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Toward Rapid Wild Yeast Isolation: Low-Nanomolar SELEX-Derived Aptamers for Whole-Cell S. cerevisiae Detection
Audrey Wayne M Abinal1,2,3, Mei-Yeh Jade Lu1, Lin-Chi Chen4, Isheng Jason Tsai1,2
1Biodiversity Research Center, Academia Sinica, Taipei, Taiwan
2Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan
3National Taiwan Normal University, Taipei, Taiwan
4Department of Biomechatronics Engineering, National Taiwan University, Taipei, Taiwan
The baker’s yeast, Saccharomyces cerevisiae, is a widely studied model species central to both basic research and industrial applications, and it is also found in natural environments such as tree bark, soil, and fruit surfaces. While domesticated strains are well characterized, wild strains hold untapped genetic diversity and biotechnological potential. However, current isolation methods for wild lineages are slow, inefficient, and labour-intensive, creating a major bottleneck for biodiversity research. To address this, this study identifies specific oligonucleotide aptamers to serve as rapid recognition elements for S. cerevisiae. Using a whole-cell SELEX approach coupled with high-throughput Illumina sequencing and qPCR quantification, we enriched a random ssDNA aptamer library to select aptamers that bind intact S. cerevisiae cells (unknown surface targets). Top candidates were selected from Illumina sequencing data based on enrichment trends. Binding affinity was quantified using a qPCR-based cell-binding assay across an aptamer concentration series, followed by isotherm model fitting to estimate the equilibrium dissociation constant (KD). Flow cytometry provided an orthogonal, fluorescence-based confirmation of aptamer binding to S. cerevisiae cells. Our analysis identified multiple candidates, with the top aptamer reaching estimated KD of 14.26 nM. Here, we take initial steps toward developing a specialized, cost-effective aptamer-based tool to reduce detection latency and support the discovery of wild S. cerevisiae in natural environments.
Aptamers as Versatile Recognition Elements in Modern Biotechnology
Florian K. Blaser1,2, Nico Dreymann1, Thomas Hirsch1,2, Marcus M. Menger1, Antje J. Baeumner1,2
1Competence Team Bioanalytics, Fraunhofer IZI-BB, Am Mühlenberg 13, 14476 Potsdam, Germany
2Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Universitaetsstrasse 31, 93053 Regensburg, Germany
Through chemical and biochemical methods aptamers can be brought to their full potential. At Fraunhofer, high-end facilities enable even specialized applications including cell and toxin studies, supported by an extensive landscape of analytical instruments like NGS, SPR and ITC. The basis of many applications is the immobilization of aptamers on diverse materials. We successfully established this expertise for surfaces ranging from glass, over nanofibers and nanoparticles to plastic coated with TruContact®. Another conjugation strategy involves the strong binding of biotin to NeutrAvidin. This system is applied in our most sensitive dual-aptamer sandwich assay detecting urokinase plasminogen activator (uPA). The dynamic range of the assay can be adjusted by simply switching between a fluorescent and enzymatic label covering an impressive range from 5 pM to 100,000 pM. Because of its sandwich format, the assay exhibits high specificity and demonstrates matrix‑effect–free performance in human urine. Expanding beyond diagnostic applications, we generated a His-tag aptamer for high‑throughput tag availability screening. By probing the accessibility of His-tags on recombinant proteins directly in microtiter plates, hundreds to thousands of variants can be assessed rapidly and efficiently, streamlining early-stage recombinant protein discovery workflows. We also applied aptamers in innovative sensing concepts, illustrated by an ATP-responsive aptamer conjugated to upconversion nanoparticles (UCNPs). Upon ATP binding, the aptamer forms a G‑quadruplex that recruits an intercalating fluorophore into FRET proximity, enabling robust lifetime-based readout even in complex intra cell environments. The UCNP probes operate effectively across physiological ATP levels, remain biocompatible and provide a modular platform easily adaptable to other structure-switching aptamers.
Expanding Aptamer Functionality via Transcriptional Incorporation of Bio‑Inspired Nucleotide Modifications
Laia Civit1, Kevin Neis1, Jørgen Kjems1, 2, Julián Valero1,2
1Interdisciplinary Nanoscience Center, Aarhus University DK-8000 Aarhus, Denmark
2Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus, Denmark
With the growing interest in RNA‑based therapeutics and diagnostics, expanding the chemical diversity of RNA has become essential for generating aptamers with enhanced stability and novel binding capabilities. Here, we present an enzymatic transcription strategy using a mutant T7 RNA polymerase to incorporate a broad range of amino acid– and glycosyl‑inspired nucleotide modifications, alongside stabilizing ribose chemistries such as 2′‑fluoro and 2′‑deoxy groups. We systematically evaluated the compatibility of these modified nucleotides with transcription and downstream amplification, confirming efficient incorporation and generally low misincorporation rates. Crucially, the modified nucleotides were well tolerated throughout the sequencing process, demonstrating their robustness in aptamer evolution workflows. As a proof of concept, we selected a Histidine‑U–modified aptamer that binds Influenza hemagglutinin with low‑nanomolar affinity, demonstrating that introducing bio‑inspired chemical functionalities during transcription can enhance aptamer performance and expand their molecular recognition capabilities. This work establishes a versatile platform for selecting next‑generation chemically modified aptamers and highlights the potential of transcriptionally introduced modifications to broaden the scope of targets and functions accessible to RNA aptamers.
Utilizing aptamer technology for life science enzyme control and production
James Elliott, Esta Tamanaha, Brenda Baker, Lisa Maduzia, Michael Sproviero, Jennifer Ong, Nathan Tanner
New England Biolabs, Ipswich, Massachusetts, USA
Single-stranded DNA aptamers offer a versatile and highly selective approach for controlling and processing enzymes used ubiquitously across life science and biotechnology applications. Their ability to bind specific target molecules with high affinity enables the development of aptamer-based affinity chromatography systems when immobilized onto solid supports. These systems provide efficient and reproducible separation and purification of enzymes, proteins, nucleic acids, and other biomolecules. Through representative case studies, aptamer chromatography demonstrates enhanced selectivity, improved purity, increased yield, and greater process reproducibility compared with conventional chromatographic approaches. In addition, innovative column formats and integration with analytical techniques extend functionality beyond purification, enabling post-separation characterization and improved understanding of target structure and function. Together, these advances highlight the potential of aptamer technology to improve enzyme control, downstream processing efficiency, and production performance in life science and biomanufacturing workflows.
Isolation of aptamers against RANKL
Evely Estrada-Benítez, Reyna A. Padilla-Vázquez, José G. Vázquez-Jiménez, Maria J. Navarro-Ibarra, Natalia Martínez-Acuña, Ana G. Leija-Montoya
Facultad de Medicina y Nutrición Mexicali, Universidad Autónoma de Baja California, Mexicali, Baja California, México
Receptor Activator of Nuclear Factor B Ligand (RANKL) is the principal protein regulating osteoclast activity; when it predominates, it leads to overstimulation that is reflected in bone destruction, playing a key role in the pathophysiology of diseases such as osteoporosis, rheumatoid arthritis, and periodontitis. The main objective of this study was to isolate aptamers with high specificity for RANKL. A 75 nt combinatorial DNA library, consisting of a randomized central region flanked by two fixed regions, was amplified by asymmetric PCR to obtain the ssDNA pool. Subsequently, the SELEX technique was performed, including three cycles of negative selection, in which sequences that did not interact with a complex protein mixture present in serum and fetal bovine serum were recovered, followed by five cycles of positive selection using functional murine sRANKL. During the selection process, target and counter-selection proteins were immobilized on PVDF membranes; after incubation, unbound sequences from the counter-selection were collected from supernatant, while sequences bound to RANKL were recovered by heat denaturation. Individual sequences were cloned into the TOPO-TA vector and transformed into E. coli TOPO cells. After colony growth, nine plasmids were obtained, containing vectors with inserts of both expected and higher molecular weights, as visualized on a 2% agarose gel. Finally, four samples were selected for sequencing. The obtained sequences constitute a suitable basis for subsequent qPCR-based interaction assays aimed at evaluating specificity toward RANKL. This approach represents an initial step toward the development of aptamer-based tools for studying and potentially modulating RANKL-mediated osteoclast activity in bone-related disorders.
