Cell surface proteins are key disease biomarkers and therapeutic targets, yet high-throughput methods for aptamer discovery targeting these proteins in situ remain limited. We introduce single-cell perturbation-driven aptamer recognition and kinetics sequencing (SPARK-seq), a high-throughput platform integrating single-cell messenger RNA and aptamer sequencing with CRISPR-based surface protein perturbation. In a single experiment, SPARK-seq simultaneously mapped 5535 distinct aptamers to eight surface proteins, capturing interactions across more than two orders of magnitude in protein abundance and spanning diverse biophysical classes. The method discriminated closely related paralogous proteins with no detectable cross-reactivity and provided kinetic information that enabled the prioritization of aptamers with slow dissociation rates. Leveraging this kinetic diversity, we engineered variants with improved off-rate properties. SPARK-seq establishes a platform for high-efficiency discovery and rational variant design of aptamers and functional nucleic acids, unlocking possibilities in diagnostics and therapeutics.

High-throughput sequencing has revolutionized aptamer discovery; however, the process is still limited by the lack of effective methods to extract structural insights from diverse sequences, crucial for aptamer truncation, optimization, and molecular design. Herein, we present a machine learning-based framework that decodes aptamer secondary structures directly from single-round selection data, enabling detailed structural insights without the requirement of iterative enrichment. By employing an unsupervised autoencoder clustering (UAE-Clustering) algorithm, our method identified conserved structural motifs in aptamers targeting a model CD8, a key immune regulatory protein. The resulting optimized aptamer exhibited an order-of-magnitude enhancement in binding affinity. We further validated the generalizability of this approach using fibroblast activation protein (FAP), revealing common sequence-structural binding patterns and successfully generating additional optimized aptamers. This approach enabled the rational truncation and optimization of high-affinity aptamers without relying on conventional multi-round selection protocols or experimental structural determination methods such as nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography. By predicting functional secondary structures directly from primary sequences, our strategy streamlined aptamer engineering and bypassed the need for traditional structure-function analyses. Overall, this strategy not only markedly accelerates aptamer discovery and optimization, but also provides new paradigms for mechanistic investigations of aptamer-target interactions.

Programmed death-ligand 1 (PD-L1) critically relies on extensive N-glycosylation at four conserved sites to regulate its immune-checkpoint function. However, the distinct roles of individual glycans remain poorly understood because of a lack of site-specific tools, which also limits the efficacy of current PD-1/PD-L1 blockade therapies. Here, we developed a glycoprotein-targeted Systematic Evolution of Ligands by EXponential enrichment platform (Glyco-SELEX) using an indole-incorporated DNA library to screen aptamers against epitope-specific glycans. Using natively glycosylated PD-L1 isolated from cell membranes as the selection target, we identified a panel of aptamers that can discriminate glycosylation sites on PD-L1. Our results demonstrated that aptamers targeting glycans at the N35 or N192 site effectively disrupt the PD-1/PD-L1 interaction. Moreover, by engineering a bivalent aptamer directed against both sites, the antitumor activity of CAR-T cells was significantly enhanced. This work not only resolves the functional ambiguity of PD-L1 glycosylation but also establishes a versatile platform for developing therapeutics against epitope-specific glycans.

Afterglow luminescence imaging materials have evolved from inorganic to organic systems, and further from macromolecular polymers to small molecules, highlighting the imperative of developing safe and efficient organic afterglow molecules for bioimaging. However, flexible organic afterglow molecules with well-characterized structure-luminescence intensity relationships for high-contrast activatable imaging remain scarce. Here, we report for the first time that coumarin derivatives exhibit bright afterglow luminescence and reveal that rigidified coumarin structures display stronger emission than their flexible and backbone analogues. Further, we designed MP molecules bearing boronic ester and methylenecyclobutane moieties and integrated them with coumarin to construct the activatable afterglow probe C545-MP NPs. Oxidation of the boronic ester by ONOO- primes the probe, whereas subsequent irradiation induces coumarin-mediated singlet oxygen generation that reacts with the methylenecyclobutane unit to initiate afterglow emission. By virtue of the elimination of autofluorescence and high activation contrast, C545-MP NPs enable precise assessing therapeutic efficacy of ulcerative colitis and Parkinson's disease.

