Thank you for considering presenting your work as a poster at this conference.
Thank you for considering to present your work as a poster at this conference.
Before uploading your poster, you must make sure that you follow ALL of the instructions above!
Please keep an eye on this page for any future updates.
Before uploading your poster, you must make sure that you follow ALL of the instructions above!
(Presenters in Bold)
If your abstract has been accepted for presentation but it does not appear in the list below, please let us know as soon as possible by emailing AptamersOxford@gmail.com.
SELEX of modified aptamers to study the catalytic mechanism underlying peptidoglycan polymerization by transpeptidase
Yan Badji1,2, Mélanie Etheve-Quelquejeu1, Laura Iannazzo1, Marcel Hollenstein2
1Team “Chemistry of RNAs, Nucleosides, Peptides and Heterocycles”, CNRS UMR8601, Université Paris cité, 45 Rue des Saints-Pères, 75006 Paris, France
2Team “Bioorganic chemistry of nucleic acids”, CNRS UMR 3523, Institut Pasteur, 75015 Paris, France
Antibiotics are known to treat serious bacterial infections, such as pneumonia, tuberculosis but also more benign infections such as angina. These compounds can do so kill or prevent bacterial growth. Nevertheless, bacteria can quickly mutate, and bacterial resistance against antibiotics can occur. Because of antibacterial resistance, the treatment of some infections becomes increasingly difficult. In a recent assessment, antibiotic resistance will soon become one of the main causes of death worldwide. To fight against this phenomenon, it is very important to understand the mechanism and mode of action of bacterial resistance. In this context our group is interested in Penicillin binding protein (PBP) transpeptidases which are involved in the biosynthesis of the peptidoglycan, the most important constituent of the bacteria cell wall. Our understanding of the cross-linking reaction catalysed by PBPs and the identification of inhibitors of these enzymes have been limited by the absence of a versatile assay. The SELEX approach will be applied to the identification of peptide-DNA aptamers that will act as soluble surrogates of the complex substrates of the transpeptidase. This is mandatory for (i) understanding the mechanism of the transpeptidation reaction, which is largely unknown due to limited access of chemically defined substrates, and (ii) developing assays to determine the efficacy of transpeptidase inhibitors. Our objective is to obtain peptide-nucleotides conjugates and raise aptamers via SELEX against transpeptidases. The peptidic substituent will mimic the amino acids of the natural substrate while the role of the glycan chain will be adopted by the aptamer. The bioconjugation between peptide and nucleic acid will be carried out by application of click chemistry. The synthetic pathway developed in the group to access to peptide-nucleotides conjugates will be presented here.
Synthetic small molecule-binding aptamers as versatile regulatory elements
Leon Boettger, Beatrix Suess
Synthetic RNA Biology, TU Darmstadt, Schnittspahnstraße 10, 64287 Darmstadt, Germany
One of the most exciting areas of synthetic biology is to control cellular behavior using engineered genetic circuits. To combine genes of interest in a building block-like manner and transfer them to organisms of interest in order to achieve desired biological functions. However, the expression levels of the corresponding genes need to be regulated and fine-tuned to avoid unbalanced gene expression and the accumulation of toxic intermediates. Synthetic RNA-based systems have increasingly been used for the regulation of gene expression. Due to their structural properties, riboswitches provide a convenient basis for the development of ligand-dependent controllable systems. Here, we present a set of five synthetic riboswitches for the control of gene expression using small molecules as a ligand. Our devices can be used for the control of translation initiation, pre-mRNA splicing, RNA self-cleavage as well as fluorogenic aptamers. Thereby, we offer modular investigative tools for the design and study of complex regulatory systems, metabolic pathways, and biosensors.