Aptamers as next-generation smart therapeutics for breast cancer
Débora Ferreira1,2, Ligia R. Rodrigues1,2
1CEB Centre of Biological Engineering, University of Minho, 4710 057 Braga, Portugal
2LABBELS Associate Laboratory, Braga/ Guimarães Portugal
Cancer remains a major therapeutic challenge due to its biological complexity and extensive heterogeneity, which often limit the effectiveness of conventional treatments and cause toxicity in healthy tissues. Consequently, there is a pressing need for novel therapies capable of selectively targeting cancer cells based on their molecular signatures. In this regard, aptamers have emerged as promising targeting ligands in cancer therapy owing to their high affinity and exceptional specificity for molecular targets. Compared with antibodies, aptamers offer several distinct advantages, including straightforward chemical synthesis, ease of modification, low toxicity, enhanced tissue penetration, and minimal immunogenicity. Aptamers are short single-stranded nucleic acids generated from large random libraries through the Systematic Evolution of Ligands by EXponential Enrichment (SELEX) process, which involves iterative cycles of selection and amplification. Among SELEX methodologies, the cell-SELEX approach enables the direct selection of aptamers against native cell-surface molecules by using intact living cells as selection targets, preserving the physiological context of target epitopes. In the present study, cell-SELEX was combined with next-generation sequencing and bioinformatic analyses to identify aptamer candidates capable of selectively recognizing metastatic cancer cell lines, including breast cancer models. Lead aptamer sequences were subsequently validated, and their binding affinity and specificity were confirmed using flow cytometry and fluorescence microscopy. To further assess their translational relevance, the binding performance of selected aptamers was evaluated on tumour tissue sections. Bioinformatic approaches were employed to characterize aptamer–target interactions. Collectively, these findings highlight the considerable potential of aptamers as versatile, highly selective, and effective tools for targeted cancer diagnosis and therapy.
Development of Target-specific Aptamers for Pancreatic Ductal Adenocarcinoma Detection
Aurelia Fraticelli1,2, F. Ingenito3, S. Verde1,2, G. Petrillo3, G. De Luca4, M. Mascolo3, C. Pignataro3, K. Tkalčec3, Y. Veneruso3, D. Fiore3, C. Quintavalle4, A. Affinito5 and G. Condorelli3,4
1Department of Biomedicine and Prevention, University of Rome “TorVergata”, Rome, Italy
2AKA Biotech, Naples, Italy
3Department of Molecular Medicine and Medical Biotechnology, “Federico II” University of Naples, Via Pansini 5, Naples, Italy
4Istituto di Endocrinologia ed Oncologia Sperimentale, Consiglio Nazionale delle Ricerche (CNR), Naples, Italy
5IRCCS SYNLAB SDN, Naples, Italy
Pancreatic Ductal Adenocarcinoma (PDAC) is among the most lethal malignancies, characterized by extremely low 5-year survival rate and the absence of effective early-diagnostic biomarkers. The molecular complexity of PDAC requires the development of highly specific molecular probes for both diagnostic imaging and targeted therapy. To address these limitations, we employed SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology used to isolate high-affinity and high-specificity oligonucleotides. A combined Robotic SELEX and stringent manual selection strategy enabled the isolation of aptamers against extracellular vesicles (EVs) derived from PDAC cells, ensuring high affinity and selectivity for the PDAC-specific secretome.Furthermore, given the key role of EphA2 in metastasis and chemoresistance of pancreatic cancer, we evaluated the use of A40s, an aptamer previously identified and characterized to selectively bind the EphA2 receptor in glioblastoma, in PDAC. RT-qPCR and flow cytometry–based binding assays demonstrated that both the newly selected pools and the A40s aptamer exhibit high recognition of PDAC cell surfaces and their derived EVs.These findings highlight the potential of both these aptamers as versatile molecular platforms for the development of next-generation liquid biopsy strategies and targeted delivery systems, representing a significant step forward overcoming current limitations in the diagnosis, clinical management, and treatment of PDAC patients.
Real-Time Kinetics of Internalization of Anti-EGFR DNA Aptamers and Aptamer Constructs into Cells Derived from Glioblastoma Patients
Valeria L Ivko1,2, Olga M Antipova1,2, Boris M Ivanov1, Vadim N Tashlitsky1, Fatima M Dzarieva2,3, Nadezhda S Samoylenkova2, Dmitry Y Usachev2, Galina V Pavlova2,3, Alexey M Kopylov1,2
1Lomonosov Moscow State University, Moscow, Russia
2Federal State Autonomous Institution “N.N. Burdenko National Medical Research Center of Neurosurgery” of the Ministry of Health of the Russian Federation, Moscow, Russia
3Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, Moscow, Russia
WHO considers Epidermal Growth Factor Receptor (EGFR) as key biomarkers of glioblastoma (GB), the aggressive brain tumor. EGFR is identified and targeted using molecular recognition elements (MoREs), like aptamers, covalent aptamer–drug conjugates (ApDCs), and non-covalent aptamer-drug complexes (ApDCo). For the first time, a novel approach was implemented to study real-time kinetics of the aptamer uptake by measuring the cellular index (CI) using impedance (xCELLigence Real Time Cell Analysis, RTCA). Doxorubicin (DOX) was used as indicator drug. DOX non-covalently intercalates into aptamer DNA double helix; when adding to the cells, anti-EGFR ApDCo interacts with EGFR, it is internalized into the cell, and after dissociation it attacks chromosomal DNA and stops cell proliferation. For truncated anti-EGFR DNA aptamer GR20 (46 ns), additional duplex was constructed by synthesizing extra region (h) yielding GR20h (66 ns) and via hybridization with the complementary oligonucleotide (h’, 18 ns) to form a duplex (hh’), thus creating the aptamer construct with complementary oligonucleotide (ACCO) GR20hh’ (84 ns). The original HPLC method quantified the complete assembly of ACCO. ACCO GR20hh’ retained affinity for recombinant extracellular domain of EGFR, shown with Biolayer Interferometry. According to cytofluorimetry, the ACCO GR20hh’ interacts with cells of continuous culture from GB patient (CCGBP) surgical sample 107 from BioBank of Burdenko MSRC. DOX–ACCOGR20hh’ complexes are faster and more efficient internalized by both EGFR+ cells lines A-431 and CCGBP107 than DOX alone, as it was clearly shown by xCELLigence RTCA. Moreover, some interesting early cellular behavior was found fairly before conventional endpoints of static cell viability tests, like MTT. This work was financially supported by the Ministry of Science and Higher Education of the Russian Federation (agreements no. 075-15-2024-561, 24.04.2024).
