Search Results (366)

Therapeutic agents targeting the PD1–PDL1 interaction, commonly called PD1 blockade, are of great clinical value; however, predicting which patients will benefit has been inconsistent, in part, due to a lack of reliable biomarkers. Quantifying PD1 saturation by PDL1 in tumor tissue has the potential to serve as a biomarker; unfortunately, few diagnostic technologies are available to reliably quantify PD1 saturation in clinical biospecimens. Here, we report on a novel bioassay based on RNA aptamers, called the PD1 LIRECAP assay, that allows for quantification of the saturation of PD1 by PDL1 in formalin-fixed, paraffin-embedded (FFPE) tumor biospecimens. The assay is technically straightforward, high-throughput capable and reproducible. Results showed that quantification of PD1 saturation determined by PD1 LIRECAP assay correlates closely with PD1-mediated signaling and PD1–PDL1 proximity. Analysis of sarcoma FFPE biospecimens confirmed the assay to be consistent and revealed significant differences between patients as well as considerable intratumoral heterogeneity in PD1 saturation by PDL1. We conclude that this novel PD1 LIRECAP platform is technically feasible, reproducible and has the potential to be a superior predictive biomarker assay to predict the outcome of PD1/PDL1-based therapy. Similar assays based on this platform could be used in other systems and settings to quantify the interaction between two molecules. Full article

Nanobiosensors, with their unique physicochemical properties, are transformative tools for diagnosing and monitoring neurodegenerative diseases and mental disorders. This article systematically reviews the latest progress of nanomaterial systems and integrated sensing modalities in neurological disease diagnosis. First, we clarify the multiple functional roles of nanomaterials in biosensors, including signal amplification, interface optimization, and spatial positioning, and compare the applicable scenarios of various sensing principles based on different nanomaterials. Second, we evaluate the design and integration strategies of molecular recognition elements (antibodies, nucleic acid aptamers, molecularly imprinted polymers, and CRISPR-Cas systems) and discuss their synergistic integration mechanisms for improving detection performance. In terms of detection targets, we focus on three applications: high-sensitivity quantification of established protein biomarkers, real-time monitoring of dynamic neurochemicals (dopamine, serotonin, glutamate), and emerging liquid biopsy targets such as exosomal cargo and circulating microRNAs. Finally, to address the core challenges of biofouling, sensitivity–selectivity trade-offs, and multiplex detection in complex matrices, we propose three breakthrough directions for next-generation diagnostics: deep integration of multimodal and multiplexing platforms, closed-loop chemical brain–computer interfaces (cBCIs), and AI-driven predictive diagnostic models, collectively enabling a transition from passive detection to active sensing and intervention for precise, rapid, and non-invasive neurological disease management. Full article

RNA aptamer sensors are promising for environmental and clinical detection, but their poor stability limits practical application. Here, we developed a tRNA-fused strategy to enhance the stability of unmodified RNA aptamer sensors. The tRNA scaffold was fused to the 3′ end of Spinach aptamer to construct tRNA-Spinach. In vitro stability assays showed that tRNA-Spinach retained 50% of its initial fluorescence for 84 days at 25 °C (60% humidity), a 7-fold improvement compared with native Spinach (12 days). The tRNA-fused strategy also doubled the in vivo half-life of Spinach from 20 min to 40 min in KM mice. Based on this strategy, a tobramycin sensor was constructed, which exhibited a LOD of 30 nM, a linear range of 30–100 nM (R² = 0.9905). The biosensor could be detected with a handheld UV lamp within 10 min. This tRNA-fused strategy enables room-temperature storage of RNA aptamer sensors without chemical modification, providing a scalable and cost-effective platform for point-of-care diagnostics in resource-limited settings. Full article

