Both p53 and hMSH2 are essential for maintaining genomic integrity, yet the regulatory link between them remains poorly understood. This study investigates the transcriptional repression of the DNA mismatch repair gene hMSH2 by wild-type p53 in normal human oral keratinocytes (NHOKs). We observed that inducing p53 with the alkylating agent led to a significant, time-dependent downregulation of hMSH2 mRNA and protein levels. Conversely, HPV-16-transformed oral keratinocytes, characterized by low p53 levels, exhibited dramatic overexpression of hMSH2 driven by a 27- to 38-fold increase in promoter activity. Transient transfection assays confirmed that wild-type p53 directly represses hMSH2 promoter activity, while antisense p53-mediated knockdown in NHOKs increased hMSH2 expression, mimicking the transformed phenotype. Sequencing of the hMSH2 5'-flanking region indicated that this regulation is mediated through trans-acting mechanisms rather than cis-acting mutations. These findings identify hMSH2 as a novel target of p53-mediated transcriptional repression and suggest that p53 may modulate mismatch repair capacity to coordinate the cellular decision between DNA repair and apoptosis in response to genotoxic stress.

Effective tumor cell apoptosis critically depends on the efficient delivery of pro-apoptotic agents to mitochondria. While mitochondria-targeted peptide-based nanoassemblies offer a promising platform for controllable delivery, achieving mitochondria-specific responses and programmable subcellular apoptotic regulation remains a critical yet underexplored challenge. Herein, we report controllable mitochondria-targeted DNA-peptide nanoassemblies co-assembled from an enterokinase (ENTK)-responsive cationic peptide-incorporating a DDDDK substrate motif targeted to the perimitochondrial ENTK-and a multifunctional DNAzyme chimera (DC) through DNAzyme-catalyzed dityrosine crosslinking, thereby enabling mitochondria-specific and programmable subcellular apoptotic regulation. In addition, the DC integrates a hemin-intercalated G-quadruplex (G4/hemin) that catalyzes H2O2-dependent reactive oxygen species (ROS) generation and an antisense oligonucleotide (ASO) targeting antisense long noncoding mitochondrial RNAs (ASncmtRNAs). Upon selective peptide cleavage by perimitochondrial ENTK, the DNA-peptide nanoassemblies undergo subcellular-specific disassembly and payload release, resulting in mitochondria-localized ROS generation and synergistic ASO-mediated ASncmtRNA knockdown, accompanied by suppression of the anti-apoptotic protein survivin. These coordinated effects collectively induce mitochondria-mediated apoptosis and achieve effective tumor suppression. This work establishes a DNAzyme-catalyzed covalent assembly strategy that integrates the molecular programmability of nucleic acids with the biological specificity of peptides, providing a modular and highly tunable platform for organelle-targeted nanosystems and enabling sophisticated spatiotemporal combination therapy.

Bacterial infection is a major cause of delayed wound healing, while overuse of antibiotics has accelerated the emergence of antimicrobial resistance. Developing multifunctional antibacterial biomaterials that eradicate bacteria without relying on conventional antibiotics is therefore highly desirable. Herein, we report an injectable polyethylene glycol (PEG)-based hydrogel incorporating gold nanorods (ARs) and a DNA nanostructure-mediated silver and antisense oligonucleotide delivery system (A-C@Ag) to achieve synergistic antibacterial therapy and enhanced skin regeneration. The hydrogel is formed by rapid thiol-maleimide crosslinking of PEG precursors containing AR@A-C@Ag, providing excellent injectability, biocompatibility, and mechanical stability. Upon near-infrared irradiation, the AR generates mild photothermal heating to directly damage bacteria and simultaneously promotes the on-demand release of Ag[+] and antisense oligonucleotides from the DNA nanostructure, resulting in markedly enhanced antibacterial activity against both Staphylococcus aureus and Escherichia coli by facilitating bacterial membrane disruption. In a S. aureus-infected full-thickness wound model, AR@A-C@Ag/Gel under NIR irradiation significantly accelerated wound closure by efficiently eliminating bacteria, suppressing inflammation, promoting angiogenesis and collagen deposition, and facilitating the regeneration of skin appendages. This work demonstrates that integrating DNA nanostructure-enabled drug delivery with mild photothermal therapy represents an effective strategy for constructing antibiotic-free antibacterial hydrogels for infected wound repair.