Selection and Characterization of ssDNA Aptamer Targeting a Celiac Disease Epitope from Wheat High Molecular Weight Glutenin and Its Application in a Gold Nanoparticle–Based Assay
Jon J Kalita1,2, Utpal Bora2
1Department of Food Science and Nutrition, The Assam Royal Global University, Betkuchi, Guwahati-781035, Assam, India
2Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati-781039, Assam, India
Celiac disease–triggering peptide epitopes occur in both the gliadin and glutenin fractions of gluten proteins found in cereals including wheat, rye, and barley. Although many aptamers have been reported for gliadin protein and its immunoreactive peptide sequences, epitopes originating from high molecular weight glutenin subunits (HMW-GS) remain unexplored in aptamer development. Herein, we report the in vitro selection and characterization of a DNA aptamer against a 14-mer peptide, GQGQQGYYPTSPQQ, derived from the HMW-GS 1Bx13 subunit of wheat, containing the celiac disease epitope QGYYPTSPQ. The peptide was synthesized through Fmoc solid-phase synthesis and immobilized on magnetic beads, to carry out Mag-SELEX using a 56-mer ssDNA library. The selected aptamer, apt_J91P (56 bp), showed a dissociation constants of 2.26 μM (primary site) and 4.385 mM (secondary site) in binding buffer as determined by isothermal titration calorimetry. The interaction was found to be both enthalpically and entropically driven. Circular dichroism analysis revealed local conformational changes upon binding. The limit of detection evaluated through direct ELAA was found to be 16.08 μM. To assess the preliminary utility of the selected aptamer, a gold nanoparticle–based colorimetric assay was employed. The aptamer, apt_J91P, was surface absorbed on the gold nanoparticles, and upon addition of a high salt concentration, aggregation of the nanoparticles occurred in the absence of the respective peptide target. The aptamer demonstrated specificity against unrelated peptides. The assay was successful in experimental buffer; however, further studies are required to validate detection in complex food matrices.
Designing Oscillatory and Irreversible Light Responses with Optoribogenetics
Annabelle Kamitz1, Christian Renzl1,3, Andreas Möglich2, Günter Mayer1,3
1Chemical Biology and Aptamers, Life and Medical Sciences Institute (LIMES), University of Bonn, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany
2University of Bayreuth, Department of Biochemistry, Universitätsstraße 30, 95447 Bayreuth, Germany
3Center of Aptamer Research & Development, University of Bonn, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany
Periodic expression of regulatory proteins in cells is crucial for the correct clocking of the cell cycle, circadian rhythms and intracellular signaling networks. Oscillations are regulated by biological circuits that e.g. involve negative feedback and time-delay. Precise manipulation of these circuits is necessary to understand and explore the features, that modulate these. Thereby, light would be a preferred trigger, as it can be applied in a spatio-temporal manner. NmPAL is a LOV photoreceptor, which binds specifically to a short RNA stem loop (aptamer) in blue light and reverts to the unbound state in darkness, making it a promising candidate for establishing such a light-inducible oscillating system. Previously, a light-controlled hammer-head ribozyme (optozyme) was generated, which enables light-dependent control of mRNA stability. We hypothesize that the insertion of a NmPAL expressing mRNA upstream to the optozyme will introduce a negative feedback loop, that attenuates its own mRNA stability and thereby should modulate its own expression in an oscillatory fashion. This approach will be generally applicable and will help to study oscillations of proteins in different cellular pathways. Advancing our understanding of such regulatory mechanisms could reveal novel mechanisms behind diseases that may lead to improved therapies for cancer and inflammatory disorders. In addition, we introduce a new PAL variant with an altered switching character, which allows for an extended on-time without the need to have a constant light source applied. This expands the functional diversity of PAL-based photoreceptor variants and provides new opportunities to study dynamic RNA regulation.
Aptamer as molecular tool for monoclonal antibody drug monitoring
Feifei Long1, Louisa H. Y. Lo2, Julian A. Tanner1,2
1School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
2Advanced Biomedical Instrumentation Centre, Hong Kong Science and Technology Park (HKSTP), Hong Kong SAR, China
Rheumatoid arthritis (RA) is a chronic autoimmune disorder that necessitates long-term therapeutic intervention with biologic agents. Adalimumab, a monoclonal antibody (mAb) targeting tumor necrosis factor-alpha (TNF-α), has been extensively utilized in the clinical treatment and management of inflammatory conditions, including RA. However, interindividual variability in pharmacokinetic profiles and therapeutic responses to adalimumab remains a critical challenge in clinical practice. Consequently, the monitoring of circulating adalimumab concentrations is of substantial clinical significance for evaluating treatment efficacy and guiding individualized dose adjustments. Over the past few decades, DNA aptamers have emerged as promising tools in therapeutic drug monitoring (TDM), owing to their inherent advantages such as high stability, structural versatility, and cost-effectiveness compared to traditional recognition elements (e.g., antibodies). In the present study, DNA aptamers specific to adalimumab were selected via in vitro bead-based systematic evolution of ligands by exponential enrichment (SELEX). The lead aptamer candidate exhibited high specificity and binding affinity toward adalimumab, with a dissociation constant (Kd) of less than 100 nM. This lead aptamer holds potential not only as a real-time monitoring tool for adalimumab in both clinical therapeutic monitoring and monoclonal antibody manufacturing processes, but also to be a ‘molecular switch’ to regulate the in vivo biological activity of adalimumab, thereby potentially mitigating treatment-related adverse effects.
Aptamer-based targeting for cancer-associated aberrant O-glycans in pathological MUC1
Alessandra Martucci1,2, Ana Díaz-Fernández1,2, Noemí de los Santos-Álvarez1,2, María Jesús Lobo-Castañón1,2
1Department of Physical and Analytical Chemistry. Universidad de Oviedo. Av. Julián Clavería, 8, 33006 Oviedo, Spain
2Health Research Institute of Principado de Asturias, Av. Roma s/n, 33012, Oviedo, Spain
Glycosylations are a class of post-translational modifications of proteins that play a role in maintaining cellular functions. It consists of the addition of chains of carbohydrates at specific sites. Their aberrant dysregulation has been the subject of debate in recent years due to its significant connection to the development of tumour conditions. Both N- and O-glycan types undergo morphological alterations that changes their functional characteristics, contributing to tumour progression by modulating cell adhesion, immune evasion, and signalling pathways. Their presence is a hallmark of cancer, which make them a promising non-invasive biomarker more specific than the total concentration of the protein. Among the recognition tools, aptamers are valid alternatives to antibodies. MUC1 is a transmembrane glycoprotein already validated in the clinical setting as a tumour-associated antigen. Its extracellular domain contains a region of repeated motifs called Variable Number Tandem Repeats (VNTR) highly O -glycosylated. In physiological conditions, O-glycans are very long while cancer-associated O-glycans show truncations and hyper-sialylations. Specific tumour-associated carbohydrate antigens (TACA) are correlated with a poor prognosis in multiple cancers. While aptamer selection against N-glycoproteins has already been described in literature and performed in our group against PSA, targeting O -glycans with aptamers remains a field largely unexplored. In this work, we show the selection of aptamers against one of the aberrant TACA in MUC1 by using specific counter-selection steps and elution molecules. The goal is to provide a guidance to rationally direct the SELEX toward these specific glycans and to have a more specific tool to be used in low-cost screening in cancer diagnostics platforms and new biomarkers’ development. Ack: Financially supported by STRIM EU 101169504, Spanish (PID2024-156199NB-I00) & Principado de Asturias (IDE/2024/000677) Governments.