In prokaryotes, translation initiation orchestrates protein synthesis through a network of dynamic interactions among the ribosome, mRNA, initiator tRNA^fMet, and initiation factors (IFs). Traditional approaches that rely on radioactive labeling or surface immobilization are hindered by inherent safety risks and methodological constraints. We present a fluorescence-based analytical platform that integrates microscale thermophoresis (MST) as a unified, multiparametric toolkit for comprehensive interrogation of bacterial translation initiation at the molecular level. By systematically applying MST to a panel of fluorescently labeled components—initiator tRNA^fMet, mRNAs, and initiation factors—we quantify assembly pathways and equilibria as initiation progresses from simple bimolecular interactions to higher-order, multicomponent complexes. To broaden the fluorescence toolbox for ribosomal studies, we developed a robust BODIPY-labeling protocol for 70S ribosomes and confirmed preservation of structural integrity and function by nano differential scanning fluorimetry, stopped-flow kinetic assays, and peptide-synthesis activity tests. Our microscale fluorescent system facilitates probing initiation at a variety of steps, since the role of magnesium ions and initiation factors upon 30S initiation complex formation. The same platform can be applied to investigate the effects of different compounds on translation initiation, as demonstrated for a number of antibiotics, aptamers, and antimicrobial peptides. Using this approach, we determined the antibiotic streptomycin dissociation constant for both 30S and 70S ribosomes, which proved identical at 0.3 ± 0.1 μM, and demonstrated the effect of the antimicrobial peptide rumicidin-1 on translation initiation. Offering a cost-effective and high-sensitivity alternative to conventional methods, this approach advances mechanistic understanding of prokaryotic translation and provides a versatile framework for the discovery of novel protein synthesis inhibitors. Full article

G-quadruplexes are guanine-rich DNA or RNA structures comprising two or more stacked guanine tetrads (G-quartet) with nucleobases in the loops linking the G-quartets. The thermal stability of the thrombin-binding aptamer G2 d(G₂T₂G₂TGTG₂T₂G₂) G-quadruplex was quantified using m-values in aqueous glycine betaine (GB), proline, TMAO, and urea solutions using UV-absorbance spectroscopy. The thermal stability of the G2 variants was also explored with nucleobase substitutions in the TGT loop (TAT, TCT, TTT, UGU, and UUU), in the T₂ loops (T₄, U₂), or in both loops (UGU and U₂, UUU and U₂). GB and TMAO strongly stabilized G2 and its variants. Urea weakly destabilized G2 and its variants, while proline acted as a weak stabilizer or destabilizer depending on G2 variant nucleobase sequence. Both the unfolding enthalpy and entropy were positively correlated with cosolute molality for all cosolute and G-quadruplex combinations. Analysis of the change in solvent-accessible surface area (ΔASA) after unfolding G2 suggested the stability of G2 and its variants in aqueous cosolute solutions was driven by favorable interaction of cosolutes with G-quartet ΔASA and net-favorable or unfavorable interactions with the loop ΔASA. Our results reinforce a general mechanism of folded DNA and RNA stability modulation in cosolute solutions. Full article

Cancer therapy is increasingly shaped by the need for agents that are both mechanistically precise and clinically tolerable. Sulforaphane (SFN), a dietary isothiocyanate enriched in cabbage-family vegetables such as cauliflower and Brussels sprouts, has emerged as a pleiotropic modulator of tumor biology. This review synthesizes current evidence that SFN regulates diverse cancer-relevant processes, including redox homeostasis, cell-cycle progression, apoptosis, autophagy and epigenetic remodeling, largely through coordinated effects on transcriptional (for example, Nrf2, MAPK, NF-κB and AP-1), post-transcriptional (microRNAs and messenger RNAs) and epigenetic (DNA methyltransferases and histone deacetylases) networks. We then examine how functional nucleic acids, including aptamers, small interfering RNAs, microRNAs and tetrahedral DNA nanostructures, can be engineered to guide SFN to tumor cells, amplify pathway-specific effects and overcome resistance. Particular emphasis is placed on nanotechnology-enabled delivery platforms that enhance SFN stability, bioavailability and tumor selectivity. Finally, we outline key challenges, such as context-dependent Nrf2 activity, inter-individual variability in metabolism and incomplete clinical validation, and propose priorities for translating SFN-based functional nucleic acid systems into rational, combination-ready strategies for precision oncology. Full article