Astragali Radix, the dried root of Astragalus mongholicus, is a prominent traditional Chinese medicinal material, with isoflavonoids serving as its primary bioactive components. Light serves as a pivotal environmental cue modulating isoflavonoid biosynthesis, its regulatory influence on A. mongholicus remains poorly understood, and the underlying biosynthetic pathways have yet to be fully elucidated. This study investigated the impacts of different light on isoflavonoid accumulation through integrated transcriptome and metabolome analyses. Compared to white light, red and blue light treatments significantly altered the expression of 93 genes within the isoflavonoid biosynthetic pathway, 52 of which were up-regulated. Metabolomic analysis revealed 35 differential isoflavonoid metabolites, with 23 compounds, including the primary medicinal components calycosin (CA) and formononetin (FO), showing increased accumulation. Notably, blue light exhibited a significantly stronger promoting effect on CA and FO accumulation than red light. Through integrated multi-omics analysis, we revealed the molecular network underlying this light quality-specific regulation. Correlation analysis revealed that multiple structural genes were significantly positively correlated with these key active components, among which AmCHR, AmCHS, AmCHI, and AmIFS were identified as core regulatory genes. The biochemical roles of candidate genes AmCHR, AmCHS, AmCHI, and AmIFS were validated via in vitro enzymatic assays. Furthermore, utilizing a newly established nonsterile hairy root transformation system, functional validation through overexpression and RNA interference experiments demonstrated that these candidate genes positively regulate the accumulation of key isoflavonoids, including CA, calycosin-7-O-β-D-glucoside, FO, and ononin. These findings were further corroborated by transient gene silencing using antisense oligodeoxynucleotides. This study elucidates the regulatory network of light-induced isoflavonoid biosynthesis in A. mongholicus and provides essential genetic resources for the metabolic engineering and sustainable production of these medicinal compounds.

Chronic hepatitis B (CHB) remains a major global health burden despite the availability of potent nucleos(t)ide analogue (NA) therapy. Although NAs effectively suppress hepatitis B virus (HBV) DNA replication and reduce the risks of cirrhosis and hepatocellular carcinoma, they have limited effects on covalently closed circular DNA and hepatitis B surface antigen (HBsAg) production, and most patients require prolonged treatment. Bepirovirsen is an antisense oligonucleotide that targets conserved HBV RNA sequences, promotes RNase H1-mediated RNA degradation, and reduces viral antigen expression and replication. In August 2026, Japan approved bepirovirsen (Hibsago) for selected adults with chronic HBV infection receiving NA therapy, representing its first global regulatory approval. Approval was supported by the phase III B-Well 1 and B-Well 2 trials, in which a functional cure after treatment withdrawal was achieved in 20% and 19% of bepirovirsen-treated patients, respectively, versus 0% with a placebo. Response rates were higher among patients with lower baseline HBsAg levels. The overall safety profile was acceptable; however, elevated alanine aminotransferase levels were observed in 6% of bepirovirsen-treated participants. Ongoing studies will further define long-term durability, broader applicability, and optimal treatment strategies.

Therapeutic nucleic acid (TNA)-based gene silencing strategies, such as antisense oligonucleotides (ASOs), have been explored for cancer treatment. However, no ASO-based anticancer drugs are currently available, largely because of limited therapeutic efficacy. Conventional ASO-based cancer therapies typically target oncogenes or cancer-associated genes because constitutively active ASOs may also damage normal tissues. We hypothesized that this limitation can be overcome by conditionally activating ASOs only in response to cancer-specific biomarkers. In this paradigm, therapeutic efficacy is determined by the potential of the released ASO rather than by the cancer specificity of its target, allowing any gene essential for cell survival to serve as a target. Our aim was to identify the most cytotoxic ASO sequences for cancer cells. We designed 37 ASOs targeting 13 candidate genes and evaluated them in human ovarian adenocarcinoma (SKOV3) and lung adenocarcinoma (A549) cell lines. An ASO targeting DARS1 exhibited the strongest activity, inducing apoptotic cell death in both cell types. Additional ASOs targeting DYNC1I2 and EIF2S3 were identified as effective candidates in SKOV3 and A549 cells, respectively. Importantly, the selected ASOs exhibited low toxicity toward the tested noncancerous cell line. These findings identify DARS1, DYNC1I2, and EIF2S3 as promising targets for marker-dependent ASO-based cancer therapy.