Targeting Hypoxic Glioma Stem Cells with miRNA-340-5p/A40s aptamer via HIF-1A–CA9 regulation
Martina Mascolo1, G. Roscigno1, G. Petrillo1, G. De Luca2, S. Nuzzo3, S. Verde4, A. Fraticelli4, K. Tkalčec1, Y. C. Veneruso1, C. Pignataro1, D. Fiore1, A. Affinito3, L. Ricci Vitiani5, S. Pellecchia2, P. Pallante2, G. Condorelli1 and, C. Quintavalle2
1Department of Molecular Medicine and Medical Biotechnology, “Federico II” University of Naples, Italy
2Institute of Endotypes in Oncology, Metabolism and Immunology “G. Salvatore (IEOMI), Consiglio Nazionale delle Ricerche (CNR), Naples, Italy
3IRCCS SYNLAB SDN, Naples, Italy
4Department of Biomedicine and Prevention, University of Rome “Tor Vergata”, Rome, Italy
5Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy
Glioblastoma multiforme (GBM) is a highly aggressive brain tumour, characterized by recurrence and resistance to conventional therapies. Glioma stem cells (GSCs), particularly enriched in hypoxic niches, play a central role in tumour progression and adapt to hostile microenvironments, including low oxygen and acidic conditions. Carbonic anhydrase IX (CA9) is overexpressed in GSCs and upregulated under hypoxia via HIF-1A, contributing to an acidic microenvironment and poor clinical outcome.In this study, we identified a regulatory mechanism in which miR-340-5p, a tumour-suppressor microRNA downregulated in GBM, directly targets CA9, as predicted by bioinformatic analysis and confirmed through robust experimental validation. This interaction reduces CA9 expression and limits the stem-like aggressiveness of GSCs. Hypoxic conditions suppress miR-340-5p through HIF-1A, as we discovered that HIF-1A binds to the miR-340 promoter, demonstrated by chromatin immunoprecipitation (ChIP). Building on this, we developed an innovative therapeutic strategy to target hypoxic niches in GBM using a pioneering therapeutic platform: an aptamer-microRNA based system. We used A40s aptamer as a carrier to deliver miR-340-5p directly into GSCs by generating a chimeric construct. The RNA aptamer A40s selectively binds GSCs over differentiated GBM cells by engaging the erythropoietin-producing hepatocellular receptor A2 (EphA2) and is rapidly internalized. This chimeric system efficiently binds and internalizes into GSCs, downregulates CA9, and markedly inhibits stem-like features like cell growth, viability and induces apoptosis. It reduces microenvironment acidification and promotes a shift toward normoxia-like conditions. These findings highlight the therapeutic potential of the A40s–miR-340-5p chimera as a targeted delivery system to disrupt GSC maintenance and counteract GBM aggressiveness.
Targeting the CT-Domain of CTGF: A Superior Therapeutic Strategy for Idiopathic Pulmonary Fibrosis
Meishen Ren1#, Xue Xue1#, Huarui Zhang2#, Shanshan Yao2, Xin Yang1, Luyao Wang1, Weizhe Huang3, Wen Zhao4, Zongkang Zhang2, Yuanyuan Yu1, Aiping Lyu1, Sifan Yu1*, Baoting Zhang2*, Ge Zhang1*
#Co-first authors
*Co-corresponding authors
1Institute for Advancing Translational Medicine in Bone & Joint Diseases, School of Chinese Medicine, Hong Kong Baptist University, 34 Renfrew Road, Kowloon City District, Hong Kong Special Administrative Region, China
2School of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories District, Hong Kong Special Administrative Region, China
3Department of Cardiothoracic Surgery, The Second Affiliated Hospital of Shantou University Medical College, China
4Department of Pulmonary and Critical Care Medicine, Shenzhen University General Hospital, China
Idiopathic pulmonary fibrosis (IPF) is a fatal disease with an urgent need for effective therapies. Connective tissue growth factor (CTGF), a key mediator of fibrosis, contains four functional domains (IGFBP, VWC, TSP1, CT). Both VWC- and CT-domains contribute to CTGF’s fibrotic activity, highlighting their therapeutic potential. FG-3019, a human monoclonal antibody targeting VWC-domain, showed safety and efficacy in a Phase II trial of IPF patients with >55% predicted forced vital capacity (FVC). However, it failed to meet primary efficacy endpoints in a subsequent Phase III trial enrolling patients with >45% predicted FVC. Our in vitro data showed the VWC-domain deletion reduced pro-fibrotic signaling by only 20–25%, while CT-domain deletion suppressed it by 80–85%. It indicates CT-domain contributes more to CTGF’s fibrotic activity than VWC-domain. Notably, TGF-β1 level was significantly elevated in CTGFKO fibroblasts expressing CTGF-ΔVWC, but not in those expressing CTGF-ΔCT. It suggests VWC-domain targeting may trigger a compensatory fibrotic feedback loop via TGF-β1. To validate the CT-domain as a superior target, we developed Apc003OA using our aptamer platform. This long-lasting aptamer exhibits high affinity and specificity for CTGF CT-domain. In bleomycin-induced IPF mouse models, Apc003OA reduced fibrosis markers by 80–85% without ascending TGF-β1. In contrast, an equivalent dose of FG-3019 achieved only ~25% reduction and elevated TGF-β1. Additionally, no pathological lesions were detected in vital organs following Apc003OA treatment in vivo. The anti-fibrotic effect of Apc003OA was completely abolished by co-administration of a CT-domain blocking peptide, confirming its efficacy depends on specific CT-domain engagement. In conclusion, these findings raise the CTGF CT-domain as a superior therapeutic target compared to the VWC-domain and present Apc003OA as a potent, targeted candidate for the treatment of IPF.
Cell-based SELEX against the Glucagon-Like Peptide-1 Receptor using Fluorescence-Activated Cell Sorting.
Timothy Noel, Ioanna Mela
Signalling by the Glucagon-Like Peptide-1 Receptor (GLP1R) has been under intense investigation over the last decade, owing to its therapeutic potential for treating Type II Diabetes Mellitus and obesity. As a Class B1 G Protein-Coupled Receptor (GPCR), the GLP1R is endogenously activated by peptide ligands, and exogenous peptides have been used as therapeutic interventions. The GLP1R, like all GPCRs, couples to both G proteins and b-arrestins, the latter inactivating the receptor and causing internalisation. Evidence suggests that the b-arrestin pathway prevents the therapeutic benefits caused by GLP1R signalling. Previous aptamers raised against GPCRs have been able to differentiate between a receptor’s conformations and, therefore, also their signalling profiles. This project set out to raise aptamers against an mCherry-tagged human GLP1R construct expressed in a Human Embryonic Kidney cell line by Systematic Evolution of Ligands by EXponential enrichment (SELEX). Live cells expressing the receptor were selected by fluorescence-activated cell sorting (FACS), from which aptamers were selected. This project has involved a lot of troubleshooting with the FACS-SELEX system and PCR to expand the selected aptamer library: different fluorophores were selected for, different wash buffer components were trialled, and a wide variety of PCR conditions were screened. PCR proved to be one of the larger issues, due to the emergence of high-molecule weight oligonucleotides, likely caused by aptamers binding primer binding sites.
