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Significant difference in affinity of aptamers interacting with glioblastoma CD tumor markers alone vs CD+ cell lines and continuous cell cultures
Olga Antipova1, Valeria L Moiseenko1, Fatima M Dzarieva2, Ekaterina A Savchenko3, Dmitry Y Usachev3, Galina V Pavlova2, 3, Alexey M Kopylov1, 3
1 Lomonosov Moscow State University, Leninskie Gory Str. 1, Bld.3, Moscow, 119234, Russia
2 Institute of Higher Nervous Activity and Neurophysiology of RAS, Butlerova Str., Bld. 5A, Moscow, 117485, Russia
3 Federal State Autonomous Institution «N.N. Burdenko National Medical Research Centre of Neurosurgery» of the Ministry of Health, 4th Tverskaya-Yamskaya Str. Bld. 16, Moscow, 125047, Russia
Translating aptatheranostics into biomedicine requires knowledge of the direct interactions of aptamers with cells. Most measurements aimed to establish specificity of aptamers, requiring blocking nucleic acids, which may not be applicable in treatment. This research focused on a studying two tumor markers: EGFR is WHO glioblastoma (GB) marker of proliferation, and CD133 is a tentative marker of stem-like cells. Known aptamers (anti-EGFR: ME07, CL4, U31, GR20, U2, Gol1; and anti-CD133: A15, B19, Cs5 and Ap1M) were used for flow cytometry assessments of direct interactions with both cell lines (A431, U87, MCF7 for EGFR; Caco-2 for CD133) and continuous cell cultures developed from GB patients (107, G01, Sus, ets.). It turned out that flow cytometry signal per se could not be an indicator of specific the aptamer-target interactions, the signal reflects both target-driven interactions, and membrane-mediated events; therefore, only titration experiment could reveal specific interactions. Even in this case the half-saturation concentrations are about 150 nM or so. Our bio-layer interferometry data shows that interactions of aptamers with recombinant EGFR have Kd in the nM range, matching published data. Thus, an intriguing discrepancy exists between aptamer-protein and aptamer-cell interactions.
DNA aptamers to distinguish Dengue and Zika NS1 protein
Soma Banerjee1 and Marit Nilsen-Hamilton1,2
1 Aptalogic Inc, USA
2 Roy J Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, USA
The diseases caused by dengue viruses (DENV), mosquito-borne members of the family Flaviviridae,have emerged as the most important arthropod-borne viral diseases of humans. The frequency, geographicaldistribution, and severity of DENV epidemics have increased dramatically over the past decade. DENV isspread by mosquito species that also harbour Zika virus (ZIKV). Although these virusesare endemic in tropical countries, they have also been reported in the southern states of the USA and are likely to move northward as global climate change increases their range. Early symptoms of DENV infection overlap with symptoms caused by many other haemorrhagic viruses including ZIKV, Ebola and yellow fever. Cross reactivity and prolonged presence of virus specific antibodies bring challenges to serological tests in distinguishing ZIKV and DENV infections. This is a serious problem particularly in areas where DENV and ZIKV are endemic. As an alternate to antibodies, nucleic acid aptamers are emerging as a new generation of molecular recognition elements for diagnostics based on their synthetic nature, stability under a wide range of temperatures and amenability to different sensing platforms. Aptamers and antibodies often bind to different epitopes on a target protein, which can result in aptamers recognizing regions on an antigen that are not bound by antibodies and that therefore might be less likely to evolve rapidly as the virus avoids antibody detection. Therefore, we selected DNA aptamers that can specifically bind the DENV NS1 but not the Zika NS1 protein. Interestingly our studies show that the specificity exists only at NS1 protein concentrations higher than 250 nM. At lower concentrations the selected DNA aptamers bind both proteins and cannot distinguish DENV NS1 from ZIKV NS1. Understanding the molecular basis of this result is expected to reveal the structural features of Zika NS1 protein that alter aptamer binding in a concentration dependent manner.
DNA aptamers as allosteric modulators of G protein-coupled receptors, namely of the Turkey β1-adrenergic receptor
Federico Bosetto1, Timothy Noel1, Thomas Harman2, Daniel Nietlispach2, Graham Ladds1, Ioanna Mela1
1Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK
2Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK
G protein-coupled receptors (GPCRs) constitute the largest group of membrane proteins in the human body capable of transmitting extracellular information to intracellular signaling. One-third of all pharmaceutical agents on the market target GPCRs for treating a wide range of health issues. However, the selectivity of these drugs can be problematic, leading to off-target effects. The use of allosteric ligands to bind sites that are not preserved within a family can exceed these limitations. In this work DNA aptamers are selected so that they can act as allosteric modulators of the Turkey β1-adrenergic receptor. Starting from a DNA library with a random region of 40 nucleotides, protein-SELEX is used to select aptamers simultaneously in 3 different conditions of the receptor: absence of ligand (apo state receptor), agonist-bound (active state) and antagonist-bound (inactive state), to generate aptamers that are selective for individual receptor conformations. The agonist and antagonist used are isoprenaline and propranolol, respectively. The receptor is purified and reconstituted into phospholipid bilayer nanodiscs stabilized by membrane scaffold protein (MSP). Six rounds of selections are initially performed, during which the counter-selection is performed with empty magnetic beads and empty nanodiscs. Selected putative aptamers, from each condition, are cloned in pCR™4-TOPO® vector and sequenced by the Sanger-method. The resulting clones are tested for their binding affinity and specificity to the target and screened for similar secondary structures and motifs. Interestingly, preliminary data obtained from a pharmacologically based assay indicate that some aptamers interact with the receptor and increase the agonist efficacy more than the control. The outcome of the functional analysis will inform the experimental design of the next rounds of aptamer selection.
