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J Venom Res
Aptamers 2019
03-04 April 2019, Oxford
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Thank you for considering to present your work as a poster at Aptamers 2020. The current deadline for poster submission is advertised on the homepage. However, we may stop accepting poster abstracts earlier, as soon as we reach the capacity of 36.

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Invitation to submit an article to the journal Aptamers

We invite you to submit your research to the journal Aptamers (ISSN: 2514-3247), which is a new official open-access journal of the International Society on Aptamers, dedicated to publishing peer-reviewed research and reviews on all aspects of aptamer research.

We are happy to waive basic open access publication fee until 30 September 2020 for Aptamers 2020 delegates, as long as the manuscripts are prepared according to the guidelines for authors.

Accepted Posters

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 email on aptamersoxford@gmail.com.

We are delighted to announce that International Journal of Molecular Sciences will offer three cash prizes each to poster and flash-talk winners.

(+F) = Poster plus flash-talk

(+S) = Poster plus a short presentation

(Presenter in bold)

G-quadruplex based DNA aptaswitches for detection of small molecules and proteins: a critical evaluation (+S)

Yasaman Ahmadi, Krista Rathammer, Laura Eibler, Regina Soldo, Ivan Barisic

Molecular Diagnostics, Center for Health and Bioresources, AIT Austrian Institute of Technology GmbH, Giefinggasse 4, 1210 Vienna, Austria

DNA G-quadruplexes (GQ) are getting increasingly popular as reporter systems for biosensor development. The hemin/GQ complex mimics the catalytic activity of HRP enzyme and catalyses the peroxidase reaction of ABTS/H2O2 generating a colorimetric signal. In addition, the GQ is a light-up aptamer and binds to Thioflavin T forming a fluorescent complex. By conjugating the GQ (as a DNAzyme and as a fluorescent light-up probe) to an aptamer, a class of biosensor called “aptaswitch” can be developed. A few GQ-based aptaswitches have been reported for the detection of lysozyme, ATP, ochratoxin A, exosome and CCRF-CEM cells. Many aptaswitches have hairpin structure, in which a blocking tail captures a part of an aptamer and GQ sequences in the stem region of the hairpin structure…

Aptamers as alternative affinity reagents for precision oncology

Michelle L Bauer1, Mazher-Iqbal Mohammed1,2, Alister C Ward1, Sarah L Shigdar1

1 School of Medicine, Centre for Molecular and Medical Research, Deakin University, Geelong, VIC, 3216, Australia

2 Digital Design and Fabrication Research Group, Digital Design School, Loughborough University, UK

Recent years have seen intense appreciation for the heterogeneous and dynamic nature of solid tumours. Consequently, the field of oncology has seen a swift evolution away from a ‘one-size-fits-all’ approach, toward the relatively tailor-made approach that is precision oncology. However, in order to realise the full potential of individualised treatment strategies, there is urgent need for sensitive, specific and precise methods of sampling tumour heterogeneity at the molecular level. Aptamers represent a promising alternative to traditional antibodies for the detection of clinically-relevant tumour biomarkers due to their relatively small size, ease of functionalisation and low batch-to-batch variability. Using aptamers against the cancer stem cell-associated membrane proteins, EpCAM and CD133, we utilised ‘aptahistochemistry’ to identify this…

Development of Cubane-modified Aptamers to specifically recognise a fundamental malaria biomarker (+F)

Federico Bosetto1, Fabienne Levi-Acobas2, Giuseppe Firrao1, Julian A. Tanner3, Marcel Hollenstein2

1 Department of Agricultural, Food, Environmental and Animal Sciences (DI4A), University of Udine, Udine, Italy 2Institut Pasteur, Department of Structural Biology and Chemistry, Laboratory for Bioorganic Chemistry of Nucleic Acids, Paris, France

3 School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China

Plasmodium falciparum (Pf) is the most virulent parasite among Plasmodium spp. that causes malaria in humans. The Pf lactate dehydrogenase (PfLDH) is considered as a potential molecular target for antimalarials, due to the parasite dependence on glycolysis for energy production. Therefore, PfLDH is also an important biomarker for the detection of malaria. An efficient diagnostic tool should be able to specifically distinguish the same molecular biomarker among the different parasite species, but canonical DNA aptamers are not so sensitive. In this work, Cubane-modified aptamers or Cubamers have been introduced to try to reach this goal since a Cubamer highly specific for Plasmodium vivax LDH (PvLDH) was recently isolated. Starting from a DNA library with a random region of 35 nucleotides, we used SELEX to select Cubamers with PfLDH as target. Negative selection was performed first with free magnetic beads then in presence of PvLDH