Gapmer antisense oligonucleotides (ASOs) represent a clinically validated therapeutic platform, with a 2'-O-methoxyethyl modification commonplace within the flanking regions. An alternative sugar modification, incorporation of 4'-thioribose, has demonstrated enhanced nuclease resistance in siRNAs and aptamers, but incorporation of this motif into gapmer architectures remains underexplored. Herein, we report the synthesis of a 4'-thiouridine phosphoramidite building block and its incorporation into gapmer ASOs targeting PTEN. Evaluation of sequences containing 4'-thiouridine in the flanking regions (compared with 2'-O-methoxyethyl) and varying phosphorothioate linkage composition demonstrates that the modified gapmers maintain equivalent knockdown efficacy under transfection and gymnotic delivery. These findings indicate 4'-thioribose as an alternative sugar modification for wider exploration within gapmer ASOs.

Therapeutics based on nucleic acids are emerging as a transformative drug class in the era of personalized medicine. They include antisense oligonucleotides (ASOs), aptamers, small interfering RNAs (siRNAs), small activating RNAs (saRNAs), microRNAs (miRNAs), and long non-coding RNAs (lncRNAs), in addition to messenger RNAs (mRNAs). ASOs are short synthetic molecules of nucleic acids designed to bind to specific RNA molecules to allow for precise modulation of gene expression in a personalized medicine context. This review will summarize the state of the art in in silico tools and methods that are used for the design, 3D structural prediction, molecular docking, and simulation of ASOs, including their applications and limitations. By leveraging these computational approaches, the review will provide in silico methodologies toward improved specificity and efficacy of ASO therapeutics. These methodologies can enhance understanding of biological mechanisms of ASOs to allow for improved accuracy and efficacy of ASOs in the clinic while also providing new pathways toward the development of targeted therapies.

Variants that disrupt pre-mRNA splicing represent a historically underrecognized proportion of pathogenic alleles in Mendelian neurological disorders, accounting for an estimated 15-30% of disease-causing variants. Conventional clinical genomic pipelines frequently overlook these variants because analysis and reporting have traditionally focused on protein-coding regions, with intronic and synonymous positions assigned lower priority. The central practical problem this review addresses is therefore not whether splicing matters in neurogenetic disease - that is well established - but how a splice-altering variant identified on a diagnostic report can be reliably identified, experimentally validated, and translated into a clinical diagnosis or therapeutic decision. Deep learning-based splice prediction tools such as SpliceAI and Pangolin, RNA sequencing, long-read transcriptomics, and minigene functional assays have collectively improved diagnostic yield in previously unsolved neurogenetic cohorts, particularly by identifying deep intronic pseudoexon variants and leaky canonical splice-site alleles. Nevertheless, accurate variant classification remains challenging: the Pathogenic Very Strong 1 (PVS1) criterion of the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) framework requires careful gene- and variant-specific adaptation when applied to splicing, prediction tools disagree with one another and are least reliable outside the core splice-site consensus, and RNA-based validation is constrained by the tissue- and developmental-stage-restricted nature of splicing itself. This review synthesizes the molecular basis of splice-altering variants, current standards for their nomenclature and computational prediction, the practical strengths and limitations of RNA-level validation, and their contribution to epilepsy, hereditary neuropathy, ataxia, dystonia, mitochondrial disease, neuromuscular disorders, and the newly recognized spliceosomal small nuclear RNA (snRNA) neurodevelopmental disorders. We place particular emphasis on the diagnostic and therapeutic decisions that depend on getting this interpretation right, on the unresolved limitations of current prediction and validation tools, and on priorities for embedding RNA-level diagnostics more routinely into clinical neurogenetic practice.