Development of DNA aptamers as therapeutic tools against Pseudomonas aeruginosa
Federico Bosetto, Tongyuan Wei, Maria Zacharopoulou, Ioanna Mela
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK
Antibiotic resistance is posing a serious threat to human health. Therefore, innovative therapeutic tools are needed to efficiently treat resistant bacteria. Aptamers are small synthetic oligonucleotides which show high specificity and affinity for a designated target. They have multiple applications in the biotechnological field and can be considered as biological drugs to target resistant microorganisms. In this work we selected DNA aptamers to specifically target the hemophore of the haem assimilation system (HasA) from Pseudomonas aeruginosa. Starting from a DNA library with a random region of 40 nucleotides, we used SELEX to select DNA aptamers with HasA as target. Twelve rounds of SELEX were performed, and the selection was monitored by qPCR and melting curve analysis. From round 1 to 12, the main fluorescence signal peak at 70°C, typical of the library, gradually shifted to 84°C. Selected putative aptamers were sequenced by Next Generation Sequencing and, the most abundant and relevant candidates are currently being tested for their binding affinity and specificity. At the same time, our group is also working on the aptamer selection towards the receptor of the haem assimilation system (HasR). Since iron is an essential micronutrient for P. aeruginosa, as for many bacteria. Targeting and potentially blocking elements of the bacterium’s iron acquisition mechanisms, such as HasR and HasA, can put the bacterium in significant metabolic disadvantage.
Ultrasensitive and label-free detection of hepatitis C virus core protein using graphene field-effect biosensors based on in vitro selected aptamers
Carlos Briones1, Irene Palacio2, Miguel Moreno1, Beatriz Torres-Vázquez1, Telma Domingues3,4, Jérôme Borme3, Marzia Marciello5, Jesús Ignacio Mendieta-Moreno2,6, José Ignacio Martínez2, María Francisca López2,
Mar García-Hernández2, Luis Vázquez2, Pavel Jelínek6, Pedro Alpuim3,4, José Ángel Martín-Gago2
1Department of Molecular Evolution, Centro de Astrobiología (CAB, INTA-CSIC), 28850 Torrejón de Ardoz, Madrid, Spain
2Institute of Material Science of Madrid (ICMM-CSIC), Canboblanco, 28049 Madrid, Spain
3International Iberian Nanotechnology Laboratory (INL), 4715-330 Braga, Portugal
4Centro de Física das Universidades do Minho e Porto (CF-UM-UP), Universidade do Minho, 4710-057 Braga, Portugal
5Department of Chemistry in Pharmaceutical Sciences, Faculty of Pharmacy, Complutense University (UCM), 28040 Madrid, Spain
6Institute of Physics, Czech Academy of Sciences, 16200 Prague, Czech Republic
Aptamers are short, single-stranded and structured nucleic acids (RNA or ssDNA) that can bind to different targets (from small molecules to tissues), showing high affinity and specificity. They are increasingly being used as biological recognition elements in biosensing, disease diagnosis and therapy, with broad applicability in virology. We obtained ssDNA and RNA aptamers targeting six variants of the HCV core protein belonging to genotypes 1 to 4. Their affinity constants (Kd) were quantified by colorimetric ELONA and ELONA-(RT)qPCR. Notably, two high-affinity DNA aptamers (termed AptD-1312 and AptD-1932 and characterized by Kds in the low nanomolar range) were prominently represented in all selection processes. They were assayed in Huh-7.5 reporter cell lines infected with HCV, demonstrating a reduction in both the viral progeny titer and the extracellular HCV RNA level [Torres-Vázquez et al., 2022, Journal of Molecular Biology, 434, 167501; European Patent EP4124659A1]. Afterwards, AptD-1312 was used as the biosensing probe in a graphene-based sensor. Biosensors that use graphene solution-gated field-effect transistors (g-SGFETs) have emerged as a promising tool for detecting a wide range of analytes, though their performance is significantly influenced by the graphene functionalization method. As an alternative, we developed a controlled in-vacuum physical protocol for the covalent functionalization of graphene enabling the construction of novel aptamer-based biosensors. The AptD-1312-based, g-SGFET aptasensor exhibited exceptional specificity and robustness, achieving attomolar (10xE-18 M) detection of HCV core protein in both buffer solution and human blood plasma. Such an improved sensitivity paves the way for the use of this kind of biosensing platforms for ultrasensitive, real-time and label-free diagnosis of various diseases caused by RNA or DNA viruses [Palacio et al., 2023, Biosensors and Bioelectronics 222: 115006; European Patent EP4124855A1].