Early diagnosis of acute diseases is fundamentally constrained by the lack of chemically tractable methods for biomarker discovery directly from a complex plasma. Here, we report PSABD (plasma-SELEX-enriched aptamer-based biomarker discovery and diagnosis), a chemical platform that integrates carboxylate magnetic-bead-mediated plasma protein capture within situ aptamer SELEX, enabling molecular-recognition-driven identification of disease-associated biomarkers. With this strategy, enriched aptamers not only serve as affinity probes but also function as structurally programmable molecular handles to pull down their targets, thereby linking selection chemistry with proteomic discovery. With acute myocardial infarction (AMI) as a model, PSABD identified malate dehydrogenase 2 (MDH2) as an unexpected AMI-associated plasma protein, which was significantly elevated in AMI patient samples and exhibited a high binding affinity to selected aptamers. On the basis of this interaction, we developed an aptamer-based analytical platform capable of distinguishing AMI patients from healthy individuals. Collectively, PSABD establishes a general chemical strategy that bridges molecular recognition, biomarker discovery, and diagnostic development, highlighting aptamers as programmable chemical probes for the acute disease diagnosis and clinical translation.

Lysosomes maintain a highly acidic lumen to regulate H+-dependent hydrolase-mediated degradation, but how protons are ‘leaked’ out to regulate organellar functions through cytosolic effectors remains unknown. Here we developed DNA nanodevices on the cytosolic leaflet of lysosomal membranes to monitor juxta-organellar pH in cells. Unexpectedly, we revealed a radiating acidic layer (up to 21 nm in thickness) on the outer surface of all lysosomes, typically 0.2–0.7 pH units more acidic than the neutral cytosol. This acidic nanolayer is established and maintained primarily by TMEM175, a lysosomal H+ efflux channel associated with Parkinson’s disease. Activation of TMEM175 causes opposite pH changes on both sides of lysosomes; however, it is the juxta-lysosomal, not the luminal, acidity that determines lysosome positioning in cells with dynein adaptor RILP acting as a juxta-lysosomal pH sensor. Hence, through inside-out proton conduits, lysosomes create a steady acidic surrounding that acts as a nano-interface for cytosolic machineries to regulate organellar activities. Tan and colleagues develop DNA nanodevices to detect the pH of the lysosomal outer surface, observing an acidic layer generated by TMEM175 that regulates lysosome positioning in response to changes in juxta-lysosomal pH.

