Thank you for considering presenting your work as a poster at Aptamers 2020 Virtual.
Poster deadline: Prepare your poster as you would normally do for printing, and submit your final poster as both PDF and JPG/PNG files via the link below no later than 28th August 2020. Late posters may not be included in the symposium programme. Please DO NOT send your poster files by email.
Naming your poster files: Name your poster files as follows: <your surname>-APT20V-Poster.pdf | <your surname>-APT20V-Poster.png | <your surname>-APT20V-Poster.jpg, etc. For example, for David Jones, name your file as Jones-APT20V-Poster.pdf. DO NOT name your poster files as, e.g., Oxford-poster, Aptamers2020v, Oxford-aptamers-poster. Such files will be automatically rejected.
Poster presentation: Posters will be made available via a secure page to the symposium participants before the meeting. There will be two ways to interact with the poster presenters:
The poster presenters should regularly check Twitter for any questions about their posters before, during and after the meeting, and post their answers on Twitter using appropriate hashtags, as above. However, the presenters are not compelled to respond via Twitter.
We ask participants to be polite and respectful when asking questions, keeping discussions courteous and non-confrontational and based around the scientific content.
Any further information about the poster presentations at this digital meeting will be available in the future.
If your poster was accepted for presentation at the March Aptamers 2020 meeting and you would like to present it virtually, please get in touch by emailing AptamersOxford@gmail.com.
(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.
Twitter: #AptaOx20V, #MCzarnecka
Monika Czarnecka, Magda Puchała, Joanna Guzdek, Marta Radzińska, Barbara Dąbrowska, Agnieszka Sok-Grochowska, Monika Lopko, Kamilla Sołtys, Zbigniew Darżynkiewicz, Emilia Liputa, David Carter, Aleksandra Żurek, Tomasz Bąkowski
Pure Biologics Plc., Wrocław, Poland
AQP4-IgG (anti-aquaporin-4 IgG) is currently regarded as a specific biomarker of Neuromyelitis optica (NMO) and a key factor in its pathogenesis. Methods to eliminate AQP4-IgG from a patient’s blood while simultaneously not reducing IgG level, represent a promising approach 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 while leaving other blood components intact. To tackle the problem, we used our modular platform termed PureApta™ to identify chemically-modified DNA aptamers as ligands that specifically bind to AQP4-IgG. Target-specific ssDNA aptamer was obtained under stringent selection conditions. It demonstrated a high binding affinity towards the AQP4-IgG target with the simultaneous lack of interaction with other IgG. Furthermore, using the AQP4-IgG-targeting aptamer coupled to column-packed resin we have shown high capacity for clearing pathological antibodies from anti-AQP4-positive human serum without marked interference with common IgG. This proof-of-concept study represents a new formulation strategy for the use of aptamer-based medical device in targeted therapy.
Twitter: #AptaOx20V, #MAChowdhury
Md Anisuzzaman Chowdhury 1, David Gell 1, Sarah Shigdar 3, Sharn Perry 1, Michael Breadmore 2, Anna King 1
1 Wicking Dementia Research and Education Centre, University of Tasmania, TAS, Australia
2 Australian Centre for Research on Separation Science, University of Tasmania; TAS, Australia
3 Centre for Molecular and Medical Research, Deakin University, VIC, Australia
Monitoring brain health is challenging due to the isolated location of the brain within the skull and the expense and invasive nature of imaging techniques such as PET scans. However, monitoring brain heath is critical for early detection of adverse changes in the brain that can lead to neurodegenerative diseases such as dementia, the second leading cause of death in Australia, and development of preventative and therapeutic strategies. Biomarkers are determinable biological molecular indicators, which can help to assess brain health and disease stage and risk. Blood-based biomarkers offer benefits according to accessibility, invasiveness and cost compared to other fluid biomarkers to diagnose or monitor the progression of disease. Biomarkers such as the neurotrophin brain-derived neurotrophic factor (BDNF), may be useful to monitor synaptic plasticity. BDNF levels in human serum have served as an indicator of brain function and are associated with different conditions in NDD. In this study, we are developing a DNA aptamer that specifically binds BDNF protein from a DNA library containing 50-nuceotide random sequences. Following eleven cycles of systematic evolution of ligands by exponential enrichment (SELEX) procedure, we identified the best cycle that demonstrated high evolution of aptamers by restriction fragment length polymorphism. A sandwich-based assay was also performed to determine the SELEX cycle with the highest binding affinity to the BDNF. We anticipate the selected DNA aptamer(s) will be a good alternative detection biosensor for the sensitive and precise detection of BDNF. Single-molecule array (SIMOA) assay is 100 times more sensitive than standard ELISA, these aptamers will be used firstly in SIMOA based assay to determine the targeted proteins and then further developed to point-of-care devices for aptamer-based biosensing (aptasensors), which have been incorporated in clinical applications recently.