Ligand interaction and dynamics of the Ochratoxin-A-binding aptamer (+F)

Zachary R. Churcher1, Devid M. Garev1, Aron A. Shoara1, Richard A. Manderville2, Philip E. Johnson1

1 Department of Chemistry, York University, Toronto, ON, Canada

2 Department of Chemistry, University of Guelph, Guelph, ON, Canada

The interaction of the ochratoxin-A-binding aptamer (OTA-1) with its ligand ochratoxin-A is being studied using NMR, and Fluorescence spectroscopy. OTA-1 is a monomolecular G-Quadruplex centred around a two-tetrad core. Ochratoxin A is a mycotoxin produced by certain types of Penicillium and Aspergillus fungi. Found in grain, pork and several other sources, ochratoxin-A is one of the most abundant food contaminating mycotoxins. Ochratoxin-A is a strong neurotoxin thought to deplete dopamine levels in brain and cause oxidative damage to DNA. Using magnetization transfer NMR the exchange rates constants (kex) of the imino protons in the ligand bound aptamer were studied to see understand how dynamic the nucleotide bases in the aptamer are. Our data shows a rigid core, and the kex values of the bases in the two tails supports our hypothesis that the two tails of the quadruplex from a helix…

Aptamer-enabled specific removal of anti-AQP4 antibodies

Monika Czarnecka

Pure Biologics S.A., Dunska 11 street, Wroclaw, Poland

AQP4-IgG is currently regarded as a specific biomarker of Devic’s disease (NMO – Neuromyelitis optica) and a key factor in its pathogenesis. Methods to eliminate anti-AQP4 from patient’s blood while simultaneously not reducing IgGs level are a great chance for a good recovery of NMO-suffering patients. The main goal of the study is to develop a first-in-class therapeutic medical device which will selectively remove pathological antibodies present in patients’ blood and will leave all other blood components intact. To tackle the problem, we used our modular platform termed PureApta™, to identify DNA aptamers as ligands that specifically bind to AQP4-IgG. Two (?) target-specific ssDNA aptamers were obtained under stringent selection conditions. Aptamers demonstrated high binding affinity towards AQP4-IgG target with the simultaneous lack of interaction with other IgGs…

Native ion mobility mass spectrometry uncovers the role of aptamer structural dynamics in ligand binding (+F)

Elise Daems1,2, Karolien De Wael1, Frank Sobott2,3,4

1 AXES Research Group, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium

2 BAMS Research Group, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium

3 Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK

4 School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK

The range of applications for aptamers has been steadily expanding. Our understanding, however, of the mechanisms governing aptamer-ligand recognition and binding is lagging, stymieing the progress in the rational design of new aptamers and optimization of the known ones. We demonstrate how native ion mobility-mass spectrometry can contribute to the analysis of the higher-order structure and noncovalent interactions of aptamers. A set of cocaine-binding aptamers, displaying a range of folding properties and ligand binding affinities, was used as a case study. Using carefully controlled experimental conditions, we probed the conformational dynamics of the aptamers and their interactions with the high-affinity ligand quinine. The ratio of bound and unbound aptamer peaks in the mass spectra was used to rank the aptamers according to their quinine-binding affinity, matching the published ranking order…

Design of bifunctional variants of the cocaine-binding aptamer and experimental analysis using Isothermal Titration Calorimetry (+F)

Nusaibah Dawood, Ruqaiya Qureshi, Sladjana Slavkovic, Phillip E. Johnson

Department of Chemistry, York University, Toronto, Ontario, Canada M3J 1P3

The cocaine-binding aptamer has been used as a model system in the development of small molecule sensing aptamer-based technologies. This project focuses on fusing two variants of the cocaine-binding aptamer to create a bifunctional aptamer that is capable of independently binding two ligands, cocaine and deoxycholic acid (DCA). Isothermal titration calorimetry (ITC) is employed in order to explore the binding behavior of the aptamer with its ligand pair. ITC is a powerful analytical tool used to characterize binding affinities and stoichiometries of binding interactions from a single experiment. The cocaine-binding aptamer was modified to bind DCA by changing the GA base pair at the core of the aptamer to a GC base pair, which resulted in decreased affinity for cocaine and increased binding affinity for a steroid, DCA. Upon performing ITC experiments, the dissociation constant and thermodynamic profile of the resulting binding interaction will be analyzed…