Isolation of DNA Aptamers for the Specific Detection of the Circular Analog CRIVi45-51
Reyna A Padilla-Vazquez1, Evely Estrada-Benitez1, José G Vazquez-Jimenez1, Octavio Galindo-Hernandez1, Ana G Leija-Montoya1,2
1Facultad de Medicina y Nutrición, Universidad Autónoma de Baja California. Mexicali, BC, México
2Unidad de Ciencias de la Salud, Universidad Autónoma de Baja California. Mexicali, BC, México Vasoinhibin is a proteolytic fragment of prolactin that exerts antiangiogenic and antivasopermeability effects. Its biological activity is primarily mediated by the HGR motif (His46-Gly47-Arg48), identified as the main determinant of endothelial inhibitory function. These properties are particularly relevant in angiogenesis-dependent diseases including cancer, rheumatoid arthritis, and diabetic retinopathy. CRIVi45-51 is a circular retro-inverso synthetic analog derived from residues 45–51 of vasoinhibin and designed to preserve the spatial orientation of side chains while incorporating D-amino acids to confer resistance to proteolysis and enhanced structural stability. Due to its improved stability and reported preclinical efficacy, CRIVi45-51 represents a promising therapeutic candidate; however, reliable molecular tools for its specific detection and monitoring remain limited. The aim of this study is to isolate DNA aptamers with specific binding to CRIVi45-51 using a combinatorial single-stranded DNA library and the SELEX methodology. The selection strategy included three negative cycles against serum components followed by five positive cycles using CRIVi45-51 as the target molecule. Enriched pools were amplified and subjected to cloning procedures, resulting in the isolation of five individual clones. These clones will be sequenced and analyzed in two different groups to evaluate sequence diversity and enrichment patterns. Interaction assays will be performed to confirm binding affinity and specificity toward CRIVi45-51. The successful identification of specific aptamers may provide novel molecular recognition tools to facilitate its specific detection and support the development of future analytical and therapeutic monitoring strategies.
An Antibody-Based SELEX Strategy for the Enrichment of Breast Cancer Cell-Specific Aptamers
Mayur Nivrutti Pol, David-Marian Otte, Günter Mayer
Life & Medical Sciences Institute (LIMES), University of Bonn, Gerhard-Domagk-Straße 1, Bonn, Germany.
Aptamers are short, single-stranded DNA (ssDNA) or RNA molecules that bind to molecular targets with high affinity and specificity. They are identified by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and this method can be used for the identification of aptamers that bind target molecules on cell-surfaces. Cell-SELEX is often labor-intensive and requires multiple negative selection steps to reduce non-specific binding. Here, we present an alternative cell-SELEX strategy that uses a mixture of target and non-target cells during the incubation step with a ssDNA library. The specific interaction of the target cells with a Lewis Y antibody to separate the cell populations to enrich specific binders. Lewis Y-positive MCF7 cells and Lewis Y-negative HEK293T cells were used in an initial SELEX experiment. After six rounds of selection cycle, the enriched ssDNA library showed increased binding to MCF7 cells compared to HEK293T cells. In contrast, SELEX performed only with MCF7 cells resulted in elevated non-specific binding to HEK293T cells. Next-generation sequencing (NGS) of the enriched library enabled the identification of candidate aptamers, which will be further validated on target and non-target cells. These results demonstrate that the simultaneous inclusion of target and non-target cells during cell-SELEX improves selection specificity and represents an efficient alternative to conventional negative selection strategies.
The ASC-1 targeting AS1 aptamer enables selective targeting of subcutaneous white adipose tissue in vivo
Valentin K Reichenbach1,2, Ming Gao1, Ayman Mahoud1, Karoline Kadletz3, Ceren Kimna3, Yagmur A Kulcu3, Ali Ertuerk3, Siegfried Ussar1,2
1Research Unit Adipocytes & Metabolism (ADM), Helmholtz Diabetes Center, Helmholtz Center Munich, Neuherberg, Germany
2German Center for Diabetes Research (DZD), Neuherberg, Germany
3Institute for Intelligent Biotechnologies (iBIO), Helmholtz Center Munich, Neuherberg, Germany Incretin‑based anti‑obesity drugs have recently enabled measurable population‑level weight loss and addressed a major societal burden. However, patients eventually reach a weight‑loss plateau. Thus, new strategies are needed to fully restore white adipose tissue, the body’s major energy reservoir, following weight loss. Targeted delivery systems capable of selectively addressing mature white adipocytes could support sustained metabolic improvements. Aptamers offer high specificity and modularity for targeted delivery but are rarely used in the metabolic disease area. To develop a white-adipose-tissue‑specific DNA aptamer, we performed cell‑SELEX using adipocytes overexpressing the mature adipocyte surface marker Asc‑1 (SLC7A10) for positive selection and Asc-1 knockdown adipocytes for negative selection. FACS screening identified AS1 as the aptamer with the highest binding affinity to Asc-1 expressing cells. AS1 binds an extracellular epitope of Asc‑1 and shows a half‑life of 5 h in mouse serum, and selectively stains subcutaneous white adipose tissue in vivo, following intravenous injection in mice.To facilitate conjugation for drug-delivery applications, we prolonged the aptamers sequence by a DNA sticky-bridge. Dynamic light scattering measurements showed successful AS1 annealing to lipid nano particles displaying the reverse complementary sticky-bridge. AS1 conjugated LNPs carrying fluorescent mRNA displayed markedly enhanced accumulation in WAT following subcutaneous injection compared to untargeted LNPs, visualized by light‑sheet imaging of nanoDISCO‑cleared whole mice.The AS1 aptamer tissue specificity provides a unique possibility to target subcutaneous adipose tissue separate from other fat depots. This platform provides a versatile route for tissue‑specific delivery and opens new paths for metabolic disease intervention.
The optimization of the structure of kanamycin binding aptamers using computational methods and isothermal titration calorimetry
Alexey V Samokhvalov, Anatoly V Zherdev, Boris B Dzantiev
A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology, Russian Acad. Sci., Moscow, Russia
This study analyzed the binding parameters of known DNA aptamers to the widely used antibiotic kanamycin (KANA) that were found in four SELEX investigations. Using NUPACK, UNAfold, and RNAfold software, their structures were estimated. The predicted secondary structures, their Gibbs free energies, and assumptions about the location of the ligand-binding sites were used to propose different modifications of the aptamers with the potential stabilization of their ligand-binding sites. The affinities of thirteen synthesized variants of the 13_82 aptamer (proposed by Stoltenburg et al.; DOI: 10.1155/2012/415697) were measured by isothermal titration calorimetry. By shortening the chain length from 93 to 40 nucleotides and stabilizing of its structure, the dissociation constant was decreased from 470±40 to 92±11 nM. The measurements were carried out in 10 mM Tris-acetate buffer, pH 7.4, with 50 mM NaCl and 3 mM magnesium acetate. The designed superior aptamer variants were applied for KANA detection in a homogeneous competitive system with registration of Förster resonance energy transfer (FRET). The results demonstrate the efficient combination of direct affinity measurements with computational methods for optimizing aptamer design. This study was financially supported by the Russian Science Foundation (Project 25-14-00421).