Lipid nanoparticles (LNPs) have become an important platform for the delivery of RNA therapeutics, including messenger RNA (mRNA) and small interfering RNA (siRNA). However, most clinically approved LNP formulations exhibit strong liver tropism following systemic administration, which limits efficient delivery to extrahepatic tissues. This inherent biodistribution profile has therefore been recognized as a key challenge for expanding the therapeutic applications of RNA nanomedicine. Recent efforts have focused on engineering functionalized LNP systems to improve delivery specificity beyond the liver. Surface modification with targeting ligands—such as antibodies, peptides, and nucleic acid aptamers—can promote receptor-mediated uptake by specific immune cell populations, including macrophages, dendritic cells and T lymphocytes. In parallel, advances in lipid design have improved intracellular RNA delivery by facilitating endosomal escape. These developments have broadened the potential use of RNA nanomedicine for inflammatory disorders, including autoimmune diseases, neuroinflammation, and cardiovascular inflammation. Functionalized LNPs are also being investigated for in vivo engineering of immune cells. This review summarizes current strategies for designing functionalized LNP systems, highlights their emerging applications in immune and inflammatory diseases, and discusses key challenges for clinical translation. Full article

Field-effect transistor (FET) biosensors using graphene have become one of the most promising biosensing platforms for the early diagnosis of diseases with features such as high sensitivity, label-free detection and application compatibility with point-of-care systems. Herein, we critically discuss recent advances in graphene FET (GFET) biosensor development toward clinically relevant biomarkers associated with representative diseases including cancer, neurodegenerative disease, infectious disease, and inflammatory conditions. Recent progress was reviewed to evaluate GFET architectures, surface functionalization methods, and detection quality. The biomarkers explored were clusterin in Alzheimer’s disease, thrombin in coagulopathy, estrogen receptor α (ER-α) in breast cancer, Carcinoembryonic antigen in lung cancer, microRNAs for malignant tumors, exosomes derived from HepG2 for the hepatocellular carcinoma (HCC) cell line, interleukin-6 (IL-6) for chronic obstructive pulmonary disease (COPD), Polyclonal antibodies and antigens (P24) for HIV and prostate-specific antigen for prostate cancer. The developed devices demonstrate ultralow detection limits at femtomolar to attomolar concentrations with the aid of designed antibodies, aptamers and nanomaterials. Herein, this review presents the sensing mechanisms and biomedical application of various GFET platforms, focusing on their emerging potential as next-generation platforms for rapid, non-invasive and point-of-care diagnostics. Full article

Background: Gene expression and cellular identity are regulated by epigenetics that occurs through chromatin modifications, RNA changes, chromatin accessibility, and three-dimensional genome organization. Although DNA methylation has been the focus of most epigenetics studies in the past, other non-methyl epigenetic processes, including histone post-translational modifications (PTMs), epitranscriptomic marks, and chromatin remodeling, are dynamic, reversible, and context-dependent, and thus are difficult to accurately interrogate using endpoint sequencing-based assays, especially in heterogeneous tissues, developing systems, and therapeutic response environments. Scope and Approach: The present review discusses epigenetic modifications other than DNA methylation regarding sensor-based technologies that can measure live, dynamic, and spatially resolved measurements. Epigenetic sensors include any genetically encoded sensors (GECs) based on resonance energy transfer, CRISPR/dCas-derived sensors, or aptamer-based sensors, and hybrid biochemical/imaging sensors that can be used in live or semi-live settings. It lays emphasis on the technologies, which have been developed recently, that allow real-time kinetic measurements, working in three-dimensional and organoid models, and being applied to disease-relevant perturbations. On these platforms, performance properties such as specificity, sensitivity, spatial and temporal resolution, ability to perform dynamic versus locus-specific interrogation, and perturbed endogenous chromatin states are compared. Key Conclusions and Outlook: Together, these sensing strategies are complementary to the traditional methods of measuring epigenomics in that they show epigenetic dynamics unobservable with static measurements. We list the important technical issues, including specificity, quantitation, multiplexing, and chromatin perturbation, and report the barriers and solutions in development and design. Lastly, we provide a conceptual map of how live epigenetic sensing and multi-omics and translational models can be integrated, and how the two methodologies can be used to develop functional epigenetics and guide disease modeling and drug development. Full article