In vitro selection of glypican-3 specific aptamers for active targeted drug delivery applications in hepatocellular carcinoma
Magdolna Casian1,2,3, Oana Hosu-Stancioiu1, María Jesús Lobo Castañón2,3, Noemí de-los-Santos-Álvarez2,3, Cecilia Cristea1
1Department of Analytical Chemistry, Faculty of Pharmacy, Iuliu Hațieganu University of Medicine and Pharmacy, 4 Pasteur Street, 400349, Cluj-Napoca, Romania
2Departamento de Química Física y Analítica, Universidad de Oviedo, c/Julián Clavería 8, 33006 Oviedo, Spain
3Instituto de Investigación Sanitaria del Principado de Asturias, Av. de Roma s/n, 33011, Oviedo, Spain
Hepatocellular carcinoma (HCC) is one of the most common types of primary liver cancer and is characterized by rapid progression and poor survival rates. Current treatment options have limited efficacy, primarily because of the low penetration of chemotherapeutic agents into the tumor site, chemoresistance, and severe toxicity in healthy tissues, which significantly undermines therapeutic outcomes. Aptamer technology has been widely explored in various biomedical fields, due to their high affinity towards specific targets, small size and non-immunogenicity, making them suitable for efficient tissue penetration and active delivery of chemotherapeutics to the tumor site. In this study, magnetic bead-based SELEX technology was applied for the screening of novel DNA aptamers targeting glypican-3 (GPC-3), an HCC tumor biomarker. The designed strategy involved the immobilization of GPC-3 on tosylactivated magnetic beads, after which multiple selection rounds were performed using different serum proteins as counter-selection molecules to make the selection more stringent. The progress of aptamer selection was monitored by quantitative real-time PCR, melting curve analysis, and enrichment assay. After selection, the resulting oligonucleotides were sequenced for primary structure determination and their affinities were evaluated by optical measurements. The obtained aptamer will be further explored for the development of targeted delivery systems based on magnetic nanoparticles loaded with an anti-angiogenic tyrosine kinase inhibitor as a theranostic approach that combines active targeting and imaging of HCC. The funds for this work were obtained from project PID2021-123183OB-I00), financed by MCIN/AEI/10.13039/501100011033/ FEDER, UE, Romanian Ministry of Education and Research Project PN-IV-P1-PCE-2023-1104, no.62PCE/03.01.2025. and UMF internal grant no. 779/1/13.01.2025.
Active targeting of Cardiomyocytes via Aptamer-Functionalized Lipid Nanoparticles for Cardiac Regeneration following ischemic heart disease
Hiba AM Gafar1, Alexandra R Paul1,3, Adam A Walters1, Mauro Giacca2, Khuloud T Al-Jamal1,3
1Institute of Pharmaceutical Science, Faculty of Life Sciences & Medicine, King’s College London, 150 Stamford Street, London, SE1 9NH, UK
2School of Cardiovascular and Metabolic Medicine & Sciences, Faculty of Life Sciences & Medicine, King’s College London, James Black Centre, 125 Coldharbour Lane, London, SE5 9NU
3Department of Pharmacology and Pharmacy, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR
Myocardial infarction (MI) is a major cause of heart failure. Despite considerable progress in medical therapy and supportive devices, mortality for heart failure is still 50% at 5 years. A major cause of post-MI heart failure is the inability of cardiomyocytes (CMs) to regenerate the lost portion of myocardium. A recent high throughput screening has identified a series of human microRNAs that stimulate cardiomyocyte replication and induce cardiac regeneration. For a miRNA to become a viable therapeutic prospect, it must be effectively delivered to the cytosol of the CM. Lipid nanoparticles (LNPs) are potent non-viral vectors used for gene delivery, which are also suitable for miRNA delivery to CMs. However, LNPs have a natural liver tropism, and active targeting must be implemented for myocardial-specific delivery. Aptamers are usually produced by selection from a large random sequence pool of nucleotides with the technology of systematic evolution of ligands by exponential enrichment (SELEX). Aptamers can be used to functionalize LNPs to achieve CM targeting and reduce off-target effects. Here, we study the application of LNPs to deliver pro-regenerative miRNA-199a-3p for treating ischemic heart disease post-MI. The formulated LNPs will be surface modified with aptamers, acting as a targeting moiety to improve cardiac delivery. The therapeutic effects of miRNA-199a-3p LNPs will be tested first in primary cultured cardiomyocytes and then in MI animal models.
