC), compared to that in healthy controls. It is expressed at high levels in NSCLC cell lines, as revealed by qRT-PCR analysis. Functionally, we demonstrated that circ_GRHPR promotes NSCLC proliferation and invasion in vitro and in vivo by cell proliferation, transwell, cell cycle, and tumour-forming assays. Mechanistically, RNA pull-down and RNA immunoprecipitation assays showed that circ_GRHPR interacts with the RNA-binding protein poly(rC)-binding protein 2 (PCBP2) and regulates its subcellular localisation by forming the circ_GRHPR/PCBP2 complex, localizing PCBP2 mainly in the cytoplasm and reducing the proportion found in the nucleus. Furthermore, we demonstrated that four-and-a-half LIM-only protein 3 (FHL3) is a tumour-stimulating factor in NSCLC that interacts with and is influenced by PCBP2. Circ_GRHPR increased FHL3 expression in the nucleus of NSCLC cells by decreasing PCBP2 expression therein and promoting the proliferation and invasion of NSCLC cells. Therefore, our study identified that circ_GRHPR promotes NSCLC proliferation and invasion, providing a possible explanation for its mechanism of action.

DNA Origami Guided Self-Assembly of Plasmonic Polymers with Robust Long-Range Plasmonic Resonance.

Plasmonic polymers consisting of metallic nanoparticles (NPs) are able to squeeze light into the deep-subwavelength space and transfer along a highly confined nanoscale path in long range. DNA nanotechnology, particularly benefiting from the molecular programmability of DNA origami, has provided otherwise nearly impossible platforms for constructing plasmonic nanoparticle polymers with designer configurations and nanoscale gaps. Here, we design and assemble a DNA origami hashtag tile that is able to polymerize into one-dimensional chains with high rigidity. The DNA origami hashtag chains are used as frames to enable robust, versatile, and precise arrangement of metallic NPs into micrometer-long chiral and magnetic plasmonic polymers, which are capable of efficiently transporting plasmonic angular momentum and magnetic surface plasmonic polaritons at the deep-subwavelength scale. Our work provides a molecular platform for the fabrication of long, straight, and structurally complex nanoparticle polymers with emerging plasmonic properties that are appealing to a variety of fields.

Immunosuppressive and metabolic agents that influence allo- and xenograft survival by in vivo expansion of T regulatory cells.

The transplanted organs or cells survive if the recipient receives adequate long-term immunosuppressive therapy. Immunosuppressive therapy combined with cell-based strategies (eg, regulatory T cell [Treg]-based therapy) promotes graft survival. A combination of Treg-based therapy and minimal or no immunosuppressive drug therapy would have the potential to minimize the risks of the complications and side effects of these drugs. Fortunately, some immunosuppressive and other agents not only impede the effector T cell response, but also help generate new CD4+ Tregs from conventional effector T cells. These agents include IL-2, TGF-ß, agents that block the CD40/CD40L costimulation pathway, mTOR inhibitors, and histone deacetylase inhibitors. Consequently, a state of relative unresponsiveness to the transplanted organ may be induced through the expansion of Tregs. We here review the effect of these various agents on expansion of CD4+ Tregs in allo- and xenotransplantation. The expansion of Tregs might allow a dose reduction of the standard immunosuppressive drugs.

Programming the Nucleation of DNA Brick Self-Assembly with a Seeding Strand.

Recently, the DNA brick strategy has provided a highly modular and scalable approach for the construction of complex structures, which can be used as nanoscale pegboards for the precise organization of molecules and nanoparticles for many applications. Despite the dramatic increase of structural complexity provided by the DNA brick method, the assembly pathways are still poorly understood. Herein, we introduce a "seed" strand to control the crucial nucleation and assembly pathway in DNA brick assembly. Through experimental studies and computer simulations, we successfully demonstrate that the regulation of the assembly pathways through seeded growth can accelerate the assembly kinetics and increase the optimal temperature by circa 4-7 °C for isothermal assembly. By improving our understanding of the assembly pathways, we provide new guidelines for the design of programmable pathways to improve the self-assembly of DNA nanostructures.

Programming DNA Tube Circumference by Tile Offset Connection.