Twitter: #AptaOx20V, #NDawood
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. The data will be fit to both, an independent and cooperative binding model, from which the appropriate binding model will be determined. Quantifying these binding parameters is an important step to characterize the aptamer-ligand interaction. The next goal is to design new bifunctional aptamers that have cooperative binding sites, progress in this will be achieved by conducting ITC experiments on further aptamer constructs.
Twitter: #AptaOx20V, #HDunham
Hamish Dunham1, Jennifer Soundy2, Janet Pitman1
1School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand
2AuramerBio Ltd, Te Toki a Rata building, Victoria University of Wellington, Wellington, New Zealand
Small molecule aptamer development is notoriously difficult. The diminutive size of the target molecule limits the number of binding sites, and the small structural changes that occur due to the binding event only confer minor mass and energetic changes. This presents a significant challenge when attempting to characterise these small-molecule aptamer interactions and relies upon extremely sensitive methods that are capable of detecting ligand binding. Isothermal titration calorimetry (ITC) is widely regarded as the gold standard to discern molecular kinetic data. ITC is capable of providing an almost complete profile of thermodynamic parameters associated with molecular interactions, with enough sensitivity to investigate small-molecule aptamer interactions. However, published literature is lacking on the use of low volume ITC to detect these types of interactions. Herein, I will present novel strategies for characterising the binding interactions between aptamers and their small molecule targets, asparagine and glutamic acid using low volume (190 µL) ITC. The efficacy of these strategies were validated using the extensively characterised cocaine binding aptamer, MN4. Our results reveal the viability of low volume ITC to detect small molecule aptamer binding events.
Twitter: #AptaOx20V, #NGilmartin
Brian D Henderson 1,2, Steve Meaney 1,2 and Niamh Gilmartin 1,2
1 School of Biological and Health Sciences, College of Sciences and 2 Health and Environment, Sustainability and Health institute, Technological University Dublin, Dublin, Ireland
Amplification of aptamer libraries by PCR is in an integral component of systematic evolution of ligands by exponential enrichment (SELEX) protocols. However, the efficiency of target library amplification can be impacted by “off-target” products greater in size that the aptamer library. This paper describes experimental evidence of the origin of off-target products and a candidate universal approach to maximise target yields in SELEX. This approach provides a simple technical foundation to minimise off-target products during the SELEX process, via a simple modification of the PCR thermal cycling conditions which can reduce hands on time and reagent cost for selection processes.
Twitter: #AptaOx20V, #AJonczyk
Anna Jonczyk1, Marlene Gottschalk2, Lorenz Fülle2, Markus Funke1, Fabian Gondorf2, Franziska Pfeiffer1, Julia Siegl1, 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 diseases. As a ligand of CCR4, it primarily directs the recruitment and activation of leukocytes to sites of inflammation. However, if CCL17 is excessively produced, it promotes the development of autoimmune diseases, such as atopic dermatitis, asthma or allergic rhinitis, most likely through increased recruitment of pathogenic T cells. In our previous study, we reported two 2’F-RNA aptamers, MF11.46 and MF35.47, that specifically bind murine CCL17 and inhibit CCL17-mediated chemotaxis. Both aptamers were tested in a Transwell migration assay and a mouse model of contact hypersensitivity (CHS). In vitro, blocking CCL17 with aptamers specifically inhibited directed migration of CCR4 expressing thymic lymphoma cell line BW5147.3 in a concentration dependent manner. IC50 values of 2.9 pmol and 0.42 pmol were measured for MF11.46 and MF35.47, respectively. For in vivo experiments, both aptamers were modified with a 3’-dT-cap structure and a 20 kDa PEG tail at the 5’ end. In the CHS model, systemic application of MF11.46 and MF35.47 significantly prevented ear swelling and attraction of T cells during the challenge phase of CHS. We could also show that MF11.46 and MF35.47 bear no immunogenicity. Since the identified aptamers are highly specific for murine CCL17 and do not recognize human CCL17, we now aim to isolate aptamers specific for human CCL17. We are currently in the process of generating such aptamers both manually as well as by an automated in vitro selection platform, using DNA and 2’-deoxy-2’-fluoro pyrimidine-bearing RNA libraries. As our previous results showed, that inhibition of CCL17 strongly improves symptoms in the murine CHS, aptamers that recognizing human CCL17 and inhibit its chemotactic activity represent a promising therapeutic tool for the treatment of allergic and inflammatory diseases.