SELEX with methylene-blue modified nucleotide to raise aptamers against pancreatic cancer biomarker glycan CA19-9
Weisi HE*1, Ryan HoPing Siu1, Alix Bouvier-Müller3, Marcel HOLLENSTEIN3, Julian Alexander 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 Park, Shatin, New Territories, Hong Kong SAR, China
3Institut Pasteur, Université Paris Cité, CNRS UMR3523, Department of Structural Biology and Chemistry, Laboratory for Bioorganic Chemistry of Nucleic Acids, 28, rue du Docteur Roux, 75724 Paris Cedex 15, France
Pancreatic cancer (PC) has a high mortality rate with poor prognosis. The poor prognosis is partially due to late detection of PC. The only existing blood testing for the pancreatic cancer glycan biomarker CA 19-9 uses the 1116NS19.9 antibody. CA 19-9 is the only FDA approved biomarker for monitoring PC. Aptamers hold promise as an alternative to antibodies but targeting glycans with aptamers has been a challenge. In addition, aptamers used in electrochemical sensing devices typically require the post-SELEX addition of methylene blue . There are potential advantages of including the methylene blue within the selection process itself which we investigate here. Here, we report a methylene blue modified DNA aptamer SELEX against CA19-9. We have synthesised the methylene blue modified nucleotide. Different conditions, including different polymerases, extension times and concentration of methylene blue modified nucleotides, have been tested to integrate the modified nucleotide into a naïve ssDNA library. Furthermore, the strategy of CA19-9 aptamer SELEX was developed. Two CA19-9 sequences (ModSeedF000 and ModSeedF003) have been identified after SELEX and NGS analysis of enriched pools. Preliminary MST data have shown the binding trend between biotin-modified CA19-9 and two isolated sequences. Compared to ModSeedF003, ModSeedF000 has better selectivity against biotin- CA19-9 than unmodified biotin. Comparing the sequence with and without methylene blue modification, we speculate that the methylene blue on CA19-9 sequence is involved in binding. We further utilized electrochemical assay to confirm the binding and selectivity between BSA-CA19-9 and ModSeedF000. In the longer term we aim to apply the methylene blue modified CA19-9 aptamer into serum buffer systems to see the potential clinical usage of methylene blue modified aptamer.
Aptamers for detection in novel assays and delivery of therapeutic payloads
George W Jackson
Base Pair Biotechnologies, Inc., 8619 Broadway St, Suite 100. Houston, TX, USA
Aptamers represent a highly promising modality for sensing targets across a broad size range, from small molecules to whole viral particles, in “self-reporting” (i.e., label-free) biosensors. This advantage arises from their ability to undergo significant conformational changes upon target binding, often exceeding those observed with antibody-based reagents. Beyond biosensing, aptamers are increasingly utilized as both standalone therapeutic agents and as targeted delivery vehicles for other therapeutic payloads, enhancing drug specificity and efficacy. Their versatility, high affinity, and ease of modification make them attractive for applications in diagnostics, targeted therapy, and drug delivery. This presentation highlights recent advancements in aptamer-based biosensing and therapeutic strategies, showcasing their expanding role in precision medicine and biotechnology.