DNA tubes with prescribed circumferences are appealing for numerous multidisciplinary applications. The DNA single-stranded tiles (SSTs) assembly method has demonstrated an unprecedented capability for programming the circumferences of DNA tubes in a modular fashion. Nevertheless, a distinct set of SSTs is typically required to assemble DNA tube of a specific circumference, with wider tubes requiring higher numbers of tiles of unique sequences, which not only increases the expense and design complexity but also hampers the assembly yield. Herein, we introduce "offset connection" to circumvent such challenges in conventional SST tube assembly. In this new connection scheme, the boundary SST tiles in an SST array are designed to connect in an offset manner. To compensate for the offset, the SST array has to grow wider until the array can close to form a wide tube with a tolerable degree of twist. Using this strategy, we have successfully assembled DNA tubes with prescribed circumferences consisting of 8, 12, 14, 16, 20, 24, 28, 32, 36, 42, 56, or 70 helices from two distinct sets of SSTs composed of 19×4 or 19×14 tiles.

Dynamic DNA Structures.

Dynamic DNA structures, a type of DNA construct built using programmable DNA self-assembly, have the capability to reconfigure their conformations in response to environmental stimulation. A general strategy to design dynamic DNA structures is to integrate reconfigurable elements into conventional static DNA structures that may be assembled from a variety of methods including DNA origami and DNA tiles. Commonly used reconfigurable elements range from strand displacement reactions, special structural motifs, target-binding DNA aptamers, and base stacking components, to DNA conformational change domains, etc. Morphological changes of dynamic DNA structures may be visualized by imaging techniques or may be translated to other detectable readout signals (e.g., fluorescence). Owing to their programmable capability of recognizing environmental cues with high specificity, dynamic DNA structures embody the epitome of robust and versatile systems that hold great promise in sensing and imaging biological analytes, in delivering molecular cargos, and in building programmable systems that are able to conduct sophisticated tasks.

Quantitative proteomic characterization of lung tissue in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive, eventually fatal disease. IPF is characterized by excessive accumulation of the extracellular matrix (ECM) in the alveolar parenchyma and progressive lung scarring. The pathogenesis of IPF and whether the ECM involved in the process remain unknown. METHODS: To identify potential treatment target and ECM associated proteins that may be involved in the development of IPF, we employed isobaric tag for relative and absolute quantitation (iTRAQ) combined liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach to examine protein expression in lung tissues from IPF patients. RESULTS: A total of 662 proteins with altered expression (455 upregulated proteins and 207 downregulated proteins) were identified in lung tissue of IPF patients compared with control. KEGG pathway enrichment analysis showed that the altered proteins in lung tissue mainly belonged to the PI3K-Akt signaling, focal adhesion, ECM-receptor interaction, and carbon metabolism pathways. According to the bioinformatic definition of the matrisome, 229 matrisome proteins were identified in lung tissue. These proteins comprised the ECM of lung, of which 104 were core matrisome proteins, and 125 were matrisome-associated proteins. Of the 229 ECM quantified proteins, 56 significantly differentially expressed proteins (19 upregulated proteins and 37 downregulated proteins) were detected in IPF lung tissue samples. In addition to proteins with well-known functions such as COL1A1, SCGB1A1, TAGLN, PSEN2, TSPAN1, CTSB, AGR2, CSPG2, and SERPINB3, we identified several novel ECM proteins with unknown function deposited in IPF lung tissue including LGALS7, ASPN, HSP90AA1 and HSP90AB1. Some of these differentially expressed proteins were further verified using Western blot analysis and immunohistochemical staining. CONCLUSIONS: This study provides a list of proteomes that were detected in IPF lung tissue by iTRAQ technology combined with LC-MS/MS. The findings of this study will contribute better understanding to the pathogenesis of IPF and facilitate the development of therapeutic targets.

Histopathological and molecular analysis of idiopathic pulmonary fibrosis lungs from patients treated with pirfenidone or nintedanib.

AIMS: The objective of this study was to quantify the impact of pirfenidone or nintedanib treatment on lung histopathology and molecular mediators of fibrosis in patients with idiopathic pulmonary fibrosis (IPF). METHODS AND RESULTS: We collected lung tissue from IPF patients at the time of lung transplantation. Histopathological changes were quantified using a blinded scoring method. Proteins associated with senescence or active TGF-ß were quantified in lung tissues by immunoblot and immunostaining. Histopathological quantification showed similar amounts of dense collagen fibrosis, fibroblast foci and alveolar macrophages in untreated or pirfenidone- or nintedanib-treated IPF patients. There was less diffuse alveolar damage and organising pneumonia in pirfenidone-treated IPF patients. Lungs of nintedanib-treated patients had a trend towards less lymphocytic interstitial infiltration. There was no difference in expression of p-SMAD3, p21 or p16 in the lungs of untreated, pirfenidone- or nintedanib-treated IPF patients. Alveolar epithelial cells, but not fibroblast foci, were immunoreactive to p16. Pirfenidone or nintedanib treatment did not inhibit activation of senescence programming in cultured lung epithelial cells mediated by hydrogen peroxide. CONCLUSION: Pirfenidone and nintedanib do not modulate expression of senescence markers, levels of p-SMAD3 or the amount of fibrosis in IPF lungs. Treated patients have less histopathological evidence of acute lung injury at the time of lung transplantation.