DNA aptamers targeting FimH as a novel strategy to control Salmonellosis
Inês O Santos1,2, Márcia Faria2, Josué Carvalho2, Luís D R Melo 1,3,4, Débora Ferreira1,3
1Centre of Biological Engineering, University of Minho, Braga, Portugal
2ALS Life Sciences Portugal, Tondela, Portugal
3LABBELS – Associate laboratory, Braga, Guimarães, University of Minho, Portugal
4Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal
Salmonellosis is one of the most common foodborne diseases, with eggs and egg products being the main sources of outbreaks. Infections caused by Salmonella species led to significant economic losses. Restrictions on antibiotic use in poultry production due to antimicrobial resistance and EU regulations have created an urgent need for alternative solutions to control Salmonella contamination and reduce human disease. Aptamers offer a novel and promising strategy beyond antibiotics. These short, single-stranded DNA or RNA molecules fold into specific 3D structures, allowing highly specific, high-affinity binding to target molecules, including microbial proteins. In this work, aptamers were selected against the FimH protein, an adhesin in S. Enteritidis that mediates initial contact with intestinal cells. This selection was performed using Systematic Evolution of Ligands by Exponential Enrichment (SELEX). In this process, magnetic beads were coupled with FimH and incubated with a diverse single-stranded DNA library. Bound sequences were amplified and subjected to successive SELEX rounds. After 12 cycles, enriched pools from late SELEX rounds were analysed by next-generation sequencing and processed using AptaSUITE. Five major sequence clusters were identified in the final ssDNA pool. The most abundant sequences from each cluster were selected for further in silico evaluation, revealing 8 aptamers with favourable stability, as indicated by negative Gibbs free energy values, high GC content, and G-quadruplex motifs. Enrichment of these aptamers across the sequenced pools was observed. Of these, three aptamers (Apt X, Y and Z) revealed promising in vitro binding affinities, with dissociation constants (Kd) in the nanomolar range. Molecular docking and binding free energy calculations were used to characterise FimH–aptamer interactions. Overall, this work highlights the potential of aptamers as candidates for developing novel strategies to control Salmonella infections.
Sensitive, fast and affordable small molecule detection using a Aptazyme assay design made of single stranded functional DNA
Jörg Schönfelder, Merve Sorgucu, Kim Vermeulen, Lars Verschuren and Bart Keijser
TNO Health & Work, Microbiology and Systems Biology, Sylviusweg 71, 2333BE Leiden, The Netherlands
The global market for point‑of‑care (POC) diagnostics is steadily growing, driven by the increasing shortage and need for healthcare personnel and by the discovery of new applications in personal health, sports, and lifestyle monitoring. Traditionally, the majority of POC diagnostics relies on antibodies as biomarker recognition elements. They show strong performance for immunogenic biomarkers, but there are notable exceptions; particularly non‑immunogenic biomarkers and small molecules for which antibodies cannot be raised. Here, we develop a potential point‑of‑care method for these challenging biomarkers by combining split‑DNAzyme and aptamer technologies, i.e., Aptazyme technology. In our approach, the biomarker affinity of the aptamer triggers enzymatic‑like phosphodiester bond cleavage by the split DNAzyme in an RNA containing substrate. This translates biomarker presence into a readable fluorescent signal. Our Aptazyme technology is sensitive, functions at room temperature, and is applicable to a wide range of biomarker types, including those that are difficult to target with antibodies. Moreover, it is entirely based on single‑stranded DNA and therefore enzyme‑free, offering significant advantages such as low cost of goods, long shelf life, robustness, and POC platform flexibility. Importantly, our Aptazyme assay shows improved sensitivity and signal strength compared to traditional aptamer assays, as it produces a kinetically increasing fluorescent signal rather than a decreasing one. In this study, we present a proof of concept of our Aptazyme assay for the small molecule adenosine. We demonstrate that the assay has a response time of 10–15 minutes, a limit of detection in the low micromolar range, functions in various spiked matrices such as urine, serum, and saliva, and performs well across different POC platforms.
Aptamer-Based Biosensor for Smoke Taint Detection in Wine Grapes
Micaela Sequeira¹, Soniya Yambem2, Josh Hixson3, Kirill Alexandrov¹,4, Elena Eremeeva¹
¹Centre for Agriculture and the Bioeconomy, Queensland University of Technology, Brisbane, Australia
2Faculty of Science, School of Chemistry & Physics, Queensland University of Technology, Brisbane, Australia
3Wine Australia, Adelaide, Australia
4ARC Centre of Excellence in Synthetic Biology, Canberra, Australian Capital Territory, Australia
The wine industry is a cornerstone of the Australian economy, contributing over AUD 45 billion annually in Australia and more than USD 340 billion globally. It supports thousands of jobs across viticulture, production, and export. However, the rising frequency and severity of wildfires and heat-driven fire seasons pose a major risk worldwide. Smoke taint renders grapes and wine unsellable, with losses in Australia exceeding AUD 150 million during severe fire seasons, and similar impacts reported in the United States and key European wine regions, including France, Italy, and Portugal. Smoke taint is primarily associated with volatile phenols and their glycosylated forms, like syringol gentiobioside, which accumulate in grapes after smoke exposure and remain undetectable until fermentation, causing undesirable ashy or medicinal flavours. Current assessment relies on costly and time-consuming laboratory testing, with no reliable, portable device for direct smoke taint detection in vineyards. While aptamer-based sensors have transformed the detection of pesticides and environmental pollutants, their application to smoke taint has not yet been reported. Here, we report the discovery of the first high-affinity DNA aptamer specific to syringol gentiobioside, selected through a Capture-SELEX strategy. Isothermal titration calorimetry confirmed nanomolar binding with no cross-reactivity to structurally similar sugars, verifying its viability in complex grape matrices. Based on this aptamer, we developed a fluorescent sensor and generated preliminary data demonstrating its potential for an electrochemical sensor format. We are now expanding this platform to qualitative lateral flow assay and quantitative electrochemical sensors, enabling rapid, on-vineyard diagnostics that can reduce costs for growers and enhance harvest decision-making. This project has already secured over AUD 500,000 in funding to support its transition into field-deployable devices, in close partnership with the wine industry.