Combined NMR, ITC and native MS approach to unravel the AMP17 – ampicillin interaction

Anne-Mare de Vries1,2, Fabio Bottari3, Elise Daems3, Annemieke Madder2, Karolien De Wael3, José C. Martins1

1 NMR and Structure Analysis research group, Department of Organic and Macromolecular Chemistry, Ghent University, Ghent, 9000, Belgium

2 Organic and Biomimetic Chemistry Research group, Department of Organic and Macromolecular Chemistry, Ghent University, Ghent, 9000, Belgium

3 AXES Research Group, Department of Chemistry, University of Antwerp, Antwerp, 2020, Belgium

Our research interest concerns the development and use of aptamer based electrochemical sensing approaches for antibiotic residue detection and identification. In addition to using a library-selection approach (such as SELEX) for aptamer development, specific design represents an alternative approach which, however, suffers from the lack of understanding of the molecular events involved in the aptamer-target interactions to guide such bottom-up design. Therefore, we are currently investigating the use of three techniques: ITC, native MS and NMR to obtain such detailed information in a number of already reported aptamer-antibiotic systems. We here present our results on the ampicillin binding aptamer AMP17, previously described by Song et al. This DNA based 19mer reportedly binds ampicillin with good selectivity, allowing detection thresholds of 13.4 nM using a colorimetric assay involving gold NP precipitation…

Aptamers for the Forensic Detection of Human Sperm Cells (+F)

James Gooch1, Sireethorn Tungsirisurp2, Richard Napier2, Nunzianda Frascione1

1 King’s Forensics, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK

2 School of Life Sciences, The University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK

Body fluids (such as blood, semen and saliva) are often of enormous value within a forensic investigation, as they may allow offenders to be linked to an offence through DNA profiling. However, such fluids are often difficult to find at crime scenes, as they may be transparent or present in small volumes. Missing fluid evidence during a forensic examination can lead to miscarriages of justice (as seen in the murder cases of Stephen Lawrence and Damilola Taylor). A potential solution to these issues may be offered by fluorescent ‘biosensors’, molecules able to produce specific emission signals in response to biological interactions with body fluid biomarkers. When sprayed over items of evidence within an aerosolised reagent, these biosensors would bind to traces of blood, semen and saliva to make them fluorescent, with different colours of light used to establish the type of fluid present…

Printable Antibiotic binding RNA Aptamers for Goldnanoparticle based Biosensing

Jeannine Jaeger1, Florian Groher1, Jacqueline Stamm2, Dieter Spiehl2, Johannes Braun1, Edgar Dörsam2 and Beatrix Suess1

1 Synthetic Genetic Circuits, Dept. of Biology, TU Darmstadt, Darmstadt, Germany

2 Institute for Printing Science and Technology, Dept. of Mech. Engineering, TU Darmstadt, Darmstadt, Germany

The excessive use of antibiotics in food-producing animals causes a steady rise of multiple antibiotic resistance in foodborne bacteria. Next to sulfonamides, the most common antibiotics groups are fluoroquinolones, aminoglycosides, and ß-lactams. Therefore, there is a need for a quick, efficient, and low-cost detection procedure for antibiotics. We propose an inkjet-printed RNA aptamer developed for the detection of the fluoroquinolone ciprofloxacin. Due to their extraordinary high affinity and specificity, aptamers are already widely used in various applications. The ciprofloxacin-binding RNA aptamer was developed by systematic evolution of ligands by exponential enrichment (SELEX). We characterized the secondary structure of the aptamer and its short version and determined the KD, that allow detection of antibiotic contamination in a relevant range. We defined a 10 nt long region that forms the ligand binding pocket…

Selection of CCL17 recognizing aptamers to inhibit immune cell chemotaxis

Anna Jonczyk1, Marlene Gottschalk2, Lorenz Fülle2, Markus Funke1, Fabian Gondorf2, Franziska Pfeiffer1, Julia Siegl1, Frederika V. Opitz2, Silvana K. Haßel1, Irmgard Förster2, Günter Mayer1,3