Specific Targeting and Imaging of RNA G-Quadruplex (rG4) Structure using Non-G4-Containing L-RNA Aptamer and Fluorogenic L-Aptamer
Hill Lam Lau[‡], Haizhou Zhao[‡], Hengxin Feng1, and Chun Kit Kwok
[‡]These authors contribute equally to this work
Department of Chemistry and State Key Laboratory of Marine Pollution, City University of Hong Kong, Kowloon Tong, Hong Kong SAR 000000, China
2Shenzhen Research Institute of City University of Hong Kong, Shenzhen 518057, China RNA G-quadruplex structures (rG4s) play important roles in the regulation of biological processes. So far, all the L-RNA aptamers developed to target rG4 of interest contain G4 motif itself, raising the question of whether non-G4-containing L-RNA aptamer can be developed to target rG4. Furthermore, it is unclear whether an L-aptamer-based tool can be generated for G4 detection in vitro and imaging in cells. Herein, we design a new strategy using a low GC content template library to develop a novel non-G4-containing L-RNA aptamer with strong binding affinity and improved binding specificity to rG4 of interest. We identify the first non-G4-containing L-Aptamer, L-Apt.1-1 with nanomolar binding affinity to amyloid precursor protein (APP) D-rG4. We apply L-Apt.1-1 to control APP gene expression in cells via targeting APP D-rG4 structure. Moreover, we develop the first L-RNA-based fluorogenic bi-functional aptamer (FLAP) system, and engineer L-Apt.1-1_Pepper for in vitro detection and cellular imaging of APP D-rG4. Our work provides an original approach for developing non-G4-containing L-RNA aptamer for rG4 targeting, and the novel L-Apt.1-1 developed for APP gene regulation, as well as the L-Apt.1-1_Pepper generated for imaging of APP rG4 structure can be further used in other applications in vitro and in cells.
Development of multivalent aptamers targeting gastric cancer cells
Gorann Lepied1,2, Christine Varon2, Pierre Dubus2,3, Jeanne Leblond Chain1
1University Bordeaux, CNRS, INSERM, ARNA, UMR 5320, U1212, F-33000 Bordeaux, France
2University Bordeaux, INSERM, BRIC, U 1312, F-33000 Bordeaux, France
3CHU Bordeaux, Tumor Biology, F-33000 France
Multivalency refers to constructing molecular structures that incorporate two or more aptamers, either identical or distinct. This strategy has already demonstrated powerful outcomes in oncology where numerous different biomarkers are often used as entry points for targeted therapies, increasing both specificity and efficacy against cancer cells. In this project, we aim to develop a dual-aptamer-based approach targeting specific gastric cancer stem cells (GCSCs) biomarkers – EpCAM (CD326) and CD44(v9) – to enhance both diagnostic accuracy and chemotherapy precision. Here, we developed bi-specific aptamer assemblies to improve the selective recognition of GCSCs. A CD44v9-binding aptamer (Apt4) was hybridized with an EpCAM-binding aptamer (SYL3C) via a 15-nucleotide linker. Size-adjustable spacers such as hexaethylene glycol or poly-thymine strands were integrated into the sequence to evaluate length and flexibility contribution to the dual-recognition process. Assembly formation was assessed by native PAGE. The targeting ability of the aptamers was evaluated on four gastric cancer cell lines—AGS, NCI-N87, MKN74, and MKN45—via flow cytometry and compared to conventional antibodies targeting both CD44 and EpCAM receptors, allowing the selection of the most relevant model for EpCAM+/CD44(v9)+ GCSCs. Binding curves of both EpCAM- and CD44(v9)-specific single aptamers were generated via flow cytometry, demonstrating affinity values consistent with literature reports. Single aptamers were compared with different SYL3C-Apt4 assembly designs for their targeting capacity toward EpCAM+/CD44(v9)+ GCSCs. We finally demonstrated receptor specificity through flow cytometry competition assay between dual assemblies and their respective antibody, revealing distinct aptamer binding kinetics for the two receptors. The next steps of this study will focus on improving assembly specificity, diagnostic capabilities, and developing aptamer-drug conjugates for targeted delivery.