Diffuse parenchymal pulmonary amyloidosis associated with multiple myeloma: a case report and systematic review of the literature.

BACKGROUND: Pulmonary is an uncommon site of extramedullary involvement in multiple myeloma (MM). Diffuse parenchymal amyloidosis as pulmonary manifestation of MM is even rarer. We report a rare case of diffuse parenchymal pulmonary amyloidosis associated with MM diagnosed by video-assisted thoracoscopic lung biopsy (VATLB). CASE PRESENTATION: A 58-year-old woman complained of cough and shortness of breath. HRCT disclosed diffuse ground-glass opacifications with interlobular septal thickening in bilateral lungs. A lung-biopsy sample obtained by VATLB revealed Congo Red-positive amorphous eosinophilic deposits in the alveolar septa. Surgical biopsy of abdominal wall skin and subcutaneous fat was also performed, which showed the apple-green birefringence with polarized light on Congo red stain was demonstrated in dermis. The serum immunoelectrophoresis showed monoclonal lambda light chains. A bone marrow biopsy specimen comprised 11.5% plasma cells. She was therefore diagnosed with diffuse parenchymal pulmonary amyloidosis accompanied by MM. The patient was referred to the hematology department for further chemotherapy. CONCLUSIONS: It is important to recognize diffuse parenchymal pulmonary amyloidosis to avoid misdiagnosis.

Constructing a Multiplexed DNA Pattern by Combining Precise Magnetic Manipulation and DNA-Driven Assembly.

There is an urgent demand to construct multiplexed biomolecular patterns to obtain more biological information from a single experiment. However, with only limited reports focusing on defective top-down approaches, challenges remain to develop a bottom-up strategy for multiplexed patterning. To this end, a novel strategy has been proposed to fabricate multiplexed DNA patterns via macroscopic assembly through combined precise magnetic manipulation and DNA hybridization-driven self-assembly. Therefore, a multiplexed DNA pattern composed of glass fibers loaded with multiple specific strands of DNA was constructed, and its potential application in simultaneous detection of multiplex target DNA was demonstrated. Moreover, the fabricated multiplexed DNA pattern shows an erasable behavior because the hybridized DNA can be disassembled by strand displacement.

Tuning the Aggregation/Disaggregation Behavior of Graphene Quantum Dots by Structure-Switching Aptamer for High-Sensitivity Fluorescent Ochratoxin A Sensor.

The design of graphene quantum dots (GQDs)-aptamer bioconjugates as the new sensing platform is very important for developing high-sensitivity fluorescent biosensors; however, achieving new bioconjugates is still a great challenge. Herein, we report the development of a new high-sensitivity fluorescent aptasensor for the detection of ochratoxin A (OTA) based on tuning aggregation/disaggregation behavior of GQDs by structure-switching aptamers. The fluorescence sensing process for OTA detection involved two key steps: (1) cDNA-aptamer (cDNA, complementary to part of the OTA aptamer) hybridization induced the aggregation of GQD (fluorescence quenching) after cDNA was added into the GQDs-aptamer bioconjugate solution, and (2) the target of OTA triggered disaggregation of GQD aggregates (fluorescence recovery). Such new fluorescent sensing platform can be used to monitor OTA with a linear range of 0 to 1 ng/mL and very low detection limit of 13 pg/mL, which is among the best in all the developed fluorescent nanoparticles-based sensors. Such sensing strategy is also successful in analyzing OTA in practical red wine sample with 94.4-102.7% of recoveries and relative standard deviation in the range of 2.9-5.8%. The present works open a new way for signaling the target-aptamer binding event by tuning aggregation/disaggregation behavior of GQDs-bioconjugates.

Serum Metabolomic Profiling Identifies Characterization of Non-Obstructive Azoospermic Men.