1 Chemical Biology and Chemical Genetics, Life and Medical Sciences (LIMES) Institute, University of Bonn, 53115 Bonn, Germany

2 Immunology and Environment, Life and Medical Sciences (LIMES) Institute, University of Bonn, 53115 Bonn, Germany

3 Center of Aptamer Research & Development, University of Bonn, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany

The chemokine CCL17 is associated with the pathogenesis of several autoimmune and inflammatory disorders. As ligand of CCR4, it predominantly directs the recruitment and activation of leukocytes to provide host defence during infection and inflammation. However, CCL17 causes pro-inflammatory function by inducing infiltration of T cells towards inflamed tissue (e.g. atopic dermatitis, asthma, allergic rhinitis). In our previous study, we reported two 2’F RNA aptamers, MF11.46 and MF35.47, that recognize murine CCL17 and inhibit CCL17-mediated chemotaxis. Both aptamers were modified with a 3’-dT-cap structure and a 20 kDa PEG tail at the 5’ end. In transwell-migration assays the directed migration of CCR4+ BW5147.3 lymphoma cells towards CCL17 was inhibited in a concentration dependent manner. IC50 values of 2.9 pmol and 0.42 pmol were measured for MF11.46 and MF35.47, respectively…

Anti-EGFR aptamers for glioblastoma

Alexey Kopylov1, Olga Antipova1, Anastasia Novoseltseva1, Ekaterina Savchenko2, Alexander Revishchin3, Elena Zavyalova1, Andrey Golovin1, Galina Pavlova3,4

1 M.V. Lomonosov Moscow State University, Moscow, Russian Federation,

2 Apto-Pharm Ltd, Moscow, Russian Federation,

3 Institute of Gene Biology, RAS, Moscow, Russian Federation,

4 N.N. Burdenko Neurosurgery Research Institute, Moscow, Russian Federation

Immunotheranostics applies monoclonal antibodies as molecular recognizing elements (MoRE). MoRE made of nucleic acids are aptamers. For glioblastoma (GBM), some aptamers were described, which are able to interact with glioma cells gaining to block proliferation (82 publications vs 6,861 for antibody): aptamers to EphB2/3 receptors, to PDGFRβ, to U87-EGFRvIII cells, to tenascin-C, AS1411 to nucleolin, ets. EGFR is the target to select aptamers gaining to halt proliferation of EGFR positive clones of GBM. Despite hundred papers for EGFR aptamers were  published till now (vs 19,000 for antibody), many aptamers do not exhibit properties for translational study; results are diverse and could not be easily compared. Therefore, there is a need for smart aptamers to develop and translate them into theranostics…

Aptamer selection by Capture-SELEX

Leon Kraus, Marc Vogel, Beatrix Suess

Department of Biology, Technical University Darmstadt, Schnittspahnstrasse 10, Darmstadt D-64287, Germany

Aptamers are short single-stranded nucleic acids that bind their target with high affinity and selectivity. They are selected in vitro and can be used for a variety of applications. For example, they can be used as synthetic riboswitches to regulate gene expression. These artificial regulators can be applied in different organisms, from Escherichia coli to mammalian cells, controlling all levels of gene expression like transcription, translation and pre-mRNA splicing. The in vitro selection method used to find these aptamers is called SELEX. With SELEX aptamers can be selected for a variety of different targets (small molecules, peptides, proteins, viruses or whole cells). However, it is still quite challenging to find aptamers that can be engineered into riboswitches. Aptamers for this specific application should undergo a conformational change upon ligand binding. This problem can now be overcome by…

Targeted Imaging of Cancer Cells Using Polydopamine (PDA)-Based Aptamer Signal Amplification

Hawon Lee, Miso Kim, and Young-Pil Kim

Department of Life Science, Hanyang University, Seoul, 04763, Republic of Korea

Despite the development of imaging strategies for cancer cells, the demand for higher quality and intensity still remains. In this study, we suggest a polydopamine (PDA)-based signal amplification method of aptamers for cancer cells. This detection strategy was enabled by HRP-accelerated rapid PDA decomposition on the aptamers attached on the cell surface. The biotinylated aptamers were specifically bind to the cell surface target and then the streptavidin-conjugated HRP was treated to localize HRP near aptamers. When the dopamine monomers are added and reacted with HRP, the rapid generation of PDA is initiated near the target of aptamers. Then, the primary amine-functionalized quantum dots (NH2-QDs) is added and the high intensity of fluorescence is decomposed and accumulated near the PDA. By using this method…