Rational Design of Structurally Stable DNA Aptamer Screening Libraries Guided by Empirical Sequence Patterns and Thermodynamic Motifs
Steven G Ochoa1, Valeria T Milam1,2
1School of Materials Science and Engineering, Georgia Institute of Technology, 771 Ferst Dr. NW, Atlanta, GA 30332-0245, USA
2Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, 771 Ferst Dr. NW, Atlanta, GA 30332-0245, USA
Aptamers are typically identified from combinatorial libraries with random sequences. Structured oligonucleotide libraries offer advantages by increasing conformational complexity to promote target binding and selection efficiency. To explore these advantages, three DNA libraries were rationally designed by integrating empirical sequence biases and experimentally validated thermodynamic motifs: (1) an Empirical library enriched in guanine and depleted in cytosine, reflecting nucleotide composition patterns observed in functional nucleic acids; (2) a Tetraloop library, embedded with known DNA hairpin motifs experimentally identified for exceptional thermodynamic stability; and (3) a Standard library composed of conventional random sequences serving as a control. Computational modeling predicted a hierarchy of thermal stabilities with the Tetraloop library exhibiting highest stability followed by the Standard, then Empirical libraries. By implementing libraries with distinct structural stabilities in high-throughput selection, this study establishes a framework for investigating the role of structural stability in aptamer functionality. Experimental validation via UV-vis melting analysis confirmed these predictions. Additionally, quantitative polymerase chain reaction (qPCR) demonstrated efficient and unbiased amplification across all libraries. Finally, an adapted protocol for next- generation sequencing (NGS) analysis confirmed compatibility with high-throughput Illumina platforms. By integrating empirical nucleotide biases with experimentally validated thermodynamic motifs, this approach enables precise control over structural features and demonstrates the potential of rationally designed aptamer libraries to enhance discovery of superior aptamers.
New solution for long oligonucleotide synthesis: A combination of chemical synthesis and enzymatic ligation accelerates high quality manufacturing of long oligo
Toru Okamatsu, Masato Sanosaka, Natsumi Sakamoto, Emi Saito, Harei Sakurai, Hirokazu Nankai
Ajinomoto Bio-Pharma Services, GeneDesign, Inc, Japan
Industrial scale synthesis of long-chain oligonucleotide including aptamers is one of the long-standing issue to commercialize oligonucleotide products in market. Recently demands for the synthesis of exceeding 100 mer long chain RNAs, such as single guide RNA (sgRNA) for genome editing, are increasing. Many challenges can be encountered during chemical synthesis of long-chain RNA such as the steric hindrance of the TBDMS protecting group on RNA phosphoramidite monomers. In vitro transcription is widely used in long-chain RNA synthesis but cannot be utilized when any partial base modifications are required.To address these challenges, we have developed efficient method for manufacturing a high-quality long chain RNA by enzymatic ligation from multiple chemically synthesized short-chain RNA fragments. Using this method we were able to synthesize 100 base length long sgRNAs with chemical modifications partially or heavily and a 560 base length mRNA which successfully produced a functional protein was produced in a cell-free translation system. This solution for production of long-stranded RNA allows for chemical modifications and is expected to lead to the development of nucleic acid drugs which have previously not been possible as drug discovery targets due to chemical synthesis limitations.