Abstract Background: Pyroptosis, a lytic and highly inflammatory form of programmed cell death, has emerged as a powerful mechanism for augmenting anti-tumor immunity. Although antibody drug conjugate (ADC)-mediated near-infrared photoimmunotherapy (NIR-PIT) can induce tumor-specific pyroptosis and enhance immune responses, the downstream mediators that bridge pyroptotic cell death to immune activation remain undefined. Here, we investigated whether pyroptosis-derived extracellular vesicles (Pyro-EVs) constitute a key immunostimulatory output of tumor pyroptosis and sought to define their molecular composition and immune-activating functions. Methods: Pyroptosis was induced in SCC7 murine carcinoma cells using Cetuximab-IR700, a clinically-approved NIR-PIT ADC. EVs released during pyroptosis were isolated by ultracentrifugation and characterized by NTA and nano-flow cytometry. Deep proteomic profiling with organelle-origin mapping, GO enrichment, and nuclear-cytoplasmic signature scoring was performed to define Pyro-EV identity. DNA cargo was examined by nano-flow cytometry, DNase-protection assays, and confocal microscopy. Immunological activity was evaluated by exposing macrophages to Pyro-EVs, followed by RNA-seq, ELISA, and Western blot assessment of nucleic-acid sensing pathways. Results: NIR-triggered pyroptosis induced rapid release of a distinct EV population, generating Pyro-EVs at 2-3x the abundance of basal EVs. Proteomics identified a pyroptosis-specific signature with 726 emergent proteins enriched for ribosomal subunits, chromatin components, and DNA-binding regulators, absent in apoptotic or necrotic EVs. These molecular hallmarks indicate nuclear rupture and cytoplasmic mixing, establishing Pyro-EVs as a unique class of lytic cell-death-derived vesicles. Pyro-EVs carried elevated levels of double-stranded nuclear DNA, confirmed by DNase resistance and colocalization with chromatin-binding proteins. Functionally, Pyro-EVs were strong activators of innate immunity, inducing broad M1-polarizing programs in macrophages. RNA-seq showed induction of TLR7/9, MyD88, IRAK1/4, and NF-κB-related genes; Western blot validated activation of the TLR7/9-MyD88-IRAK-p65 axis, and ELISA demonstrated increased secretion of TNF-α, IL-6, and other cytokines, confirming DNA-sensing-dependent innate immune activation. Conclusions: This study identifies Pyro-EVs as a previously unrecognized, immunogenic vesicle class that emerges during NIR-PIT. Pyro-EVs are rich in nuclear DNA and chromatin-associated factors and serve as high-potency activators of macrophage TLR7/9 signaling, establishing them as a central mediator connecting pyroptotic tumor death to innate immune amplification. These findings redefine the immunobiology of pyroptosis and uncover Pyro-EVs as a mechanistic driver of pyroptosis-induced anti-tumor immunity. Citation Format: Fan Chen, Yuxuan Li, Ye Lu, Peng Guo, Weifeng Qian, Wei-Hong Tan. Pyroptosis-derived extracellular vesicles as key immunoactivators in near-infrared photoimmunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3353.

Albumin-based therapeutics are widely used in cancer treatment for their biocompatibility and hydrophobic drug-loading capacity, but their efficacy is often limited by poor target specificity and weak binding affinity. Inspired by the multivalent and cooperative binding of immunoglobulin M (IgM), we developed a biomimetic tandem Y-shaped Sgc8 aptamer-albumin delivery system (TYS-HSA). By grafting two hydrophilic aptamers onto each hydrophobic C18 side chain of poly(maleic anhydride-alt-1-octadecene) (C18PMH), we constructed well-defined multivalent aptamer units that self-assembled with human serum albumin (HSA) into an IgM-like ligand corona. Quantitative analysis revealed a high ligand valency of approximately 240 aptamers per TYS-HSA nanoparticle, which, combined with its tandem Y-shaped topology, enables cooperative multivalent recognition. This design achieved high-affinity, stable target recognition while maintaining strong loading capacity for hydrophobic drugs such as paclitaxel. Compared with lower-valency conjugates (MS-HSA, approximately 95 aptamers per nanoparticle), TYS-HSA showed enhanced target binding, prolonged cellular retention, and potent antitumor efficacy after a single intravesical administration in an orthotopic bladder cancer model. With broad drug compatibility and excellent physiological stability, this platform offers a promising and generalizable strategy for the targeted design of albumin-based nanomedicines.

Damage-associated molecular patterns (DAMPs) are key mediators of inflammatory disease, among which HMGB1 is a prototypical extracellular alarmin and an attractive therapeutic target. Here, we report ZH-1a, a high-affinity DNA aptamer (Kd = 2.1 nM) identified through SELEX and sequence optimization, that preferentially recognizes the proinflammatory B-box region of HMGB1. ZH-1a functions as an extracellular HMGB1-neutralizing aptamer and suppresses HMGB1-induced inflammatory signaling, including cytokine secretion and NF-κB activation in macrophages. In vivo, ZH-1a reduced late-phase systemic inflammation and multiorgan injury in LPS-induced endotoxemia, improved survival in polymicrobial sepsis, and attenuated inflammatory responses and organ damage in an HMGB1-challenge model. In addition, ZH-1a alleviated joint inflammation and structural damage in collagen-induced arthritis, and further enhanced the therapeutic efficacy of methotrexate. Together, these findings establish ZH-1a as a promising anti-inflammatory aptamer targeting HMGB1 and support aptamer-based neutralization of pathogenic extracellular HMGB1 as a therapeutic strategy for inflammatory disease.