Opening the aptamer selection black box: A computational approach for SELEX optimization
Santiago Chaillou1, Jorge Andres Moncada Escudero2, Vitor B. Pinheiro1.
1Department of Pharmaceutical and Pharmacological Sciences, Rega Institute for Medical Research, KU Leuven, Herestraat 49, 3000 Leuven, Belgium
2 Applied Mechanics and Energy conversion (TME), KU Leuven, Celestijnenlaan 300, 3001 Leuven, Belgium
The Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology has allowed the identification of oligonucleotide sequences with high affinity for diverse molecular targets. Despite being widely used, SELEX’s inherent complexity presents significant challenges, often resulting in low success rates and considerable resource consumption. In response, we introduce a computational model designed to predict the enrichment outcome of the SELEX process. We created an agent-based model that uses binding affinity data obtained from a small sample of nucleic acid strands from the naive population. Additionally, the model takes as an input some key SELEX parameter values, such as the number of washes, washing time, incubation time, selection rounds, and concentrations of the target and nucleic acid population. The model outputs the affinity of the final nucleic acid population, allowing for the obtention prediction of high affinity towards the targets. This model allows for the systematic adjustment of selection parameters and the performance of in-silico SELEX simulations. In instances where enrichment for high-affinity binders is not achieved, our model permits the iterative exploration of alternative selection conditions. Through this process, researchers can modify the aptamer selection conditions until observing enrichment for high-affinity binders. Through the application of this computational tool, our aim is to accelerate the optimization of aptamer selection processes and to increase the likelihood of identifying high-affinity binders.
Magnetic beads SELEX technology: advancing aptamer selection towards personalized antibiotic treatment
Magdolna Casian1,2, Oana Hosu-Stancioiu1, Ioana Manea1, Dimas Suárez2, Natalia Díaz2, María Jesús Lobo Castañón2,3, Noemí de-los-Santos-Álvarez2,3, Cecilia Cristea1
1 Department of Analytical Chemistry, Faculty of Pharmacy, Iuliu Hațieganu University of Medicine and Pharmacy, 4 Pasteur Street, 400349, Cluj-Napoca, Romania
2 Departamento de Química Física y Analítica, Universidad de Oviedo, c/Julián Clavería 8, 33006 Oviedo, Spain
3 Instituto de Investigación Sanitaria del Principado de Asturias, Av. de Roma s/n, 33011, Oviedo, Spain
Glycopeptide antibiotics are the first-line treatment for severe infections, particularly methicillin-resistant Staphylococcus aureus. Vancomycin has a narrow therapeutic window, and it is rather difficult to ensure safe dose determination, reason why therapeutic drug monitoring is highly required. In the field of precision medicine, aptamers have emerged as an innovative class of biorecognition elements owing to their stable three-dimensional structure, high specificity, and versatility. This study presents the main strategy and results regarding the selection of a novel aptamer for vancomycin through SELEX technology. A total of 9 selection rounds were performed using magnetic beads, due to their good dispersion properties and quick responsiveness, facilitating a fast and effective separation process between the immobilized target and DNA sequences. To make the selection more stringent, negative- and counter-selection steps were introduced. The amplification yield and DNA purity were monitored by fluorimetry and gel electrophoresis. The evolution of the selection was assessed via UV-VIS-based enrichment assay, and round 7, showing the highest affinity towards vancomycin, was further cloned and sequenced. The affinity of the most promising aptamers was evaluated using surface plasmon resonance, obtaining dissociation constants in the nanomolar range. For a more comprehensive understanding of the aptamer–vancomycin interaction, docking analysis and molecular dynamics calculations were performed. Further steps envision the development of a portable electrochemical aptasensor for fast and specific quantification of vancomycin from clinical serum samples. This work was supported by a grant of Romanian Ministry of Education and Research PN-IV-P8-8.1-PRE-HE-ORG-2023-0076 contract no. 26 PHE/2022, Spanish Ministerio de Ciencia e Innovación Project PID2021-123183OB-100 MICIN/AEI/10.13039/501100011033/FEDER UE and Iuliu Hațieganu UMF internal grant no. 648/1/11.01.2024.
From SELEX to biosensor
Emily Hoi Pui Chao1, Yunus A Kaiyum1, Lashaun Coote1, Zachary R Churcher1, Minh-Dat Nguyen2, Philippe Dauphin-Ducharme2, Philip E Johnson1
1Department of Chemistry, York University, Toronto, Ontario, Canada
2Département de chimie, Université de Sherbrooke, Sherbrooke, Québec, Canada
Aptamers are short single-stranded nucleic acid molecules that bind ligands and are widely used in biotechnology as biosensors. Electrochemical aptamer based (E-AB) sensors are composed of a redox-reporter-modified aptamer immobilized on a gold electrode surface that specifically binds to a target molecule. It is often difficult to translate a selected aptamer to a functional biosensor and we aim to figure out rules on how to reliably do this. Isothermal titration calorimetry (ITC) was utilized to investigate the binding relationship of aptamer and ligand interactions while nuclear magnetic resonance (NMR) spectroscopy was performed to determine if ligand binding induces structure formation in an aptamer. In this study, we have investigated the theophylline, caffeine, dopamine, glucose, and methotrexate aptamers with a varied distance between the site of structure changes and the surface of the sensors. The results showed aptamers have to bind to the targeted ligand and induce structural formation in order to translate the ability to bind a ligand to obtain working biosensors. In addition, the structure formation needs to occur within approximately three base pairs from the gold surface.