Twitter: #AptaOx20V, #NKomarova
Natalia V Komarova, Daria V Barkhova, Alexander E Kuznetsov
Scientific-Manufacturing Complex Technological Centre, 1–7 Shokin Square, Zelenograd, Moscow 124498, Russia
Capture-SELEX is a specially designed SELEX protocol for the isolation of aptamers against small molecules which is devoid of target immobilization. In Capture-SELEX, the oligonucleotide library is designed with a special fixed sequence region for hybridization with a capture probe immobilized onto a solid support. This work was aimed to estimate the impact of capture fragment primary sequence on the outcome of aptamer selection. Three libraries of random DNA sequences were designed with different GC composition of the capture fragment, providing different binding energies of hybridization. These libraries were used for Capture-SELEX against acetylcholine in the independent parallel experiments. The samples of the enriched libraries from the selection cycles 3-14 were sequenced with Illumina technology. NGS results indicated that the enrichment proceeded better for the library with the highest GC-content, but the library with the medium GC-content was enriched poorly within the selection. Aptamer candidates for each library were identified based on bioinformatics analysis of NGS data, synthesized and screened for the binding ability using a range of acetylcholine concentrations. The selection using the library with the medium CG content which was poorly enriched resulted in no binding oligonucleotides. Thus, the selection with this library rendered unsuccessful. Two other selections resulted in the isolation of acetylcholine binding oligonucleotides. The best binders were identified using the library with the highest GC-content. The results indicate that an increase in the GC content of the capture fragment in the library of random DNA sequences accelerates the selection process, reduces the number of minimum required selection cycles and enables isolation of more effective aptamers. This work was supported by Russian Foundation for Basic Research (RFBR), grant #18-34-20020.
Twitter: #AptaOx20V, #JLChain
Anthony Codbreuil, Laurent Azéma, Jeanne Leblond-Chain
University of Bordeaux, CNRS, INSERM, ARNA, UMR 5320, U1212, F-33000 Bordeaux, France
Specific drug delivery to the target site is a still major challenge today. Unfortunately, many drug delivery systems lack specificity and exhibit side effects. In this work, we consider drug-binding aptamers as specific drug carriers, thanks to their high affinity for their cognate drug. Since high molecular weight is required to prevent from renal filtration and improve blood circulation time, the goal of this project is to design a supramolecular assembly of DNA aptamers, called nanotrain, as a drug delivery system. Quinine was selected as a model drug to be transported as the quinine aptamer is well described. Aptamer sequence was appended with hybridation sequences to trigger the nanotrain assembly. Two strategies were investigated: first, a continuous hybridization using two complementary sequences that associate one after the other in a polymer-like structure and secondly, the controlled association of specific sequences for each aptamer. In each case, the sequences were designed, the thermal stability and the affinity for quinine were investigated by UV and fluorescence, respectively. These two methods allowed to build a supramolecular assembly, identified by electrophoresis, which suggested improved affinity for quinine and better thermal stability.
Twitter: #AptaOx20V, #MMengoni
Martina Mengoni 1, Faming Tian 2, Nicholas Dale 1,2
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. Furthermore, response to ATP and downstream purines have been tested. The lowest measured concentrations were all well above the biologically significant values. As an effective and sensitive sensor for measurements of adenosine already exists, the two responses were compared showing that the enzyme-based sensor is superior in terms of both sensitivity and response time.