Reactivating the STING pathway in PTK7-positive acute leukemia cells using DNA-based theranostic nanodevices
Elisa Ottalagana1,2, Andrea Marranci1, Aldo Moscardini2, Francesco Olimpico1, Fabio Beltram1,2, Stefano Luin2,# & Andrea Ghelli Luserna di Rorà1,#
1Fondazione Pisana per la Scienza Onlus, San Giuliano Terme, Italy
2NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Pisa, Italy
#Equal contributions
In eukaryotic cells, the STING pathway primarily responds to cytosolic DNA coming from viral/bacterial infection or damaged genomic DNA. Once activated, this pathway boosts the immune response by triggering the production of interferon type-I and other pro-inflammatory cytokines. In cancer cells, the STING pathways can be downregulated or silent because of the lack of cytosolic dsDNA, allowing them to evade the immune system and resist eradication. Thus, in oncology, reactivating the STING pathway is a promising innovative therapeutic strategy to enhance host immune response against cancer cells. For this reason, the aim of this project is to develop and test in vitro an aptamer-based nanodevice (hereafter StingApt) for activating the STING pathway selectively in cancer cells and, in particular, in acute lymphoblastic and myeloid leukemia (ALL and AML, respectively) models. StingApt is an oligomer comprehending a ssDNA aptamer targeting PTK7 receptor, and a 90-bases dsDNA sequence that activates the STING pathway; this version of StingApt should work therefore for PTK7+ cell lines. Firstly, we tested the basal protein/gene expression of several STING pathway components in a panel of cell lines. We then characterized in these cells, by immunoblotting and/or rtPCR, the effect of reactivating the STING pathway using a commercially available STING agonist (STING agonist-4, SA-4), the electroporated StingApt and 90-base dsDNA, finding similar results. Using confocal microscopy and flow cytometry analyses, we demonstrated that StingApt selectively binds to, and is internalized by, PTK7+ ALL cells more efficiently than the 90-base dsDNA. Finally, to increase the efficacy of our nanosystem in activating the STING pathway, we are currently developing an endosomal escape strategy based on nigericin conjugation to StingApt. Our preclinical data sustain the use of aptamer-based nanodevices for selectively delivering STING agonists against PTK7+ cells.
The UTexas Aptamer Database: Lessons Learned and an AI-Driven Future
Gwendolyn M Stovall, Dhruv Kumar, Shriya Swamy, Einez Wu, and Amrut Pennaka
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 in future iterations. 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 validation. 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. Our work focuses on training OpenAI’s GPT-4o Mini using a curated subset of aptamer publications to ensure high-quality training data. We will evaluate model performance on an independent subset of analyzed papers, refining extraction accuracy through API integration, prompt engineering, dataset curation, and iterative training. Performance will be assessed using standard evaluation metrics (precision, recall, and F1 score) to optimize accuracy while reducing manual workload. This work will guide the development of a framework and toolkit for systematically aggregating aptamer selection experiments and sequence data. By enhancing the accessibility and interoperability of aptamer data, we aim to expand research opportunities and improve the visibility of aptamer information.
Development of an aptamer against the immune checkpoint protein B7-H4 using Cell-SELEX
Kien T Vien1,2, Derek Richard1, Laura Croft1
1School of Biomedical Science, Faculty of Health, Queensland University of Technology; 60 Musk Avenue, Kelvin Grove, Brisbane, 4059, Queensland, Australia
2Training Center, Pasteur Institute in Ho Chi Minh City; 167 Pasteur Street, District 3, Ho Chi Minh City, Viet Nam
The immune checkpoint protein B7-H4 plays a crucial role in regulating T-cell responses by inhibiting T-cell activity when expressed in cancer cells, making it a promising target for anticancer therapy. Recently, aptamers have emerged as potential candidates to target this checkpoint due to their specific binding capabilities and cost-effective production. This study utilized Cell-SELEX to develop an aptamer specific to the membrane protein B7-H4 in human breast cancer cell line SK-BR-3. Through 20 rounds of Cell-SELEX between SK-BR-3 and MCF-7 cells, aptamer candidates were enriched and selected. The top 17 candidates were analyzed, and the top 3 were validated via flow cytometry using both wild-type (WT) and B7-H4 knock-out (KO) SK-BR-3 cells. Two-way ANOVA analysis revealed statistically significant differences in binding rates between WT and KO cells across all 3 candidates. The aptamer with the strongest binding affinity, characterized by the lowest dissociation constant (KD) of 59.06 ± 60.69 nM to B7-H4, was selected for further development. Future work will involve aptamer modification and Electrophoretic mobility shift assay (EMSA) validation, but this candidate shows promise as a potential therapeutic agent to block B7-H4 and initiate an antitumor immune response.