Gold Nanoparticle-Assisted SELEX with Colorimetric based Monitoring Platform for Facile Selection of Target-Binding Aptamers

Eun-Song Lee and Young-Pil Kim

Department of Life Science, Hanyang University, Seoul 04763, Republic of Korea

Aptamers developed via systematic evolution of ligands by exponential enrichment (SELEX) against interested small molecules are invaluable tool for target specific biosensor development or biomedical research. For successful aptamer selection, fine monitoring for SELEX procedure is essential requirement. Here we report a SELEX process incorporated by gold nanoparticle (AuNPs) for facile monitoring of aptamer library enrichemt, which requires neither target modification nor additional analysis of SELEX product. Under the presence of target, target bound single-stranded DNA (ssDNA) aptamers are detached from the surface of citrate-stabilized AuNPs. Binding affinity was visualized for every SELEX round product right after target-aptamer binding step. It could be simply achieved by colour change of aggregated bare AuNP caused by treatment of high concentration salt…

Robotic-assisted selection of RNA aptamers targeting small molecules

Tjaša Legen, Günter Mayer

The Center of Aptamer Research and Development (CARD), Life and Medical Sciences Institute (LIMES), Bonn, Germany

Aptamers are a promising tool for small molecules detection due to their small size, chemical stability, and cost-effectiveness over conventional bioreceptors such as antibodies. Sensors developed with such technology are usually selected in an in vitro process named Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Conventionally, the target is immobilized onto a matrix to enable the separation of binding and non-binding sequences. However, the immobilization of a small molecule will change its electron distribution or may generate new epitopes for the binding. Hence, a Capture-SELEX approach was developed, where the library is immobilized on a matrix through a complementary oligonucleotide. We show that by using the Capture-SELEX approach we can adapt the selection to automatedly select RNA aptamers. Thus, small molecules binding aptamers can be selected already in a few days…

Towards the discovery of novel aptamerogenic biomarkers by on-blot SELEX

Tjaša Legen1, Günter Mayer1,2

1 The Center of Aptamer Research and Development (CARD), Life and Medical Sciences Institute (LIMES), Bonn, Germany

2 Clickmer Systems company in foundation, Gerhard-Domagk-Str. 1, 53121 Bonn

Western blot analysis is an analytical technique used to detect and quantify specific proteins in a given sample of tissue or cell homogenate. Proteins transferred to a specific membrane are usually detected with specific antibodies. Here, we show that with the implementation of an on-blot selection process, clickmers can be generated that recognize specific protein targets obtained from protein preparations from cancer cells. The clickmers were directly selected on the membrane and the on-blot selection process yielded enriched libraries containing several clickmers for different proteins. In a first attempt, we used protein preparations from two prostate cancer cell lines, namely PC3 and LNCaP cells, as positive and negative selection matrix, respectively. Clickmers were enriched that strongly and specifically bind only to individual proteins of the PC3 cells lysate as revealed by western blot analysis…

Label-free electrochemical biosensor for real-time continuous detection of ATP and adenosine (+F)

Martina Mengoni1, Faming Tian2, Nicholas Dale1,2

1 School of Life Sciences, University of Warwick,Gibbet Hill Road, Coventry, CV4 7AL, UK

2 Sarissa Biomedical Limited, Unit 4B, Vanguard Centre University of Warwick Science Park, Coventry, CV4 7EZ, UK

Detection of small molecules has always been an issue as antibodies lack sensitivity and often specific enzymes do not exist. Aptamers, with their small size, fast response and in vitro synthesis, constitute a promising alternative for biosensing applications. In this project, three aptamers are employed to achieve electrochemical detection of adenosine and adenosine-triphosphate (ATP). By functionalising the oligonucleotide with a methylene blue (MB) molecule, we can measure the change in the response upon binding of its target molecule. The signal is generated by a change in both the distance between the MB and the electrode surface and in the electrical layer at the solution-electrode interface, thus its conductive properties. The sensors are fabricated via self-assembling monolayer technique based on thiol chemistry. The sensor produced has achieved detection of adenosine in phosphate buffer saline (PBS) down to a concentration of 10 μM…