Aptamer-based system for Early-stage Pancreatic Cancer Exosome Enrichment and Diagnosis
Jingyu Cui 1, Lin Wang 1,2, Simon Chi-Chin Shiu 1, Andrew B Kinghorn 1, Julian A Tanner 1
1 School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
2 Advanced Biomedical Instrumentation Centre, Hong Kong Science Park, Shatin, New Territories, Hong Kong SAR, China
Pancreatic cancer is one of the deadliest cancers, with most cases diagnosed late, leading to a median survival of less than five years. Early detection biomarkers are critical, and exosomes, small vesicles containing cellular materials, are potential non-invasive diagnostic tools. However, traditional exosome isolation methods like ultracentrifugation and chromatography are inefficient and can damage exosomes. Therefore, there’s a need for better methods. Aptamers, which are specific nucleic acids that bind their targets with high affinity, offer a promising solution. They are stable, non-immunogenic, and cost-effective, making them ideal for developing new exosome isolation techniques for pancreatic cancer diagnosis. In this work, we have developed a SELEX strategy target to intact exosomes, termed Exo-SELEX. We isolated exosomes from cell culture medium and subjected them to 12 rounds of Exo-SELEX. Given the similar characteristics of exosomes secreted by cancer cells, we identified a universal aptamer, Apt2, capable of recognizing exosomes. Among the sequence obtained, we optimized the length of Apt2; however, the original length demonstrated the best recognition performance. Consequently, our experiments focused on the full-length Apt2, we then conjugated Apt2 with magnetic beads to facilitate the gentle and effective isolation of exosomes from complex matrices such as cell culture media or serum samples, without the need for elaborate operations or equipment. The efficacy of the exosome enrichment process was verified through methods like Western blotting. For the specific identification of pancreatic cancer exosomes, we elected to measure certain miRNAs within the pulled-down exosomes, distinguishing between different cancers for the specific detection of pancreatic cancer. It is anticipated that this approach will offer novel insights for the isolation of exosomes and the specific detection of cancers.
Impact on electrochemical aptamer-based sensor calibration by physiological-scale variations in cations, pH, and temperature
Lisa C. Fetter1,4, Matthew H. McDonough2, Kevin W. Plaxco3,4
1Program in Biomolecular Science and Engineering, University of California Santa Barbara, Santa Barbara, CA 93106, USA
2Department of Statistics and Applied Probability, University of California Santa Barbara, CA 93106, USA
3Department of Chemistry and Biochemistry, University of California Santa Barbara, Santa Barbara, CA 93106, USA
4Center for Bioengineering, University of California Santa Barbara, Santa Barbara, CA, 93106, USA
Electrochemical aptamer-based (EAB) sensors combine continuous measurement capabilities with a generalizable biorecognition element of ample selectivity for in vivo deployment, resulting in a uniquely powerful biosensing device. Consequently, several EAB sensors are reported for use in real-time, seconds-resolved pharmacokinetic monitoring of therapeutics and metabolites, indicating a potential application towards biomedical research and disease monitoring. However, a feasible complexity of widespread utilization of EAB sensors in situ in the living body arises from environmental fluctuations that exist in the body. Variable physiological factors such as pH and temperature, for example, are known to impact EAB sensor signal response to a given concentration of target by influencing both aptamer-analyte interactions and electron transfer rates of the redox reporter. Thus motivated, here we expand on the extent to which the signal responses of three test-bed sensors are affected by physiological perturbations in cation composition, pH, and temperature, ultimately in search of corrective measures for deleterious parameters. Doing so, we find that variability within the physiological ranges of cation composition and pH in human plasma have relatively limited impact on sensor response. We also show that fluctuations in temperature cause substantial error in sensor measurements. Fortunately, we lastly demonstrate a mathematical means of correction for temperature-induced error capable of reducing the mean relative error in sensor measurements by upwards of twenty percent.
Can novel aptamer-drug conjugates successfully deliver drug payloads demonstrating therapeutic potential against Glioblastoma?
Breanna L Giles1,2, Maryam Nakhjavani1,2, Rasika M Samarasinghe1,2, Sarah L Shigdar1,2
1 School of Medicine, Deakin University, Geelong VIC 3220, Australia
2 Institute for Mental and Physical Health and Clinical Translation, School of Medicine, Deakin University, Geelong VIC 3220, Australia
The limited success of current treatment options for glioblastoma, an aggressive brain cancer with poor survivability can be attributed to the blood-brain barrier (BBB). This barrier prevents entry of most therapeutic drugs to treat brain cancers thus, a novel targeted therapeutic capable of crossing the BBB for drug delivery is essential. Aptamers are small single-stranded oligonucleotide sequences that not only can bind specifically and selectively to desired targets but can be modified as a drug delivery vehicle for therapeutic purposes. We previously generated a bifunctional aptamer-drug conjugate by combining the transferrin receptor and epithelial cell adhesion molecule (EpCAM) aptamers together and intercalated the chemotherapeutic doxorubicin (DOX) for treatment of brain metastases. This bifunctional aptamer-DOX conjugate, termed TEPP-DOX, was successfully able to deliver drug payloads across an in vitro and in vivo BBB to EpCAM positive brain metastases, reducing metastatic spread and tumourigenicity. For the first time, we aim to assess the therapeutic efficacy of bifunctional aptamer-DOX conjugates in treating glioblastoma. Firstly, the binding affinity of two bifunctional aptamer-DOX candidates against the transferrin receptor was determined by flow cytometry where a strong binding affinity towards glioblastoma (16.8nM) was observed. Next, we assessed the cytotoxicity of DOX and both aptamer-DOX conjugates towards glioblastoma by performing an MTS assay on cell monolayers, and a trypan-blue exclusion assay on 3D tumourspheres. While there was an initial delay in drug delivery, upon 48 and 72-hours, the inhibitory concentrations (IC50) calculated of aptamer-DOX conjugates (0.49µM and 0.65µM; 72-hours) showed similar therapeutic efficacy against glioblastoma compared to free-DOX (0.93µM). Therefore, with the specificity aptamers bring, this demonstrates the potential these delivery vehicles have as a therapeutic candidate for future studies.