Aptamers, short single-stranded DNA or RNA oligonucleotides, are emerging as transformative tools in cardiology for the diagnosis, treatment, and theranostics of cardiovascular diseases (CVDs). This review highlights their dual utility. In diagnostics, aptamers enable the construction of highly sensitive biosensors for key cardiac biomarkers (e.g., troponins, myoglobin, C-reactive protein, natriuretic peptides), outperforming conventional assays and enabling early detection and point-of-care testing. Therapeutically, aptamers offer targeted, controllable, and reversible anticoagulation, as demonstrated by clinical-stage candidates like BT200 (anti-vWF) and NU172 (anti-thrombin), whose action can be rapidly reversed with antidote oligonucleotides. Furthermore, aptamers serve as precision delivery vehicles (e.g., Gint4.T, RNA-Apt30) for transporting therapeutic peptides or nucleic acids specifically to cardiomyocytes. Recent integration with nanomaterials (quantum dots, graphene, liposomes, DNA origami) has led to advanced biosensing and drug delivery platforms. Despite challenges like stability and the polyethylene glycol (PEG) immunogenicity, ongoing clinical trials underscore the significant potential of aptamer technology to bridge precise diagnostics and targeted therapy, paving the way for innovative, personalized CVD interventions.) Full article

Therapeutics based on RNA are commonly produced via biocatalytic approaches using RNA polymerases. The most frequently applied enzyme is the RNA polymerase of Enterobacteria phage T7. However, this enzyme has unfavorable properties, like the formation of double-stranded RNA (dsRNA). This undesired by-product can activate the innate immune system via pattern recognition receptors and cause inflammation. Removal of the contaminant is time-consuming and expensive. In this work, we applied a genome mining approach to identify unidentified single-subunit RNA polymerases with minimal dsRNA generation. A large meta database was screened, and 74 sequences were selected. Two RNA polymerases generating barely detectable amounts of dsRNA were identified from the initial sequence portfolio. Their promoters were detected via a fluorescent RNA aptamer screening, and slightly acidic transcription conditions were established. Further activity characterization showed a significant reduction of dsRNA to 0.001% and 0.02%. Due to these beneficial attributes, these RNA polymerases generate mRNA with enhanced stability, which most likely lowers the immune response towards the desired mRNA. This could be especially useful for producing long RNAs, such as self-amplifying RNA, as these typically require improved stability and low dsRNA content. Full article

Breast cancer continues to be a major challenge in global health, in part due to significant inequalities in access to costly diagnostic and therapeutic technologies based on antibodies. Their manufacturing requires complex and expensive bioproduction systems, resulting in limited availability of these tools—essential for early detection and targeted treatment—in many regions, particularly in Latin America. This gap has highlighted the need for cost-effective and scalable theranostic alternatives, increasing interest in aptamers. Obtained through SELEX technology, aptamers are synthetic DNA or RNA oligomers that fold into functional structures. Among their advantages are high affinity for their target, low immunogenicity, and chemical synthesis, which assures reproducible production. Aptamers have expanded the landscape of diagnostic platforms through the development of sensitive aptasensors, liquid biopsy strategies, and imaging systems based on nanomedicines. They also contribute to targeted therapy by recognizing cancer biomarkers selectively and enabling controlled drug delivery. This review presents a critical summary of advances in aptamer-based theranostics for breast cancer, addressing molecular mechanisms, structural folding, selective ligand binding, and nanomaterial interfacing. We also discuss applications in extracellular vesicle capture, cancer stem cell detection, and therapeutic conjugates, emphasizing their advantages and limitations relative to approaches based on antibodies. Overall, current advances show aptamers as emerging tools capable of democratizing precision oncology, particularly in regions where access to advanced technologies remains limited. Full article

Aptamers are powerful tools for detecting and analyzing biomolecules that consist of proteins or nucleic acids. However, their application to aptamers against viroids—highly structured self-replicating RNAs—has not yet been explored. In this study, a magnetic bead- and high-throughput sequencing-based SELEX (MB-HTS-SELEX) protocol for selecting potential aptamers against potato spindle tuber viroid (PSTVd) is presented. Full-length biotinylated-PSTVd RNA was transcribed in vitro, immobilized on streptavidin-coated magnetic beads, and incubated with a library of ~3.32 × 10¹⁴ molecules of random single-stranded oligo-DNAs (oligo-ssDNAs) of 20, 30, or 40 nucleotides (L20, L30, or L40, respectively) flanked by primer binding sites for downstream PCR amplification. Simultaneous biotin labeling of the anti-aptamer strand of the resulting double-stranded DNA (dsDNA) amplicons facilitated strand separation using streptavidin-coated magnetic beads. After 10 selection rounds, high-throughput sequencing, followed by bioinformatics analysis of the generated sequences, allowed for the detection of several enriched sequences, representing putative PSTVd-binding aptamers. Subsequent pull-down assays showed that the most abundant oligo-ssDNA in L30 was docked on PSTVd molecules. This combination method may ameliorate the selection of high-affinity aptamers against PSTVd, reduce the number of selection cycles, time, and other costs of aptamer production, thereby promoting future massive and cost-effective viroid detection and characterization. Full article