Spiegelmer-based cardiac troponin detection (+S)

Zoltán Tolnai, Judit András, Zsuzsanna Szeitner, Krisztina Percze, Tamás Mészáros

Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University. Üllői út 24., Budapest 1085, Hungary

Cardiac troponin I (cTnI) is the standard biomarker of myocardial injury. Similarly to most biomarker protein detecting methods, the presently applied cTnI measuring devices rely on highly-selective antibodies. Although the sensitivity, selectivity, and processing time of current apparatuses meet the demand of the point-of-care diagnostics, the fragility of antibodies limits their application. Aptamers could be reasonable alternatives of antibodies as sensor molecules of these devices but their susceptibility to ubiquitously present nucleases may result in their rapid degradation in blood samples. Leveraging the complete nuclease resistance of enantiomers of natural RNA and DNA, the so called spiegelmers, could address this drawback of aptamers…

Antiproliferative properties of bi-HD1 aptamer for human lung cancer and glioblastoma cells

Galina Pavlova1,2,3, Elena Zavyalova4, Nadezda Samoilenkova5, Alexander  Gulin6, Alexander Revishchin1, Varvara Kolesnikova7, Andrey Golovin3,4, Victor Nadtochenko6,  Alexey Kopylov4

1 Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia

2 Federal State Autonomous Institution «N.N. Burdenko National Scientific and Practical Center for Neurosurgery» of the Ministry of Healthcare of the Russian Federation, Moscow, Russia

3 Sechenov First Moscow State Medical University, Institute of Molecular Medicine, Moscow, Russia

4 M.V. Lomonosov Moscow State University, 119991, GSP-1, Leninskie Gory, Moscow, Russia

5 Apto-Pharm Ltd, Moscow, Russia

6 Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow, Russia

7 Pirogov Russian National Research Medical University (RNRMU), Moscow, Russia

Some G-quadruplex DNAs (G4-DNA) exhibit antiproliferative activity, and could be currently considered as cryptic aptamers for G4-DNA binding proteins. HD1 G4-DNA, originally selected for thrombin, has anti-proliferative effect as well. Putative structure of covalent dimer of HD1, bi-HD1, has two G-quadruplexes linked via T residue, and antiproliferative properties of bi-HD1 is not known yet, not mentioning specificity for type of cancer. In this study, a panel of cell lines of malignant tumors of various etiologies was used, including breast cancer, glioblastoma, neuroblastoma, prostate cancer, rectal carcinoma. Effect of bi-HD1 on cell lines was assessed by MTT method as indicator of decreasing of proliferative potential. bi-HD1 reduces proliferative activity in a number of cell lines of different tissue specificity…

Designing SELEX assays for Aptamers compatible with Machine Learning (ML) Analysis – Robust & Compressed High Throughput Systematic Evolution of Ligands by Exponential Enrichment (RC-HT SELEX) (+S)

Annalisa Pawlosky

GAS Labs, 1600 Amphitheater Parkway, Mountain View, CA 94043, USA

Machine learning is excellent for making predictions, yet hasn’t robustly generated consistent aptamers candidates with desired features. In some part, the delta of ML success and its ability to predict better aptamer candidates is due to the lack of ideal protocols for machine-readable empirical datasets. Here we’ve built an ML-inspired SELEX protocol to resolve this gap and discuss the deficiencies of current SELEX protocol to generate training data for ML. Our results demonstrate that we have developed an accelerated high-throughput process of generating aptamers to peptide targets that is compatible with machine learning (ML) requirements for two candidate targets – Bradykinin and vasopressin – which are major pharmacological targets for regulating blood pressure…

Aptamer-based Lateral Flow Assay and the Detection of C-reactive Protein for Point of Care Testing (+F)