DNA and Methylene-blue Modified Non-Natural DNA SELEX against Pancreatic Cancer Biomarkers
Weisi HE 1, Marcel HOLLENSTEIN 3, Julian Alexandar TANNER 1,2
1 School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
2 Advanced Biomedical Instrumentation Centre, Hong Kong Science Park, Shatin, New Territories, Hong Kong SAR, China
3 Institut 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. Now in the market, blood testing for the pancreatic cancer glycan biomarker CA 19-9 uses 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. Here, we report two aptamer SELEX to select out aptamers which could be used in a PC aptasensor. We report a natural DNA aptamer SELEX against asprosin, one of the early-stage pancreatic cancer biomarkers. We also report a methylene blue modified DNA aptamer SELEX against CA19-9. For the asprosin aptamer SELEX, we discovered three classes of asprosin aptamer sequence. Class I were A rich sequences, which contains over 50% of A in random region. Class II is AC rich sequences and Class III are diverse sequences. Ten asprosin aptamer candidates showed nanomolar level of binding to our expressed asprosin and eight candidates have high specificity in ELONA. Other characterization assays including electrochemical assays are ongoing. For the CA19-9 aptamer methylene-blue modified non-natural SELEX, 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 ssDNA library. Furthermore, the strategy of CA19-9 aptamer SELEX was developed and preliminary aptamers have been selected. In the longer term we aim to develop these aptamers into multiplexed aptasensors for pancreatic cancer.
Aptamer-functionalized microelectrode arrays for simultaneous detection of multiple neurotransmitters
Ziheng Hu, Andreas Offenhäusser, Dirk Mayer
Institute of Biological Information Processing, Bioelectronics (IBI-3), Forschungszentrum Jülich GmbH, Jülich, Germany
Neurotransmitters (NTs) play a crucial role in regulating signals within the nervous system and their abnormal levels are implicated in a series of neuronal disorders, including Alzheimer’s and Parkinson’s diseases. Therefore, it becomes vital importance to develop a feasible method for sensitive and specific detection of NTs. Aptamers, mostly single-stranded DNA or RNA molecules, exhibit a unique affinity for their target molecules, making them valuable and competitive for biosensor applications. Herein, we present a microelectrode arrays (MEAs) device functionalized with aptamers for the analysis of multiple NTs. The MEAs possess 64 individual microelectrodes and are electrodeposited with 3D gold nanostructures. To achieve the modification of different aptamers at different microelectrode, a potential-pulse-assisted method was applied for the formation of receptors layer. By pulsing between potentials that are more positive and more negative with respect to the potential of zero charge, not only is the kinetics of aptamer immobilization substantially enhanced, but the different aptamers can be site-selective functionalized on the individual microelectrodes. In our design, aptamers are terminally modified with thiol groups and redox tags. They are then immobilized onto different microelectrodes on one chip. Upon target-binding, the aptamers undergo a conformational rearrangement that repositions the redox tag relative to the electrode surface. The variation in the efficiency of electron transfer caused by analyte binding can be monitored by alternating current voltammetry (ACV). The proposed electrochemical aptasensor exhibits high sensitivity and selectivity to the individual target molecule. Meanwhile, the different aptamers modified MEAs show the feasibility of the simultaneous monitoring of multiple targets in a single chip with minimal crosstalk and interferences, suggesting its potential to study the co-release of multiple NTs in neuroscience.
Aptamer-based nanoplatform for the electrochemical detection of cortisol in biological samples
Maria-Bianca Irimes, Alexandra Pusta, Mihaela Tertis, Radu Oprean, Cecilia Cristea
Analytical Chemistry Department, Faculty of Pharmacy, Iuliu Hatieganu University of Medicine and Pharmacy, 4 Louis Pasteur St., Cluj-Napoca, Romania
Cortisol (COR), a glucocorticoid hormone, plays a crucial role in several physiological and pathological processes, influencing the immune response and stress adaptation. Its fluctuating levels in biological fluids underscore the significance of detecting this analyte for diagnosis and monitoring. Electrochemical sensors show promise for quick, cost-effective detection in unconventional biological samples. Their high sensitivity, specificity, and potential for miniaturization make them suitable for wearables and on-site analyses. The objective of this study was to develop an aptasensor for COR detection in human serum and saliva. First, the electrochemical cells were printed in-lab, followed by the functionalization of the working electrodes with a nanocomposite based on Au and Pt to increase the sensitivity. To enhance specificity, an aptamer was immobilized on the surface through thiol-Au chemistry by applying multipulse amperometry. Confirmation of the functionalization was achieved through cyclic voltammetry (CV) and electrochemical impedance spectroscopy. The detection protocol involved incubating the aptasensor with COR, and CV was employed for COR detection and was applied in the analysis of real samples. In conclusion, an aptasensor was designed for the specific electrochemical detection of COR. This work was supported by a grant of the Romanian Ministry of Education and Research, CNCS-UEFISCDI, PN–III–P1-1.1-TE-2021-1543, within PNCDI III and the Iuliu Hațieganu UMF internal grant no. 648/3.11.01.2024.