The cellular positioning of lysosomes, especially their perinuclear accumulation, plays an essential role in regulating many biological processes. In this work, we developed a non-genetic approach to drive lysosomes to the perinuclear region by displaying a nucleus-targeting DNA nanodevice on their outer surface. Our experimental results demonstrated that this nanodevice efficiently anchored onto the outer leaflet of lysosomal membrane, and then effectively promoted perinuclear clustering of lysosomes. Further analysis revealed that this repositioning significantly enhanced autophagic flux and altered the expression of autophagy-associated genes. Our study provides a versatile platform for elucidating the functional consequences of lysosomal positioning and its regulatory mechanisms in cellular physiology.

The immune system employs molecular switches to maintain dynamic homeostasis, yet malignant cells often learn from these natural switches and ultimately evade immune surveillance, leading to immune tolerance and tumor deterioration. Chemically synthetic switches designed to redirect immune signaling pathways are highly desired for reversing this pathological trajectory but are rarely reported. Herein, we develop a synthetic DNA framework (DF) switch that reprograms macrophage-mediated immune clearance of Programmed Cell Death-Ligand 1 Positive (PD-L1+) extracellular vesicles (EVs) in vivo. This synthetic switch is composed of a ligand (Man6)-terminated PD-L1-targeting aptamer (MJ5C) and a DF, termed hereinafter as MJ5C-Man6-DF, which operates through a recognition-then-recruitment mechanism. Thus, in the "off state", MJ5C stably resides within the DF, retaining Man6 in its inner cavity. However, upon target recognition, MJ5C switches to the "on state" and binds to PD-L1+ EVs, conferring conformational changes that allow coating of its terminal Man6 on EVs. Man6-coated EVs then recruit macrophages via the membrane receptor CD206, enabling efficient phagocytosis. MJ5C-Man6-DFs were shown to perform with exceptional stability and specificity, augmenting αPD-L1 therapy by 90.7% while boosting T cell activation by 55% in vivo. Therefore, our aptamer-driven DF switch provides a strategy for precise immune reprogramming in the field of DNA-based molecular engineering.

Colorectal cancer (CRC) poses a significant global health challenge, highlighting the need for better diagnostic tools and molecular targets. Compared to traditional antibodies, aptamers, which are single-stranded oligonucleotides with high affinity and low immunogenicity, offer an ideal platform for discovering novel biomarkers. Based on this approach, we used the Cell-SELEX strategy to develop a high-affinity aptamer-based probe, WHY-3E, which successfully identified the cellular prion protein (PrPC) as a key molecular target. We observed that PrPC was significantly upregulated in CRC tissues, strongly correlating with unfavorable clinical outcomes. Functionally, PrPC promotes malignant phenotypes, including migration and invasion, by regulating MSN. Mechanistically, we revealed that PrPC physically translocates from the cell surface to the cytoplasm via endocytosis. Once internalized, it interacts with the STAT3-NTD and LYN-SH domains, facilitating the ternary complex formation that enhances LYN-mediated STAT3 phosphorylation, ultimately increasing MSN transcriptional activity. Moreover, the deubiquitinase USP18 stabilizes PrPC by removing its Lys48-linked ubiquitin chains, ensuring the continuous activation of this oncogenic axis. Notably, the WHY-3E aptamer achieved 90.6% sensitivity and 89.0% specificity in detecting PrPC-positive circulating exosomes in patient cohorts. These findings deepen the mechanistic understanding of CRC progression and offer novel strategic avenues for PrPC-targeted aptamer applications in non-invasive diagnostics.