Ngoc L. Phung, Johanna G. Walter, Thomas Scheper, Cornelia Blume

Institute of Technical Chemistry, Leibniz University Hannover, Hanover, Germany

Point of care (POC) testing is relevant, especially in individualized medicine. A popular platform, the Lateral Flow Assay (LFA) test has become famous with pregnancy tests. While LFAs usually are based upon antibodies, we demonstrate for the first time an aptamer-based LFA for the detection of C-reactive protein (CRP) in patient samples, which is a clinically established biomarker for unspecific inflammation. In healthy humans, CRP concentrations range between 5-10 mg/L, while the concentration increases rapidly within inflammation.  We used microarrays to screen diverse binding conditions of CRP-aptamers and tested diverse aptamer combinations in a sandwich format before transferring optimized conditions to the LFA system. Here, aptamer pair Apt1-Apt1 were characterized to be optimal as capture and detection partner for the LFA-sandwich. The LFA platform contains a test (TZ) and control zone (CZ)…

Chasing antibody function: how to resolve avidity and affinity of multivalent aptamer interactions (+F)

Irene Ponzo, Nena Matscheko

LMU of Munich/Dynamic Biosensors GmbH, Munich, Germany

Therapeutic aptamers are gaining increasing interest due to multiple advantages over antibodies. Classical IgGs have two binding sites on each molecule, and efforts are ongoing to engineer antibodies with even more binding sites to achieve additional function, specificity and binding strength. Keeping up with this trend is the development of multivalent aptamers, which requires efficient methods for the elucidation of individual binding contributions. We have recently adapted the DNA-based switchSENSE biosensor for the study of aptamer conformational changes and interactions, yielding in-depth characterization of aptamer folding and binding rates in the presence of different salt conditions and molecular targets. Here, we show a new bivalent aptamer assay with two thrombin binding aptamers (TBAs) against individual thrombin exosites..

Aptamers as Reversible Sorting Ligands for Preparation of Cells in Their Native State

Martin D Requena, Bethany Powell Gray, Michael D. Nichols, Bruce A Sullenger

Department of Surgery, Duke University, USA

Selective isolation of cells based on cell-surface biomarkers is an invaluable tool in many research and clinical applications, including analysis of circulating tumor cells, hematopoietic stem cell transplantations, and cancer immunotherapy. Though antibody-based magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS) are effective techniques for isolation of cells from complex mixtures on the basis of expression of a specific biomarker, antibodies cannot be easily removed from cells. We have developed a MACS- and FACS-compatible method to reversibly label and purify cells using aptamers and matched oligonucleotide antidotes…

Investigation of the exact binding site location – truncation study of DOX6

Sofie Schellinck1, Anne-Mare de Vries1,2, Elise Daems3, Karen Driesen3, Annemieke Madder2, Karolien De Wael3, José C. Martins1

1 NMR and Structure Analysis research group, Department of Organic and Macromolecular Chemistry, Ghent University, Ghent, 9000, Belgium

2 Organic and Biomimetic Chemistry Research group, Department of Organic and Macromolecular Chemistry, Ghent University, Ghent, 9000, Belgium

3 AXES Research Group, Department of Chemistry, University of Antwerp, Antwerp, 2020, Belgium

Doxycycline, a small molecule target, is a widespread used antibiotic. A first step in counteracting antibiotic resistance: “one of the biggest threats to global health, food security and development” as stated by the WHO, is detection of antibiotics in, for example, waste water. Aptamers, with their high selectivity and specificity, could be applicable in on-site sensors. Our research interest concerns the development and use of aptamer based electrochemical sensing approaches for antibiotic residue detection and identification. In this respect, we are currently investigating DOX6, a DNA based 39mer that was selected against the antibiotic doxycycline using a novel, so called double library SELEX procedure…

Analysis of Aptachain: Ligand-Induced Self-Assembly in The Cocaine-Binding Aptamer (+F)

Aron A. Shoara, Miguel A. D. Neves, Philip E. Johnson

Department of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, ON, M3J 1P3 Canada

The cocaine-binding aptamer has become a widely employed model system for the development of aptamer-based biosensors for two main reasons: (i) it has a structure-switching mechanism that depends on the length of one stem, and (ii) it binds ligands, such as quinine, tighter than its originally selected cocaine ligand. We will present progress on the concept of “aptachain” formation, where an aptamer is split into two overlapping or staggered strands and assembles into an extended oligomer upon ligand binding. The assembly of aptamers can then be used to detect ligand binding by the aptamer. To demonstrate this concept, we employ the cocaine-binding aptamer as a model system, used its ability to tightly bind quinine and demonstrated its capability in a gold nanoparticle-based biosensing application…