The abnormal expansion of repetitive DNA sequences falls within the spectrum of neurological conditions driven by dual pathogenic pathways of RNA toxicity and protein toxicity. Within this spectrum, hexanucleotide repeat disorders (HRDs) represent a rare but important subgroup: C9orf72-amyotrophic lateral sclerosis/frontotemporal dementia (C9orf72-ALS/FTD), X-linked dystonia-parkinsonism (XDP) and spinocerebellar ataxia type 36 (SCA36), unified by the presence of expanded intronic repeats capable of forming non-canonical nucleic acid structures. Despite this shared repeat architecture, they differ in critical molecular and genetic respects: C9orf72-ALS/FTD is hallmarked by TDP-43 aggregation and prominent proteinopathy; SCA36 lacks TDP-43 pathology entirely; and XDP uniquely harbors a SINE-VNTR-Alu (SVA) retrotransposon insertion within the expanded locus and follows an X-linked recessive inheritance pattern. Among these, C9orf72-ALS/FTD is relatively well characterized, with established pathogenic mechanisms including G-quadruplex (G4) formation, dipeptide repeat (DPR) protein toxicity, RNA foci formation, and downstream cellular disruptions. The molecular mechanism underlying SCA36 and XDP remains comparatively less characterized. Given the mechanistic overlap with ALS/FTD, downstream disruptions such as RNA toxicity, mitochondrial dysfunction, impaired DNA repair and proteasomal dysfunction could similarly result from these core molecular processes. However, direct experimental evidence supporting these mechanisms in SCA36 and XDP remains limited. This review compares established, emerging and hypothetical mechanisms across HRDs to distinguish shared pathogenic pathways from disease-specific molecular features. Through horizontal comparison, this review identifies opportunities and limitations for translating ALS/FTD therapeutics to XDP and SCA36, establishing a unified analytical framework for mechanistic research and clinical intervention in rare repeat expansion disorders.

Ocular diseases remain a major global health challenge with substantial unmet therapeutic needs. Small nucleic acid therapeutics offer a precise strategy to regulate disease-related mRNAs or noncoding RNAs through base pairing, thereby modulating protein expression at the RNA level. Major modalities include antisense oligonucleotides, small interfering RNAs, microRNA-based therapeutics, small activating RNAs, and nucleic acid aptamers, which act through RNA degradation, RNA interference, splicing modulation, microRNA regulation, transcriptional activation, or structure-dependent target binding. These properties make them attractive for ocular diseases involving genetic defects, pathological angiogenesis, inflammation, fibrosis, or neurodegeneration. However, their clinical translation in ophthalmology remains limited by poor molecular stability, insufficient tissue retention, immune activation, off-target effects, and inefficient delivery to target ocular tissues. Rational oligonucleotide design, appropriate local administration routes, and optimized delivery platforms are therefore essential for improving stability, tissue penetration, cellular uptake, and therapeutic durability. This review summarizes the major classes, mechanisms, chemical modification strategies, ocular delivery systems, and therapeutic applications of small nucleic acid drugs in ophthalmology. We also discuss lessons from clinical successes and failures and propose future directions for safe, durable, and individualized ocular therapy.

Gene therapy and genome editing increasingly depend on the safe, effective, and cell-selective delivery of nucleic acids and protein-nucleic acid complexes. Although viral vectors remain important for applications requiring durable gene expression, non-viral vectors offer advantages in cargo capacity, modularity, transient expression, potential repeat dosing, and avoidance of vector-genome integration. Lipid nanoparticles (LNPs), polymeric nanoparticles, inorganic nanomaterials, extracellular vesicles (EVs), and biomimetic hybrid systems have consequently become central platforms for delivery of siRNA, mRNA, plasmid DNA, antisense oligonucleotides, and CRISPR-based genome editors. Among these, ionizable LNPs are currently the most clinically mature non-viral technology, supported by the clinical success of siRNA therapeutics and mRNA vaccines, as well as the emergence of in vivo CRISPR therapies. Nevertheless, efficient endosomal escape, cell-type-selective targeting, extrahepatic delivery, and repeat-dose tolerability remain substantial barriers. Polymeric vectors provide broad chemical tunability, allowing adjustment of charge density, degradability, stimulus responsiveness, intracellular trafficking, and cargo release. However, toxicity and batch-to-batch reproducibility remain key concerns. EVs provide a biologically derived alternative with favorable membrane interfaces and potential advantages for protein and ribonucleoprotein delivery, but their clinical translation is constrained by heterogeneity, loading efficiency, product characterization, and scalable manufacturing. This review summarizes recent advances in non-viral gene-delivery platforms, compares their strengths and limitations, and discusses future directions in cell-selective delivery, endosomal escape, transient delivery of genome-editing machinery, engineered EVs, hybrid vectors, and manufacturing-oriented development. The field is transitioning from organ-level delivery toward delivery of the correct payload to the correct cell type at a clinically relevant exposure and safety margin.