Exploring the Secondary and Tertiary Structure of a Minimal Dopamine-Binding Aptamer via NMR
Yunus A. Kaiyum1, Hoi Pui Chao1, Dat Nyguen3, Philippe Dauphin-Ducharm3 Cameron Mackereth2, Philip Johnson1
1 Department of Chemistry, York University, Toronto, ON, Canada
2Institut Européen de Chimie et Biologie, University of Bodeaux, 2 Rue Robert Escarpit, 33600 Pessac, France
3Département de chimie, Université de Sherbrooke, Sherbrooke, QuébecJ1K 2R1, Canada
Aptamers adopt a host of different structures in order to specifically bind their targets with high affinity. These structures can offer insight into how the aptamer may behave in more complex environments as well as their efficacy in biosensors. To date, there are a limited number of aptamer structures that have been determined which remains a large gap in our understanding of how aptamer function is dictated by its structure. Additionally, a reliable structure prediction software is yet to be developed. Here, we attempt to characterize the secondary and tertiary structure of a modified dopamine-binding aptamer that was originally selected in 2018. NMR and ITC were used to explore the binding interaction as well as structural changes the aptamer undergoes with dopamine binding. Deletions were made to the original aptamer until a minimal structure was obtained for further characterization with NMR. Binding and NMR spectra were collected for each deletion to confirm functionality. A further deletion of 3 additional bases from the 5’ end resulted in a mutant that functioned with improved affinity when compared to prior deletion mutants. Selectively labelled 13C and 15N heteronuclear experiments helped determine a preliminary structure of the dopamine-bound state. Proton exchange rate experiments were completed on the minimally functional construct to determine the imino-proton exchange rate of specific bases along the sequence which provided insight into the local structures that surrounded these bases. These results will help to generate a more detailed picture of this aptamer binding interaction with its target and provide a greater understanding of how aptamer structure is intimately tied to its function.
A Novel RNA Aptamer as Synthetic Inducer of DasR Controlled Transcription
Michael-Paul Vockenhuber1,2, Janis Hoetzel1,5, Lisa-Marie Maurer1,5, Philipp Fröhlich3,5, Sigrid Weiler4, Yves A Muller4, Heinz Koeppl3,5 and Beatrix Suess1,5
1 Department of Biology, Technische Universität Darmstadt, Schnittspahnstrasse 10, 64287 Darmstadt, Germany
2 Center for Synthetic Microbiology (SYNMIKRO), Philipps-Universität Marburg, Karl-von-Frisch-Strasse 14, 35043 Marburg, Germany
3 Technische Universität Darmstadt, Department of Electrical Engineering and Information Technology, Schnittspahnstrasse 10, 64287 Darmstadt, Germany
4 Lehrstuhl für Biotechnik, Department of Biology, Friedrich-Alexander University Erlangen-Nürnberg, Henkestrasse 91, 91052 Erlangen, Germany
5 Centre for Synthetic Biology, TU Darmstadt, 64287 Darmstadt, Germany
Progress in the synthetic biology field is driven by the development of new tools for synthetic circuit engineering. Traditionally, the focus has relied on protein-based designs. In recent years, the use of RNA-based tools has tremendously increased, due to their versatile functionality and applicability. A promising class of molecules is RNA aptamers, small, single-stranded RNA molecules that bind to a target molecule with high affinity and specificity. When targeting bacterial repressors, RNA aptamers allow one to add a new layer to an established protein-based regulation. In the present study, we selected an RNA aptamer binding the bacterial repressor DasR, preventing its binding to its operator sequence and activating DasR-controlled transcription in vivo. This was made possible only by the combination of an in vitro selection and subsequent in vivo screening. Next-generation sequencing of the selection process proved the importance of the in vivo screening for the discovery of aptamers functioning in the cell. Mutational and biochemical studies led to the identification of the minimal necessary binding motif. Taken together, the resulting combination of bacterial repressor and RNA aptamer enlarges the synthetic biology toolbox by adding a new level of regulation.
Approaches to the enzymatic synthesis of hypermodified DNA polymers and their application in development of modified aptamers
Marek Ondruš, Michal Hocek
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo nam. 542/2, 160 00 Prague 6, Czech Republic
Enzymatic synthesis of DNA is well-established method but it is highly desired to develop versatile enzymatic approaches for hypermodified DNA composed exclusively only from base-modified
2´-deoxyribonucleoside triphosphates. Base-modified dNTPs open new possibilities to explore novel bio-physical and theranostic properties of modified DNA. Therefore, we designed and synthesized two series of all four hydrophobically-modified dNTPs and used them for enzymatic synthesis of hypermodified DNA as an example of sequence-specific functionalized polymer. Due to modifications, hypermodified DNA cannot be sequenced directly by common sequencing platforms. Therefore, we developed method for replication of hypermodified DNA into natural DNA which was then easily sequenced and provided information about fidelity of DNA polymerase. Moreover, we developed new beneficial alternative for enzymatic synthesis of modified single-stranded DNA from double-stranded hybrid duplex where template and non-modified primer were efficiently removed for their application on the characterization and development of modified nucleic acids aptamers. Nucleic acid aptamers are short sequence-specific ssDNA/RNA polymers able to bind a wide variety of targets via protein-nucleic acid interactions with affinity rivalling antibodies. Despite this, there is still a major challenge for binding to “undruggable” targets such as hydrophobic proteins. To address this problem, we are developing aptamers bearing hydrophobic moieties incorporating the best attributes of two types of biopolymers: huge conformational flexibility of ssDNA combined with diversity-enhancing protein-like moieties. We have successfully selected modified aptamer bearing hydrophobic modifications, such as the 7-phenylbutyl-7-deazaadenine-modified DNA aptamer resulting in high binding affinity for the Heat Shock Protein 70 (HSP70). This work was supported by Czech Science Foundation (EXPRO), 20-00885X.