Twitter: #AptaOx20V, #ARPaul
Alexandra R. Paul 1, Michelle D. Garrett 2, Helen Lavender 3, Christopher J. Serpell 1
1 School of Physical Sciences, Ingram Building, University of Kent, Canterbury, CT2 7NH
2 School of Biosciences, Stacey Building, University of Kent, Canterbury, CT2 7NJ
3 Avvinity Therapeutics, First Floor Thavies Inn House, 3-4 Holborn Circus, London, EC1N 2HA
Aptamers can be harnessed for therapeutic agents because of their recognition capacity and high affinity. The chemistry of aptamers is largely limited to that of nucleic acids, and although non-natural modifications of nucleic acids are known to enhance aptamer affinity, there is not a technology for selecting the right modifications amongst billions of possibilities. This project aims to develop the first general method for discovery of nucleoside modifications which increase aptamer binding efficacy. A library will be created of over 1 million different chemical modifications on a known aptamer sequence (MinE07). The modifications will use different types of chemistry to see how they affect the binding and folding of the aptamer MinE07. Aptamer MinE07 binds to the Epidermal Growth Factor Receptor (EGFR) which is a protein that is over-expressed in cancerous cells, the goal is to improve the binding between MinE07 and EGFR. Our aim is to improve the affinity of MinE07 for EGFR by using flow cytometry to separate out the best bound sequences from the one-bead-one-sequence aptamer library. Here we present steps towards the aptamer library synthesis, fluorescent activated flow cytometry sorting and protein binding assays.
Twitter: #AptaOx20V, #ASamokhvalov
Alexey V. Samokhvalov 1, Irina V. Safenkova 1, Sergei A. Eremin 1,2, Anatoly V. Zherdev 1, Boris B. Dzantiev 1
1 A.N. Bach Institute of Biochemistry, Federal Centre of Biotechnology, Russian Acad. Sci., Moscow, Russia
2 Chemical Faculty, M.V. Lomonosov Moscow State University, Moscow, Russia
Due to their unique properties, aptamers are actively used in various assays. Homogeneous analytical techniques are of particular interest due to the rapid formation of detectable complexes, the minimum number of stages, and ease of use. We present here various developed analytical methods for the detection of ochatoxin A (OTA), toxic contaminant of agricultural products. The methods were applied using the earlier described OTA-specific aptamer (5´-GAT-CGG-GTG-TGG-GTG-GCG-TAA-AGG-GAG-CAT-CGG-ACA-3´).Competitive binding of OTA from a sample and fluorescein-labeled OTA provide an opportunity of the assay based on the registration of fluorescence polarization. A decrease in the rotational mobility of labeled OTA when it binds to the aptamer makes it possible to assess the concentration of native OTA molecules competing for binding. The assay under the chosen optimal conditions can be implemented in one stage for 15 min and is characterized by the limit of detection (LOD) equal to 151 nM. Gold nanoparticles (GNPs) are considered as “anchors” for aptamers immobilization that additionally slow down the rotation of the aptamer-labeled OTA complex. The screening of anchor constructions demonstrated advantages of the aptamer complexes with GNPs having diameter 8.7 nm. The LOD for this assay is decreased to 5.6 nM. Additional analytical possibilities are provided by the modulation of OTA intrinsic fluorescence caused by its binding with the aptamer. The registration of this parameter is allows the detection of OTA in concentrations up to 1.2 nM. Constructions with the application of complementary chains to different regions of the OTA-specific aptamers are tested as tools modulating fluorescent signals.This study was supported by the Russian Foundation for Basic Research (Project No. 18-08-01397_a; techniques with registration of fluorescence polarization) and the Russian Science Foundation (Project No. 20-74-00112; techniques with registration of fluorescence intensity).
Twitter: #AptaOx20V, #GTsekenis
Nikolas I. Kalavros 1,2, George Tsekenis 1
1 Center of Basic Research, Biomedical Research Foundation of the Academy of Athens, 11527 Athens, Greece
2 Department of Informatics and Telecommunications, National and Kapodistrian University of Athens, 15784 Athens, Greece
We propose a novel computational pipeline that is able to identify aptamers that bind specifically to a desired target molecule. The pipeline is composed of 4 main stages: Parametrization of the ligand and aptamer structures, nucleic acid structure prediction, rigid body docking and, finally, molecular dynamics. We examine the multiple tools that can be used for each stage of the aforementioned pipeline and their impact on the pipeline’s performance by running our pipeline on several well-researched aptamers and their ligands (thrombin, ochratoxin A and others). Furthermore, we benchmark the stages of our approach using established datasets for structure prediction and docking. Additionally, we introduce a genetic algorithm into our framework, which iteratively mutates the aptamer, refining its structure and binding affinity according to pre-defined user criteria. Finally, we show that, given the correct combination of tools, our method by and large agrees with published results. Moreover, it permits new insights to be gained through the analysis of the molecular dynamics trajectories of the aptamer-target analyte. To our knowledge, this is the first pipeline that starts with a nucleic acid sequence and performs all the necessary computations, up to the calculation of free binding energies.