Functional Selection of Molecular Aptamers Beacons
Anielle Villeronce, Jeanne Leblond Chain, Laurent Azéma
ARNA-Inserm U1212-UMR CNRS 5023-Université de Bordeaux
TAMS group-2, rue Dr. Hoffman Martinot 33000 Bordeaux
Designed for nucleic acid detection, Molecular Beacons (MB) are a powerful detection tool due to their ability to signal the target presence in real-time. To increase MB affinity and selectivity for their targets and extend their application to non-nucleic acid targets, Molecular Aptamers Beacons (MAB) were developed. Such systems gather the recognition properties of aptamers and the switching ability of MB triggering a fluorescence signal for imaging or drug release in a specific environment, with an improved signal/noise ratio. MAB designed from previous aptamers by trial-and-error or strand displacement can be tedious and do not ensure a successful candidate. Developing a SELEX method that can directly provide MAB without post-SELEX modification is valuable. In this project, we aim to develop a MAB SELEX method to detect Thrombospondin-1 (TSP1), a relevant biomarker of Glioblastoma, for theragnostic purposes. To reach that goal, we designed a library bearing FRET pair, and a two-step selection: first, 4 regular SELEX rounds were achieved using TSP1 immobilized on magnetic beads to reduce the diversity required for the next step. Then, 4 functional selection rounds were realized where monoclonal beads were produced by emulsion PCR and fluorophores grafted through NHS chemistry. The beads were incubated with TSP1; beads exhibiting fluorescence enhancement were sorted. A negative selection was used to discard sequences switching in absence of TSP1. After sequencing of all rounds, our method led to identification of Apta_1, which was synthesized and assessed by MST and fluorescence. Apta_1 exhibits moderate affinity (KD = 1.8 µM) and its fluorescence is enhanced in the presence of TSP1, compared to non-specific binding, demonstrating its structure-switching ability. Although this candidate confirmed our experimental approach, we aim to improve affinity and fluorescence switch, through some protocol optimization: further selection rounds and a Doped-SELEX library.
Re-selection using G4-SLSELEX-Seq uncovers G4-specific targeting L-RNA aptamers with unique structure features
Tianying Wu1, Chun Kit Kwok1,2,*
1Department of Chemistry and State Key Laboratory of Marine Pollution, City University of Hong Kong, Kowloon Tong, Hong Kong SAR 999077, China
2Shenzhen Research Institute of City University of Hong Kong, Shenzhen 518057, China
G-quadruplexes (G4s) are non-canonical nucleic acid structures that can adopt different folding topologies, including parallel, anti-parallel and hybrid conformations. G4-containing L-RNA aptamers have shown promise in binding various G4 targets and regulating G4-mediated gene expression. However, aptamers for selectively targeting specific G4 conformations are still limited. The binding motif structure of aptamers also needs to be broadened to enhance selectivity. To further investigate the structure diversity of L-RNA aptamer for enhanced selectivity toward specific G4 conformations, we carried out re-selection of the binding motif of existing G4-containing L-RNA aptamer. Here, we developed a new G-triplex L-RNA aptamer, L-Apt.G3, by using G4-stem-loop(SL)SELEX-Seq. We demonstrated that L-Apt.G3 exhibited strong binding affinity and selectivity to G4 targets with parallel and antiparallel conformations. Spectroscopic analysis, mutagenesis analysis and structure probing assays verified that L-Apt.G3 possessed a nonconical G-triplex structure folding from three guanine runs which is a vital motif for G4 binding. Furthermore, L-Apt.G3 could interfere the interaction between c-kit 1 dG4 and G4-binding protein DHX36. This study provides insight into the promising prospects of G4-triplex-containing L-RNA aptamers for specific G4 targeting.