A truncated multi-thiol aptamer-based SARS-CoV-2 electrochemical biosensor: Towards a variant-specific Point-of-Care Detection
Sergio R. Molina Ramirez1, Nafiseh Samiseresht2, Mateo A. Martínez-Roque1, Ferdinando Catania1, Kevin Graef1, Martin Rabe2, Krisztina Percze1, Andreas Offenhäusser1, Dirk Mayer1, Gabriela Figueroa-Miranda1
1 Institute of Biological Information Processing, Bioelectronics (IBI-3), Forschungszentrum Jülich GmbH, Jülich, Germany, 2 Department of Interface Chemistry and Surface Engineering, Max-Planck-Institut für Eisenforschung GmbH, Düsseldorf, Germany Considering ongoing challenges for the rapid and accurate detection of specific SARS-CoV-2 variants, this study presents an optimized electrochemical biosensor that employs a refined aptamer (C9t) for targeted detection in a flexible multielectrode aptasensor array with PoC capabilities. The primer binding sites of an original aptamer sequence, which detects the Spike glycoprotein (S-protein) of the SARS-CoV-2 virus, were removed. The resulting truncated aptamer was modified with dithiol phosphoramidite, containing four thiol groups, for stable immobilization on a flexible gold multielectrode array. The biosensor fabrication, optimization, and S-protein detection were verified by electrochemical methods, QCM-D, and SPR. The sensor fabrication time was reduced from 24 hours to about 3 hours without reduction in aptamer density. The analyte binding to the receptor layer was further evaluated by AFM-IR investigation, confirming the binding of the protein on the receptor layer. The aptasensor possesses a limit of detection of 1.4 fg/mL, the highest sensitivity for S protein aptasensors of 184.4 per concentration decade, and a wide dynamic range of 1.4 fg/mL- 38 ng/mL in nasopharyngeal secretion, covering the clinically relevant range for the spike proteins. The sensor possessed a high selectivity for SARS-CoV-2 S protein over biomarkers for MERS-CoV, RSV, and Influenza. Furthermore, the C9t aptamer showed a three times higher sensitivity for the Omicron variant of the S protein over the wild-type, alpha, and beta variants of the SARS-CoV-2 virus. Overall, the low costs of the truncated aptamer, the flexMEA chip, the rapid sensor assembly paired with high sensitivity and variant-specific selectivity render the reported aptasensor an affordable and reliable testing tool to manage future fast-spreading infections. With its quantification capabilities, and potential for multi-biomarker detection, it outperforms current rapid diagnosis tests.
Aptamer-based fluorescence/fluorescence anisotropy assays using complementary oligonucleotides for mycotoxins detection
Alexey V. Samokhvalov, Anatoly V. Zherdev, Boris B. Dzantiev
A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology, Russian Acad. Sci., Moscow, Russia
Short oligonucleotides can interact complementarily with the corresponding regions in aptamers, and by doing so, they can modulate the parameters of aptamer-based assays. The presented study was focused of comparison of such single-stranded DNA (ssDNA) oligonucleotides differing in length and location of contacts with aptamers. Oligonucleotides 5′-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA-3′ (that binds ochratoxin A (OTA)) and 5′-CAC GTG TTG TCT CTC TGT GTC TCG TG-3′ (that binds aflatoxin B1 (AFB1)) were taken as examples of aptamers with G-quadruplex and loop structures, respectively. The aptamer–ligand interactions were characterized using fluorescein-labeled ligands and fluorescence anisotropy (FA) technique. The measured dissociation constants were 36.3 ± 6.0 nM for the OTA case and 82.9 ± 9.8 nM for the AFB1case. A row of ssDNA preparations (24 for OTA case and 13 for AFB1) were tested. To study the interactions, a fluorescein label was introduced into aptamer or ssDNA. The complexes of complementary aptamer-ssDNA pairs were formed, and specific ligands were added to them. We have found the pairs whose complexes dissociated under such conditions and consider these pairs as tools to detect the ligands through the changed properties of the fluorophore. For FA registration the reached limits of detection were 9 ng/mL for OTA and 14 ng/mL for AFB1, and the assay time was 5 min. Additionally, the application of the Forster resonance energy transfer (FRET) technique for the AFB1 provided the detection limit of 1.3 ng/mL with 30-min testing. The developed assays demonstrated high recoveries for both analytes in food samples and the possibility to control for exceeding the officially established maximal residue levels for these mycotoxins. This study was financially supported by the Russian Science Foundation (Project No. 23-74-01080).
Split-aptamer binding assay towards in vivo continuous vancomycin monitoring in the brain
Cátia Santa1, Heather A Clark2, Bastian Hengerer3, Khulan Sergelen1
1BioMed X Institute, Im Neuenheimer Feld 515, 69120 Heidelberg, Germany
2School of Biological and Health Systems Engineering, Arizona State University, Tempe, Arizona85281, United States
3Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Str. 65, 88397 Biberach an der Riss, Germany
The continuous monitoring of drugs in the brain is essential for the understanding of their mechanism of action. Although various strategies have been developed to tackle this necessity there are still requirements that need to be addressed and improved.The ideal drug monitoring sensor in the brain should be able to detect the molecule of interest selectively, quantitatively, reversibly, robustly, and continuously at the appropriate concentration range, with the relevant spatial and temporal resolution. To enable time-course pharmacokinetic (PK) studies, we are developing a fiber optics-based in vivo biocompatible biosensor. We will employ aptamers as our selective sensing element and characterize the affinity and specificity suitable for continuous (reversible) sensing in a physiologically relevant temporal scale for a self-contained sensor. To develop the reversible, model drug detection assay, we apply a sandwich-assay by splitting Vancomycin binding structure changing DNA aptamer into two segments that interact to form a complex only in the presence of analyte. The assay is evaluated for affinity, limit of detection, stability and reversibility in standard buffer and complex matrix in physiological conditions, with the perspective of deploying it in the brain of living animals.