Background/Objectives: Interleukin-17A (IL-17A) is a key pathogenic cytokine in autoimmune arthropathies. Current monoclonal antibody inhibitors targeting the IL-17/IL-17RA axis demonstrate clinical efficacy but face significant limitations, including immunogenicity, the loss of therapeutic response, and cold-chain storage. Our study evaluated oligonucleotide aptamers targeting IL-17A and its receptor as an alternative to monoclonal antibodies to suppress an IL-17A-induced inflammatory response in cell models relevant to immunoinflammatory rheumatic diseases. Methods: We examined three aptamers: 2′-F-RNA aptamers Apt21-2 and Apt3-4 specific to IL-17A and DNA aptamer RA10-6 targeting the receptor of IL-17A. Their ability to suppress IL-17A functional activity was assessed in peripheral blood mononuclear cells (PBMCs) from healthy donors and personalized fibroblast-like synoviocytes (FLSs) from patients with axial spondyloarthritis (axSpA) and rheumatoid arthritis (RA). Inhibition was measured by quantifying IL-6 and MMP-13 secretion using ELISA and flow cytometry, using secukinumab as a reference control. Results: In PBMC, all aptamers suppressed IL-17A-stimulated IL-6 secretion and cell proliferation in a concentration-dependent manner (17–200 nM), with a 65–85% efficacy, comparable to that of secukinumab. In axSpA-derived FLS, we observed time-dependent efficacy: At 4 h, all three aptamers suppressed IL-6 to the same extent as secukinumab; at 24 h, RA10-6 maintained high efficacy while Apt21-2 and Apt3-4 showed reduced activity. A combination of receptor-targeting RA10-6 with anti-IL-17A aptamers resulted in synergistic IL-6 suppression. All aptamers reduced MMP-13 to basal levels. RA-derived FLS showed diminished responses to all inhibitors. Conclusions: Aptamers demonstrate high specificity and sustained efficacy in suppressing IL-17A signaling for an in vitro model of spondyloarthritis, with superior performance over antibodies. Disease-dependent differential efficacy in RA FLS reflects heterogeneity consistent with limited clinical anti-IL-17 efficacy in RA. These findings show the strong potential of the studied aptamers as an alternative to monoclonal antibodies for IL-17-associated inflammatory arthropathies, particularly spondyloarthritis. Full article

The development and application of therapeutic oligonucleotides, such as siRNA, miRNA, ASOs and aptamers, is a rapidly growing field in biomedicine. These molecules are undergoing extensive preclinical and clinical testing, and the market for synthetic RNA drugs is expanding. However, several challenges remain, including targeted delivery and high costs associated with development, screening and production. One significant advance has been the creation of GalNAc-conjugates, which selectively target ASGPR and deliver oligonucleotides to hepatocytes. Although these conjugates have shown promising results, their widespread use is limited by the lack of effective synthesis methods. Thus, the development of new methods for the synthesis of ligand-oligonucleotide conjugates is an important task to which this study is devoted. In this study, we created a library of siRNA conjugates with the GalNAc L-96 ligand to suppress the expression of the PCSK9 gene associated with elevated LDL and an increased risk of developing cardiovascular diseases. The selection of the most effective siRNA molecules was carried out using an algorithm previously developed by our research group, which considers thermodynamic stability, predicted specificity and effectiveness. To experimentally confirm the effectiveness of conjugates, an in vitro model based on the cultivation of hepatocyte cells was developed. Optimization of the conjugate synthesis process has significantly reduced the cost of manufacturing technology, which creates the potential for efficient scaling of synthesis for transfer and application in the pharmaceutical industry. The results of the study showed that the development of the siRNA sequence optimized in silico resulted in a significant increase in the inhibitory effect of the GalNAc-siRNA conjugate compared to a compound similar to a commercial drug. Full article