From cooperative to independent binding: A thermodynamic analysis of ligand binding by the ATP-binding aptamer

Sladjana Slavkovic, Zach Churcher, A. Aron Shoara and Philip E Johnson

Department of Chemistry, York University, Toronto, ON, M3J 1P3, Canada

The ATP-binding DNA aptamer is often used as a model system for developing new aptamer-based biosensor methods.  This aptamer follows a structure-switching binding mechanism and binds two copies of its ligand.  In this work, we demonstrate that the aptamer binds ATP, ADP, AMP and adenosine with a cooperative two-site binding model. Using both individual and global fitting methods we have determined the binding affinity and thermodynamics for both ligand-binding sites. In order to understand how cooperativity works, we separated two binding sites by inserting extra base pairs. By separating the ligand binding sites by an additional four base pairs so that they are on opposite sides of the helical structure, we engineered a variant of this aptamer that binds two adenosine ligands independently, while two ATP molecules still bind cooperatively…

Novel detection of nasty bugs: the generation of multi-reactive aptamers to hospital acquired infection causing bacteria.

Mia Strom1, Ben Wade2, Tamsyn Crowley1 and Sarah Shigdar1

1 School of Medicine, Deakin University, Geelong, Victoria, Australia

2 RMIT University, Bundoora, Victoria, Australia

A major issue affecting those in hospital are hospital acquired infections (HAIs). Studies have shown that bacteria, which are spread via person-to-person contact and contaminated instruments and surfaces, cause approximately 80% of all HAIs. Every patient in a hospital has a potential risk of contracting a HAI. However, those at most risk are patients in the intensive care unit, burn victims, transplant receivers and neonates. Studies show that HAIs contribute to increased mortality and morbidity rates but recent studies have also shown that HAIs caused by certain bacteria, such as Clostridiodes difficile have contributed to approximately $1 billion in excess medical costs as well as 14,000 deaths each year. Detection systems that are currently used can be both time consuming and expensive, they can also be prone to false negatives due to natural bacterial processes…

Developing an aptamer-based biosensor against the phytohormone auxin (+F)

Sireethorn Tungsirisurp and Richard Napier

University of Warwick, Coventry, UK

Indole-3-acetic acid (IAA) has been one of the most widely studied phytohormones due to its vital roles in plant growth and development. Techniques including mass spectrometry and genetically-encoded biosensors have provided us with valuable information on its intracellular activities. However, there is still a lack of quantitative data on its intercellular distribution with high spatial and temporal resolution. The aim of this study is to develop a novel auxin-specific, aptamer-based biosensor to provide a more sensitive and detailed IAA mapping tool. Two methods of systematic evolution of ligands by exponential enrichment (SELEX) have been evaluated to provide aptamer sequences with potential high auxin binding affinity and selectivity. Direct-SELEX captures specific sequences onto magnetic beads while solution-SELEX releases them from magnetic beads..

Optoribogenetics: A light dependent aptamer-photoreceptor system enables control of numerous RNA functions in cells

Anna M Weber1,2, Sebastian Pilsl1, Christian Renzl1, Georg Pietruschka1, Ankana Kakoti1, Jennifer Kaiser3, Thea Ziegler3, Andreas Möglich3 & Günter Mayer1,2

1 Life and medical sciences institute (LIMES), University of Bonn, Gerhard-Domagk-Straße 1, 53121 Bonn, Germany

2 Center of Aptamer Research and Development (CARD), Gerhard-Domagk-Straße 1, 53121 Bonn, Germany

3 Department of Biochemistry, University of Bayreuth, Universitätsstraße 30, 95440 Bayreuth, Germany

Optogenetic makes use of light to control cellular behaviour in a spatial and temporal manner. Several processes, such as opening of ion channels, enzymatic activity or DNA binding have been shown to be compatible with light regulation. Photoreceptor proteins, e.g. LOV (light, oxygen, voltage)-domain containing proteins play an important role in developing those processes since they are able to reversibly change their conformation upon light absorption and therefore convert the incoming signal into a specific output. However, until now it was difficult to apply optogenetic control to RNA biochemistry, e.g. RNA localization or function. Recently, the expression and the characterization of the bacterial LOV photoreceptor protein PAL facilitated the selection of a short hairpin RNA binding specifically the light state of the photoreceptor…


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