Screening and Evaluation of Aflatoxin B1-Specific Aptamers Using Gold Nanoparticle-Based HT-SELEX Technology
Shazia Shareef, Hariprasad P
Indian Institute of Technology Delhi, New Delhi, India
Aflatoxins, potent carcinogenic secondary metabolites, are produced by Aspergillus flavus and A. parasiticus. The ingestion of Aflatoxin-contaminated food/feed is associated with severe health risks, notably Hepatocellular carcinoma. Therefore, ensuring the safety of food and feed necessitates the precise detection and quantification of aflatoxins. This study, focused on selecting Aflatoxin B1 (AFB1) specific aptamers using a novel SELEX approach was employed, utilizing gold nanoparticles (AuNPs) as the separation probe without the immobilization step, facilitated by the binding interaction between single-stranded DNA (ssDNA) aptamers and AuNPs. The enrichment progress during 15 SELEX rounds was monitored through an AuNPs-based colorimetric assay for AFB1 in each cycle. Following confirmation of enrichment, the pooled aptamers from the 4th, 8th, 12th, and 15th cycles underwent Next Generation Sequencing. Analysis of the sequencing data, performed using Aptasuite and MEME suite for clustering and motif identification, respectively, resulted in the selection of 17 aptamers for further investigation. The selected aptamers underwent sensitivity and dissociation constant (kD) analysis using various methods, including AuNPs-based colorimetric assay, circular dichroism spectrometry, and SyBr Green I-based fluorescence assay. The kD of the top aptamers ranged from 1 to 2 nM, highlighting their strong binding affinity for AFB1. Fluorescence-based methods enhance sensitivity and enable real-time monitoring, facilitating dynamic tracking of binding interactions. Circular dichroism spectroscopy contributes insights into the conformational changes of aptamers induced by aflatoxin binding, thereby elucidating structural alterations crucial to the recognition process. Cross-reactivity towards other mycotoxins was also determined. To gain insights into the molecular interactions, molecular docking and simulation analyses were conducted for the top aptamer with AFB1.
UTexas Aptamer Database: the collection and long-term preservation of aptamer sequence information for research advancement (V)
Shriya Swamy, Ali Askari, Gwendolyn M Stovall
The growing interest in aptamer research, exemplified by the increasing volume of aptamer publications in recent years has underscored the necessity for a centralized resource dedicated to aptamer information. To meet this demand the UTexas Aptamer Database, a comprehensive publicly available aptamer database, serves as a repository for aptamer data designed to fulfill pivotal roles in the field. As a unifying platform for aptamer data, the UTexas Aptamer Database standardizes aptamer reporting, fosters opportunities to advance current research, and focuses on the long-term preservation of aptamer information. While several aptamer databases have emerged in previous years, many have been abandoned or removed from public access due to inherent limitations. Inspired by earlier database initiatives, the UTexas Aptamer Database aims to construct the largest comprehensive database to preserve aptamer research and knowledge while prioritizing user friendliness, interactivity, searchability, and public availability. The UTexas Aptamer Database was designed and developed through the systematic collection and peer review of aptamer information from 1990 to present day, and encompasses critical information, including but not limited to, aptamer sequences, binding characteristics, and selection methodology. All data collection is followed by an internal review process conducted by trained researchers to ensure accuracy and consistency. To maintain continual collection and review of aptamer data, sustaining mechanisms were implemented such as training protocols, an aptamer submission interface, independent data storage from the database platform, and an expanding team of researchers committed to updating and enhancing the database. At present, the UTexas aptamer database stands as the largest aptamer database in terms of the sequence entries with 1,475 internally reviewed aptamer records while holding a commitment to the preservation and advancement of aptamer research. Acknowledgement: This work was funded by The University of Texas Freshman Research Initiative, which was supported by the Howard Hughes Medical Institute (#52008124, concluded in 2021), and the College of Natural Sciences.
Developing Cadherin-11-Selective Aptamers
Alexandra Tar1, Jessica Lange2, Brigitta Kállai1, Krisztina Percze1, Thomas Karonitsch2, Felix Kartnig2, Hans Kiener2, Daniel Aletaha2, Leonhard Heinz2, Peter Mandl2, Tamás Mészáros1
1Department of Molecular Biology, Institute of Biochemistry and Molecular Biology, Semmelweis University, Budapest, Hungary
2Division of Rheumatology, Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria
Rheumatoid arthritis (RA) is an autoimmune disease that leads to joint destruction. Fibroblast-like synoviocytes (FLSs) contribute to the healthy synovial membrane; however, under pathological conditions they play a crucial role in the development of synovitis, the chronic inflammation of the synovial membrane which leads to structural joint damage and functional loss in RA. FLSs express cadherin-11 (CDH11) on their membrane, which is a vital protein in cell-cell adhesion and is considered to be a potential therapeutic target in RA. Our long-term goal is to generate TAdUTP (5-indolyl-AA-dUTP) holding CDH11-selective aptamers and evaluate their ability to inhibit FLS activation. The aptamer candidates were produced by toggle-SELEX using immobilized in vitro translated CDH11 protein and CDH11 overexpressing mammalian cell line as targets. Following SELEX, the enriched library was cloned and analysed by colony PCR, lab-on-a-chip nucleic acid electrophoresis and Sanger sequencing. In addition, the oligonucleotide pools after each selection step, including the initial library, were analysed by next-generation sequencing (NGS). The screening of aptamer candidates was carried out using AlphaScreen technology. To this end, biotinylated single stranded aptamer candidates were produced by primer blocked asymmetric PCR (PBA-PCR). The sequencing data analysed by AptaSUITE showed the enrichment of certain sequences suggesting the success of our selection strategy. Furthermore, some enriched sequence-structure motifs were identified confirming this finding. AlphaScreen interaction analysis revealed that various aptamer candidates can interact with the in vitro translated CDH11. However, further analysis is necessary for more detailed characterization of these interactions.