Aromatic camptothecins (CPTs) are clinically potent TOP1 inhibitors constrained by an "aromaticity-solubility" paradox, which leads to poor bioavailability and efflux-mediated resistance. Here, we report a modular strategy to reprogram the planar CPT scaffold into multifunctional topoisomerase I (TOP1) degraders by integrating hydrophobic tagging (HyT) with supramolecular self-assembly. Adamantane HyT introduces an orthogonal aliphatic module, converting classical inhibitors into proteasome-dependent degraders with enhanced membrane permeability while preserving TOP1-DNA binding affinity. This tag synergistically acts as a supramolecular anchor, enabling host-guest assembly with poly(β-cyclodextrin) to form stable, pH-responsive nanoparticles without pharmacophore modification. Our lead candidate, SN-38-A2, demonstrates potent TOP1 degradation and achieves superior tumor regression in xenograft models compared to clinical irinotecan. This synergistic HyT-supramolecular approach rebalances aromaticity for optimized drug-like properties, establishing a paradigm to transform solubility-limited warheads into high-performance degraders with integrated delivery.

Nucleic acid therapeutics are rapidly emerging as a transformative drug paradigm, offering precise and programmable regulation of gene expression across a broad spectrum of diseases. This review summarizes recent advances in key platforms—including antisense oligonucleotides, siRNA, miRNA, mRNA, and aptamers—emphasizing their unique mechanisms of action and therapeutic potential. We systematically outline critical contributions of chemical modification and delivery engineering, including backbone and sugar modifications, site-specific design, N-acetylgalactosamine (GalNAc) conjugation, and lipid nanoparticles, which collectively enhance stability, target specificity, and clinical applicability. Finally, we discuss persistent challenges such as immune activation, large-scale manufacturing, and long-term safety, and provide perspectives on future directions involving CRISPR-based gene editing, synthetic biology, nanotechnology, smart delivery systems, and combination therapies, aiming to offer strategic insights for the development and clinical translation of nucleic acid drugs.

Nucleic acid aptamers, often referred to as "chemical antibodies," are versatile, specific, and easily modifiable functional nucleic acids. There is a growing focus on new methods for the selection and target validation of aptamers, with the aim of expanding their biomedical applications in molecular diagnostics and therapeutics, which is currently a research hotspot. This review is composed of eight sections. In the first section, we briefly introduce aptamers and review their development in molecular diagnostics and therapeutics. The "Advantages of aptamers in molecular diagnosis and therapeutics" section summarizes and discusses the advantages of aptamers in these fields. The "New methods for screening aptamers" section presents and discusses nucleic acid aptamer screening methods, including both classical and novel approaches. In the "New methods for target validation" section, we explore new methods for target validation, covering aptamer structure validation, target recognition validation, and aptamer-target interaction validation. The "New methods for molecular diagnostics" section summarizes and discusses recent applications of aptamers in molecular diagnostics, particularly focusing on new mechanisms and detection strategies as well as their applications in various diseases. The "New methods for molecular therapeutics" section summarizes and discusses recent applications of aptamers in molecular therapeutics, emphasizing new mechanisms and aptamer-based therapy strategies, along with their therapeutic applications in different diseases. The "Challenges and future perspectives of nucleic acid aptamers" section addresses the challenges and future perspectives of aptamers in disease diagnosis and treatment. Finally, the "Conclusion" section shares our views on the future directions of aptamers in clinical disease molecular diagnostics and therapeutics.

The rapid advancement of artificial intelligence has placed increasingly high demands on intelligent perception systems. Conventional sensing frameworks adopt a separated mode of signal acquisition, transmission, and post-processing, which restricts the low-power and real-time implementation of intelligent perception. Ion-driven neuromorphic transistors, featuring learning, memory, temporal integration, threshold triggering, and reconfigurable neural characteristics, have emerged as an appealing candidate for replicating biological intelligent sensory systems. This review elaborates on the fundamental device architecture and ion-modulation principles of neuromorphic materials and transistors, and summarizes performance modulation strategies via the rational design of channel materials, electrolytes, and interfacial engineering. We then highlight the typical neuromorphic functionalities derived from ion–electron coupling, and discuss the integration of such devices with sensors for intelligent perception, as well as their promising applications in wearable health monitoring, robotic closed-loop control, and biointerface sensing. Future research directions are prospected, providing guidelines for the development and practical deployment of ion-driven neuromorphic devices toward low-power intelligent perception hardware.