Development and validation of aptamer-based diagnostic tools for malaria detection
Laura Sproge1, Gunita Paidere2, Edmunds Zutis2, Maira Elksne2, Kaspars Tars3, Una Riekstina1, Liga Kunrade1
1University of Latvia, Faculty of Medicine and Life Sciences, Department of Pharmaceutical sciences, Riga, Latvia
2Institute of Solid-State Physics, University of Latvia, Riga, Latvia
3Latvian Biomedical Research and Study Centre (BMC), Riga, Latvia
Malaria, caused by Plasmodium parasite, remains a leading cause of mortality, particularly among children and pregnant women. Current antibody-based malaria rapid diagnostic tests are limited by insufficient sensitivity at low parasitemia, and a failure to reliably differentiate Plasmodium species. We have identified two ssDNA aptamers that recognize Plasmodium falciparum and Plasmodium vivax lactate dehydrogenase (pLDH) with a potential for development of malaria diagnostic biosensor. The aim of the study was to investigate the application of P.falciparum and P.vivax aptamers Pf-LDH3t and Pv-LDH2 as a bio selective layer, in biosensor conditions. Aptamers were evaluated against in-house and commercial (SPAN Diagnostics and Creative Diagnostics) pLDH using direct ELONA method. A sandwich ELONA was employed to validate the aptamers as capture and detection tools. Additionally, a hybrid ELONA using a capture antibody and a detection aptamer was assessed. Fluorescence measurements using SYBR Gold were used to evaluate capture aptamer immobilisation on gold surfaces, reflecting biosensor interfaces. Aptamer Pf-LDH3t and Pv-LDH2 affinity to pLDH varied significantly depending on the protein source. Pf-LDH3t-NH₂/Pf-LDH3t-Biotin pair performed best in sandwich ELONA on amino plates. Target pLDH binding was enhanced using a hybrid ELONA, suggesting that optimisation of capture aptamer surface immobilisation may be required. Concentration dependent thiol functionalised aptamer immobilisation on the gold surface was successfully confirmed using fluorescence measurements, establishing the system as a representative model for aptamer-based biosensor. Acknowledgements: BioPhoT project “Smart diagnostics: an aptamer-based device for rapid malaria detection” No. OSI_PIP_BioPhoT-2025/1-0021 Reg. Nr. VPP2024/51-1-1 and UL doctoral grant “Strengthening Doctoral Research and Development Capacity” UL Project No.: 1.1.1.8/1/24/I/003”
The UTexas Aptamer Database: New Developments in Automated Curation
Dhruv Kumar, Amrut Pennaka, Abhinav Vajrala, Einez Wu, and Gwendolyn (Gwen) M Stovall
Freshman Research Initiative, University of Texas, Austin, Texas, USA
The UTexas Aptamer Database (https://sites.utexas.edu/aptamerdatabase) is a publicly accessible repository of over 1,500 aptamer sequences designed to facilitate research by integrating sequence data, selection conditions, and target information. Despite rigorous efforts to extract aptamer sequences and selection data from the literature, our research found frequent, unexplained sequence alterations in the literature itself, highlighting the need for improved validation processes. Additionally, despite peer review and researcher training, we identified discrepancies between our database and others. While cross-referencing helped resolve some inconsistencies, these challenges persist across databases and underscore the need for automated data checks. As aptamer publications continue to grow rapidly, so does the need for efficient curation of sequence and selection data. To address these challenges, we are leveraging Large Language Models (LLMs) to automate aptamer data extraction, improving efficiency and scalability. After investigating several models with and without model training, we now utilize Google Gemini 2.5 Pro through an API-based pipeline for automated extraction of relevant sequence and metadata fields from peer-reviewed publications. To date, we have evaluated our data curation process on a subset of manually analyzed papers, refining extraction accuracy through API integration, prompt engineering, and examination with and without training. Our data collection process routinely collects data from text, such as aptamer and target name (91% correct, n=50 papers), but more work is needed to refine the collection of the pool (81% correct) and the aptamer sequence (62% correct). By developing a framework and toolkit to systematically aggregate aptamer selection and sequence data, we aim to improve the accessibility and visibility of aptamer information.
Validation of GreenB1 aptamer specificity in pancreatic ductal adenocarcinoma models under static and microfluidic conditions
Kristaps Sunteiks, Karina Goluba, Vadims Parfejevs, Una Riekstina, Liga Kunrade
Department of Pharmaceutical Sciences, University of Latvia, Faculty of Medicine and Life Sciences, Latvia, Riga
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive types of cancer. GreenB1 is an integrin β1 (ITGB1) specific aptamer, and assessing its specificity on PDAC cell lines, organoids, and organ-on-chip (OoC) model systems would provide in-depth information about the GreenB1 aptamer’s ability to bind to tumour cells, depending on environmental conditions. Consequently, the aim of the current study was to characterise the affinity of GreenB1 in PDAC cell lines and organoids obtained from patient biopsies under static and microfluidic conditions. To achieve this, experimental condition optimisation and GreenB1 affinity testing for established PDAC cell lines, primary patient-derived PDAC and normal pancreatic organoid cultures were performed using ImagestreamX MarkII (Cytek Biosciences) imaging flow cytometer (FC). OoC model systems with and without endothelial channels were established using 4-channel CellBox Labs microfluidic chips. GreenB1 binding validation in microfluidic conditions was assessed using immuneluorescence analysis (IF). Fluorescence signal of GreenB1 was amplified with Alexa FluorTM 488 Tyramide SuperBoostTM kit. The resulting FC data indicate that Capan cells have a superior affinity for GreenB1. Capan cells were further used in an OoC system in which the binding of GreenB1-FAM could not be detected by IF due to low signal intensity. Signal amplification enhanced the detection of GreenB1 binding in static conditions and enabled the visualisation of aptamer binding to Capan cells in a PDAC OoC model. GreenB1 binding conditions were optimised for the Capan cell line to be further used in the OoC system. Preliminary data demonstrate that GreenB1 binding is detectable in an OoC system. These findings support the feasibility of developing a further aptamer-based targeted delivery approach in physiologically relevant conditions. This study was funded by the project No. lzp-2024/1-0206 and UL FMLS performance funding (student grants).
Targeting EphA2-Positive Extracellular Vesicles as Novel Angiogenic Biomarkers with A40s Aptamer in Glioblastoma Multiforme
Sara Verde1,2, A. Fraticelli1,2, G. Petrillo3, M. Mascolo3, K. Tkalčec3, C. Pignataro3, Y. C. Veneruso3, D. Fiore3, C. Quintavalle4, L. Ricci Vitiani5, A. Affinito6 and G. Condorelli3
1Department of Biomedicine and Prevention, University of Rome “Tor Vergata”, Rome, Italy
2AKA Biotech, Naples, Italy
3Department of Molecular Medicine and Medical Biotechnology, “Federico II” University of Naples, Via Pansini 5, Naples, Italy
4Institute of Endotypes in Oncology, Metabolism and Immunology “G. Salvatore (IEOMI), Consiglio Nazionale delle Ricerche (CNR), Naples, Italy
5 Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy
6IRCCS SYNLAB SDN, Naples, Italy
Glioblastoma multiforme (GBM) is a rare and highly aggressive brain tumor with poor prognosis with minimal therapeutic advancement. A hallmark of GBM is the extensive neovascularization, which sustains rapid tumor growth. Nevertheless, second-line treatment with anti-angiogenic drugs like Bevacizumab rarely extends overall survival of GBM patients. In addition, diagnosis also remains challenging due to the complex molecular classification and the need for less invasive procedures. In this study, we aim to address the demand of innovative therapeutic strategies and non-invasive tools for GBM early detection and treatment monitoring. GBM stem cells (GSCs), a subpopulation of GBM cancer cells responsible for tumor progression and therapeutic resistance, release small extracellular vesicles (EVs) involved in intercellular communication and molecular cargo transferring. EVs, with their protein, lipids, and nucleic acid content, represents promising candidates for liquid biopsy approaches. Furthermore, aptamers, synthetic single-stranded nucleic acids, can selectively recognize both cells and EVs. Here we studied the pro-angiogenic behaviour of GSCs and the role of EphA2 in transdifferentiation and vascular mimicry through tube formation assay. EVs were firstly isolated from GSC cultures and characterized for size distribution, concentration and molecular composition by Nanoparticle Tracking Analysis and Western blot. Moreover, we exploited the A40s aptamer, selective for EphA2, to inhibit pro-angiogenic processes and selectively recognize GSC-derived EVs by several techniques. Importantly, A40s binding was also validated on EVs isolated from sera of GBM patients, supporting the assay’s biological reproducibility and translational potential. These findings suggest that A40s may reduce GBM vascularization and enable selective detection of EphA2-positive EVs, supporting its development as both an innovative therapeutic strategy and a non-invasive diagnostic tool for GBM.