Epigallocatechin gallate (EGCG) imparts distinctive health benefits and flavor to tea, and its accumulation is modulated by plant growth regulators (PGRs). However, the regulatory mechanisms of Paclobutrazol (PAC) and Lovastatin (LS) underlying EGCG biosynthesis remain uncharacterized. We integrated quantitative analysis, transcriptomics, and metabolomics to investigate the regulatory effects of exogenous PAC and LS on EGCG accumulation in tea plants and systematically elucidate the underlying molecular mechanism governing EGCG biosynthesis. The results demonstrated that exogenous PAC and LS treatments significantly elevated the accumulation of EGCG, as well as endogenous auxin and jasmonic acid (JA) contents. The variation trend of these key metabolites was highly consistent with the accumulation patterns of upstream flavonoid components, including eriodictyol, delphinidin, and epigallocatechin. Transcriptomic profiling further verified the critical involvement of auxin and JA signal transduction pathways in PGR-induced EGCG differential accumulation, and we screened a total of 14 auxin-related and 8 JA-related core signal regulatory factors. Furthermore, integrated bioinformatic analyses and antisense oligonucleotide (AsODN) functional validation experiments revealed that the core hormone signaling genes CsARF2 and CsMYC2 regulate the expression of CsSCPL16 through the transcription factor CsMYB80, and ultimately promote the conversion of epigallocatechin to EGCG and promote the accumulation of EGCG in tea plants. Collectively, the PGRs boost EGCG biosynthesis by mediating endogenous auxin and JA signal transduction. These results support the development of targeted agronomic practices to improve tea quality and lay a theoretical basis for expanding the industrial exploitation of tea bioactive constituents.

TDP-43 proteinopathies, encompassing amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and limbic-predominant age-related TDP-43 encephalopathy (LATE), represent a heterogeneous spectrum of devastating neurodegenerative disorders. For decades, the diverse clinical presentations of these diseases have complicated antemortem diagnosis and hindered the development of disease-modifying therapies. However, recent breakthroughs in basic science are beginning to address these clinical barriers, although substantial hurdles to practical clinical application remain. Structural elucidation via cryo-electron microscopy (Cryo-EM) has shattered the single-protein amyloid dogma by revealing that TDP-43 can form hetero-amyloid filaments with ANXA11, thereby providing a molecular basis for pathological strain diversity. Concurrently, the pathogenic focus has shifted toward nuclear loss of function, which triggers a systemic "RNA crisis" characterized by aberrant alternative polyadenylation (APA) and cryptic exon inclusion (e.g., STMN2, UNC13A). Crucially, this metabolic collapse is profoundly exacerbated by patient-specific genetic risk factors, acting synergistically in a "two-hit" model of neurodegeneration. To translate these findings to the clinic, next-generation diagnostic tools are emerging. Integrating neuron-derived extracellular vesicle (EV) isolation with Seed Amplification Assays (SAAs) holds promise to help overcome the structural camouflage that limits current PET imaging, potentially offering ultra-sensitive, functional strain identification in biofluids. While these structural and diagnostic milestones provide a strong foundation for precision medicine, major challenges in assay standardization and clinical validation must be addressed. Advanced therapeutic strategies-namely, splice-switching antisense oligonucleotides (ASOs) that directly restore RNA metabolism, combined with the targeted suppression of neuronal hyperexcitability-are now entering clinical trials. This review synthesizes how decoding the structural and RNA-metabolic complexities of TDP-43 is paving a promising pathway from bench to bedside, while critically discussing current translational limitations.