Characterizing the Interactions of Methylene Blue with Different Aptamers Using Intrinsic Fluorescence Analysis
Kabisan Thavaseelan1, Aron A. Shoara2, Philip E. Johnson1
1Department of Chemistry, York University, Toronto, ON, Canada
2Canadian Blood Services, University of Toronto, St. Michael’s Hospital, Toronto, Canada
Electrochemical aptamer-based (E-AB) biosensors have become a promising tool to monitor molecules directly in undiluted complex matrices and in the body with efforts of overcoming barriers in personalized medicine. Methylene blue plays an important role in this platform as a redox-reporter by covalently attaching it to the aptamer. It was first thought that the electrochemical signal generated from the sensing platform was due to the aptamer undergoing a binding-induced conformational change which can move the redox-reporter closer to the electrode surface. But it was recently found out that for MN19, a variant of the cocaine-binding aptamer, the platform relies on a redox-reporter-ligand competition mechanism. When methylene blue is covalently bound to MN19, it binds to or near the ligand binding site, leading to a folded conformation of the aptamer. When ligand is added, it competes with the methylene blue and displaces it from the binding site, altering its electron transfer rate. Given this result, we want to see what other structures methylene blue binds on other aptamers by using the intrinsic fluorescence of methylene blue. If binding is observed between methylene blue and other aptamers, a decrease in the fluorescence intensity of methylene blue is expected. The binding affinity will also be measured to quantify how tight or weak the binding is. Some of these aptamers include but are not limited to Caff209, a variant of the caffeine-binding aptamer, Glu1, a glucose-binding aptamer, and Theo1, a theophylline-binding aptamer.
Probing the recognition site of hydrophobic 7-phenylbutyl-7-deazaadenine-modified HSP70 DNA aptamer and its dependence of modified nucleobases
Pablo Alberto Franco Urquijo, Marek Ondruš, Michal Hocek
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo nam. 542/2, 160 00 Prague 6, Czech Republic
Aptamers, short sequence-specific RNA or single stranded DNA (ssDNA) that binds to their cognate target are selected by in vitro systematic evolution of ligands by exponential enrichment (SELEX) from randomized oligonucleotide pools. Typically, natural nucleotide libraries constrain the available chemical diversity resulting in a lower structural repertoire for target interactions. To overcome this, libraries with enhancing protein-like moieties have been produced that include a range of chemical modifications conferring advantages such as increased affinity, specificity and aptamer expanded epitopes. Between this modifications, hydrophobic modifed libraries have been promisingly used for “undruggable “proteins resulting in an improved success rate and novel functionalities. Within the hydrophobic modified libraries used for SELEX, the 7-phenylbutyl-7-deazaadenine-modified DNA aptamer selected via PEX and magnetic bead-based SELEX resulted in high affinity and specific aptamers to the Hsp70 protein. Modified aptamers displayed low nanomolar range KD compared with their natural counterpart (>5 µM) demonstrating the importance of its hydrophobic modification for their binding affinity. Due to this, the impact of the chemical modification with its target and the minimal binding sequence is being tested. Modified truncations were produced by an alternative enzymatic synthesis of modified ssDNA obtained from double-stranded (RNA-DNA) hybrid duplex. By this approach, the minimal binding sequence is being determined. In addition, flexibility and length of the linker connecting the modification and their impacts on the binding properties are being tested. By using this post-SELEX modification approach, we aim to improve the performance of the previously selected aptamer and get a deeper insight about the importance of the hydrophobic moieties for their binding properties. This work was supported by Czech Science Foundation (EXPRO), 20-00885X.
Aptamer-mediated targeting of MMP14 as a potential therapeutic strategy for low bone mass
Yinuo Xie, Xiaodan Yu, Jingyu Cui, Simon CC Shiu, Kathryn SE Cheah & Julian A Tanner
School of Biomedical Sciences, The University of Hong Kong
Osteopenia, a concern in aging and congenital disorders, involves Mmp14, a matrix metalloproteinase crucial for the transition from chondrocytes to osteoblasts. Ablating Mmp14 in mice increased trabecular bone, enhancing PTH impact on osteoblastogenesis. Inhibiting MMP14 may be a potential therapeutic strategy for osteopenia. Antisense oligonucleotides (ASO), a common gene knockdown therapy, modify gene expression and mRNA splicing in genetic disorders. Stabilization strategies, like self-assembled DNA nanostructures, aid intracellular delivery. Combining aptamers with DNA nanostructures addresses several limitations, allowing specific targeting of molecules, surfaces, and cells. Herein, this study is aiming to develop aptamer-mediated gene silencing therapeutics targeting osteoblasts for bone and cartilage. We have identified a single-stranded DNA (ssDNA) aptamer targeting osteoblasts through cell-SELEX and high-throughput sequencing, utilizing the MC3T3-E1 cell line as the target and the ATDC-5 chondrocyte cell line as the control. Three aptamers were chosen based on next-generation sequencing (NGS) and phylogenetic relationships, and their binding affinity and selectivity were assessed using flow cytometry. The selected aptamers demonstrated nanomolar-range dissociation constants (Kd values), displaying robust binding affinity and selectivity under physiological conditions. Truncated versions of the aptamer lost their binding capacity, highlighting the importance of the full length for optimal binding ability. Confocal laser scanning microscope studies confirmed the aptamer’s targeting of MC3T3-E1 cell surfaces. With high affinity, specificity, stability, and specific surface recognition of osteoblasts, these aptamers exhibit potential for targeting osteoblasts in various applications.