A Quadruplex-Triplex Hybrid with a Pseudo Three-Layered Tetrad Core of a Codeine Aptamer
Yoanes M Vianney, José C Martins
NMR and Structure Analysis Unit, Department of Organic and Macromolecular Chemistry, Ghent University, Ghent
DNA can adopt a wide array of three-dimensional structures beyond the canonical B-form. One way to exploit this structural diversity for technological application is through the use of aptamers. Despite the large number of reported aptamer sequences, very few have advanced beyond proof-of-concept studies. A major obstacle in this respect is the limited structural understanding of aptamers. A key aspect of this structural diversity in aptamers is the formation of non-canonical structures. One such motif is the G-quadruplex, a class of nucleic acid secondary structures formed by guanine (G)-rich sequences that are observed in some aptamer structures. A candidate aptamer of particular interest is the codeine-binding aptamer, which has been proposed to contain a G-quadruplex element. However, the sequence proposed in the original work is prone to aggregation, complicating its structural characterization. Using the principles driving G-quadruplex folding geometry and guided by NMR data, we redesigned the codeine aptamer sequence to favour a predominant fold capable of binding codeine. Spectral assignment and subsequent structure calculations revealed that the codeine aptamer adopts a three-layered tetrad core embedded in a quadruplex-triplex (QT) hybrid structure, locating the codeine at the quadruplex-triplex interface. The glycosidic torsion angle pattern (syn-anti pattern) and thus the folding resemble those of a (3+1) hybrid topology categorized as the hybrid 1-type with a propeller, lateral, lateral (-pll) loop progression. However, unlike typical G-quadruplexes, the three-layered tetrad core is composed of two G-tetrads (upper and middle) and one GCGC tetrad at the bottom. The structure adopted by this codeine aptamer challenges and expands the current framework of G-quadruplex folding and classification.
A Long-Lasting CTGF CT-Domain Specific Aptamer Apc003OA Demonstrates Superior Anti-Fibrotic Effect in an Idiopathic Pulmonary Fibrosis Mouse Model
Xue Xue1#, Meishen Ren1#, Huarui Zhang2#, Shanshan Yao2, Xin Yang1, Luyao Wang1, Zongkang Zhang2, Yuanyuan Yu1, Aiping Lyu1, Sifan Yu1, Baoting Zhang2*, Ge Zhang1
1Institute for Advancing Translational Medicine in Bone & Joint Diseases, School of Chinese Medicine, Hong Kong Baptist University, 34 Renfrew Road, Kowloon City District, Hong Kong Special Administrative Region, China
2School of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories District, Hong Kong Special Administrative Region, China
#Co-first authors
Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal interstitial lung disease with poor prognosis. Current therapies (e.g., pirfenidone, nintedanib) for IPF merely slow the progression of fibrosis, and their clinical usage is constrained by side effects. Connective tissue growth factor (CTGF) is considered as a central driver of fibrosis progression, which is mediated by its VWC- and CT-domains. FG-3019, a VWC-domain specific antibody, showed favorable safety and efficacy profile in phase II trial, but it failed to meet the efficacy endpoints in phase III trial of IPF. Our in vitro analysis showed that genetic deletion of VWC-domain reduced pro-fibrotic signaling by only 20-25%, whereas deletion of CT-domain suppressed it by 80-85%. Notably, TGF-β1 level was significantly elevated in CTGFko fibroblast expressing CTGF-ΔVWC, while CTGF-ΔCT did not. This indicates CT-domain contributed more to the fibrosis activity of CTGF than VWC-domain in vitro. Using our aptamer platform (AptGen), we developed Apc003, a high-affinity aptamer specific for CTGF CT-domain. To extend the in vivo half-life of Apc003, we prepared the long-lasting CTGF aptamer (Apc003OA) by using our published long-lasting fatty acid modification via octadecadienoic acid (OA). Apc003OA showed 7 days of half-life in normal mice. In a bleomycin-induced IPF mice models, Apc003OA showed an 80–85% reduction in fibrosis markers without increasing TGF-β1. Comparatively, treatment with FG-3019 at an equivalent dose achieved only about a 25% drop, accompanied by a rise in TGF-β1. Moreover, no detectable pathological lesion was observed in vital organs after Apc003OA treatment in vivo. The anti-fibrotic effect of Apc003OA was completely abolished by co-administration of a CT-domain blocking peptide, confirming its efficacy depends on specific CT-domain engagement. These findings validate the CTGF CT-domain as a superior therapeutic target and present Apc003OA as a potent, targeted candidate for IPF therapy.
Inhibition of ERK/MAPK pathway by intracellular expression of C5 RNA intramer
Noushin Zahedi1, Simon Marpert1, Friederike Schlotmann1, David Marian-Otte1, Philipp Simon2, Katrin Paeschke2, Günter Mayer1,3
1Department of Chemical Biology, Life and Medical Sciences Institute (LIMES), University of Bonn, Gerhard-Domagk Str. 1, 53121 Bonn, Germany
2 Clinic of Internal Medicine III, Oncology, Hematology, Rheumatology and Clinical Immunology, University Hospital Bonn, Sigmund Freud Str. 25, 53127 Bonn, Germany
3Center of Aptamer Research & Development, University of Bonn, Gerhard-Domagk Str. 1, 53121 Bonn, Germany
Effective protein targeting is essential for the comprehensive study of signaling pathways. Intracellular aptamers, or intramers, represent a powerful tool for modulating these signaling cascades. Here, using Mitogen-Activated Protein Kinase (MAPK)-responsive luciferase and fluorescence assays, we demonstrate that expression of the ERK1/2 protein-inhibitory intramer C5 leads to inhibition of the MAPK pathway in HEK293T, HeLa, and HCT116 colon cancer cells. Moreover, expression of the C5 intramer did not induce cellular stress, as determined by analysis of the phosphorylation status of eukaryotic initiation factor 2α (EIF2α) and Ras-GTP-activating protein SH3 domain–binding protein 1 (G3BP1), two proteins involved in cellular stress responses. To identify a more potent inhibitor of the MAPK pathway, three partially randomized libraries with different mutagenesis percentages based on the C5 sequence were generated and will be used for intracellular screening. These findings highlight the potential of intramers for the targeted inhibition of intracellular interactions.
High-specificity ssDNA Aptamer Targeting Aconitine for Strand-Displacement Biosensor Development to Enhance Traditional Chinese Medicine Safety
Jin ZHAO1, Andrew B. KINGHORN2, Julian A. TANNER2, Rex Pui Kin LAM1
1Department of Emergency Medicine, School of Clinical Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China
2School of Biomedical Science, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China
Aconitum roots are extensively utilized in traditional Chinese medicine (TCM) for treating rheumatological and traumatic conditions. However, aconitine, the principal alkaloid, is a potent cardiotoxin and neurotoxin, with a human lethal dose of only 2 mg. Aconitine can persist in urine and remain detectable for up to 7 days post-overdose, highlighting the need for rapid, cost-effective detection methods. To meet this demand, we employed a capture-SELEX strategy to isolate aptamers with high specificity for aconitine. The lead aptamer exhibited a dissociation constant (Kd) of 6.974 μM, determined via thioflavin T fluorescence. We further engineered a strand-displacement biosensor by labeling the aptamer with Cy5 and hybridizing it to a quencher-tagged complementary strand, yielding an apparent Kd of 3.231 µM. The sensor discriminates aconitine from structurally related alkaloids (mesaconine, benzoylhypaconine, benzoylmesaconine) and hydrolysis products (benzoylaconine, aconine) while enabling sensitive quantification in spiked urine matrices. These findings underscore its potential utility for 1) emergency toxicological diagnosis, 2) pharmacokinetic monitoring during overdose management, and 3) quality control of Aconitum-derived herbal preparations. Our work establishes a paradigm for developing aptameric tools targeting low-functional-group phytotoxins, with direct implications for enhancing the safety profile of TCM formulations.