Male infertility is considered a common health problem, and non-obstructive azoospermia with unclear pathogenesis is one of the most challenging tasks for clinicians. The objective of this study was to investigate the differential serum metabolic pattern in non-obstructive azoospermic men and to determine potential biomarkers related to spermatogenic dysfunction. Serum samples from patients with non-obstructive azoospermia (n = 22) and healthy controls (n = 31) were examined using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Serum metabolomic profiling could differentiate non-obstructive azoospermic patients from healthy control subjects. A total of 24 metabolites were screened and identified as potential markers, many of which are involved in energy production, oxidative stress and cell apoptosis in spermatogenesis. Moreover, the results showed that various metabolic pathways, including d-glutamine and d-glutamate metabolism, taurine and hypotaurine metabolism, pyruvate metabolism, the citrate cycle and alanine, aspartate and glutamate metabolism, were disrupted in patients with non-obstructive azoospermia. Our results indicated that the serum metabolic disorders may contribute to the etiology of non-obstructive azoospermia. This study suggested that serum metabolomics could identify unique metabolic patterns of non-obstructive azoospermia and provide novel insights into the pathogenesis underlying male infertility.

Elevated sL1-CAM levels in BALF and serum of IPF patients.

BACKGROUND AND OBJECTIVE: IPF is a form of interstitial pneumonia of unknown origin that has a poor prognosis for which current treatments are limited. Recent studies have shown that EMT plays a role in IPF and tumour metastasis. L1-CAM has also been linked to EMT during tumour development and tumour metastasis. Our aim was to determine prospectively the level of L1-CAM in IPF patients. METHODS: Forty consecutive Chinese patients (with IPF, 16; LC, 12; and CC, 12), but no apparent lung or other organ's diseases were enrolled. Soluble L1-CAM (sL1-CAM), TGF-ß1, PDGF, γ-INF levels in BALF and serum sL1-CAM were measured using ELISA. RESULTS: BALF sL1-CAM levels of IPF, LC and CC patients were 10.87 ± 0.88 ng/mL, 6.34 ± 0.67 ng/mL and 5.43 ± 0.65 ng/mL, respectively. BALF sL1-CAM concentration of IPF patients was significantly higher than that in LC and in CC patients. Besides, serum sL1-CAM levels in patients with IPF, LC and CC were 9.60 ± 1.41 ng/mL, 9.82 ± 0.72 ng/mL and 5.41 ± 1.07 ng/mL, respectively. The serum sL1-CAM levels in patients with IPF and LC were significantly higher than those in patients with CC (P < 0.001, respectively). CONCLUSIONS: The concentrations of sL1-CAM both in BALF and in serum of patients with IPF are markedly increased compared with controls. This indicates that L1-CAM might be involved in the pathogenesis of IPF as well as that of LC.

Supramolecular assembly of macroscopic building blocks through self-propelled locomotion by dissipating chemical energy.

Chemical energy supplied by the catalytic decomposition of H2O2 is introduced into macroscopic building blocks, which self-propel, interact with each other, and finally assemble into ordered and advanced structures. The geometry is highly dependent on the way that the catalyst is loaded. The integration of catalyst and building block provides assembling component as well as its energy of motion.

Development of a regenerable dual-trigger tripedal DNA walker electrochemical biosensor for sensitive detection of microRNA-155.

Since microRNAs (miRNAs) are valuable biomarkers for disease diagnosis and prognosis, the pursuit of enhanced detection sensitivity through signal amplification strategies has emerged as a prominent focus in low-abundance miRNA detection research. DNA walkers, as dynamic DNA nanodevice, have gained significant attention for their applications as signal amplification strategies. To overcome the limitations of unipedal DNA walkers with a restricted signal amplification efficiency, there is a great need for multi-pedal DNA walkers that offer improved walking and signal amplification capabilities. Here, we employed a combination of catalytic hairpin assembly (CHA) and APE1 enzymatic cleavage reactions to construct a tripedal DNA walker, driving its movement to establish a cascade signal amplification system for the electrochemical detection of miRNA-155. The biosensor utilizes tumor cell-endogenous microRNA-155 and APE1 as dual-trigger for DNA walker formation and walking movement, leading to highly efficient and controllable signal amplification. The biosensor exhibited high sensitivity, with a low detection limit of 10 pM for microRNA-155, and successfully differentiated and selectively detected microRNA-155 from other interfering RNAs. Successful detection in 20 % serum samples indicates its potential clinical application. In addition, we harnessed strand displacement reactions to create a gentle yet efficient electrode regeneration strategy, to addresses the time-consuming challenges during electrode modification processes. We have successfully demonstrated the stability of current signals even after multiple cycles of electrode regeneration. This study showcased the high-efficiency amplification potential of multi-pedal DNA walkers and the effectiveness and versatility of strand displacement in biosensing applications. It opens a promising path for developing regenerable electrochemical biosensors. This regenerable strategy for electrochemical biosensors is both label-free and cost-effective, and holds promise for detecting various disease-related RNA targets beyond its current application.