Twitter: #AptaOx20V, #LZara
Lorena Zara 1,2, Emmanuelle Fiore 1, Jean-Jacques Toulmé 2, Eric Peyrin 1, Corinne Ravelet 1
1 Département de Pharmacochimie Moléculaire, Université Grenoble Alpes, Saint-Martin d’Hères, France
2 NOVAPTECH, Pessac, France
In recent years there has been a great progress in the selection of aptamers in terms of time, cost and laboriousness. From the aptamer selection to the design of biosensor, there are many check points to guarantee the quality of analytical results, such as the employment of scramble DNA sequences, the choice of homologous analytes as controls and independent methods for the determination of the equilibrium dissociation constant (Kd). To obtain reliable affinity and specificity metrics, more than one assay platform is recommended to prove the binding properties of the aptamer. In this work we proposed to assess the robustness of aptamer SS2-55 and its variant SS24-35 both targeting isocarbophos and phorate. We performed extensive binding assays using homogeneous techniques, namely Fluorescence Anisotropy (FA) and Isothermal Titration Calorimetry (ITC). The validated anti-tyrosinamide aptamer, which shows a dissociation constant in the range reported for the anti-pesticide aptamers, was included in the study. Two different FA approaches were applied, one using SYBR green displacement strategy and another one using a fluorescently-labelled complementary strand. Only SYBR green displacement method displayed some response at isocarbophos concentrations higher than 100 µM. None of the strategies showed any phorate recognition by the aptamers. Finally, in contrast to what was stated in the literature, we concluded that neither the SS2-55 aptamer nor its SS24-35 variant bind either isocarbophos or phorate in the submicromolar range.
(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.
Twitter: #AptaOx20V, #Belleperche
Micaela C Belleperche 1, Maureen McKeague 1,2
1 Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, Quebec, H3A 0B8, Canada
2 Department of Pharmacology and Therapeutics, McGill University, 3655 Prom. Sir-William-Osler, Montreal, Quebec, H3G 1Y6, Canada
Real-time detection of DNA damage and stress within a living cell would permit precise tracking of the effects of environmental and chemical stressors, with applications for drug testing and biosynthesis. Existing methods are invasive or cannot detect damage in real-time. Fluorescent RNA aptamers bind to a GFP-inspired fluorophore and cause a dramatic increase in fluorescence. When fused with a second target-binding aptamer, these constructs function as logic gates: upon target binding, the fluorophore-binding region is stabilized, creating a “turn-on” fluorescent switch. A Spinach aptamer construct has been expressed and shown to detect cyclic diGMP levels intracellularly, and the newer, higher-fluorescence Broccoli aptamer has been used to construct a similar switch. Here we present our work with designing fluorescent RNA aptamer switches for detection of DNA damage, using the Broccoli fluorescent aptamer and previously selected aptamers for DNA damage-related biomarkers. This includes design and optimization of the fused aptamer for the desired switching behaviour, and screening for sensitivity and selectivity with an eye to intracellular applications.
Twitter: #AptaOx20V, #ZChurcher
Zachary R. Churcher 1, Devid M. Garev 1, Aron A. Shoara 1, Richard A. Manderville 2, Philip E. Johnson 1
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. We are also investigating where the binding site of the ligand on the aptamer is located. Our 2D NOESY data shows NOEs from the methyl group of the ochratoxin-A to bases in the G-tetrads away from the tails of the aptamer. The binding of ochratoxin-A with several mutants of the aptamer was studied using fluorescence spectroscopy. Point mutations at several locations outside of the G-tetrad core in the aptamer were studied to see their influence on binding. These mutations generally reduced or prevented the binding of the ligand to the aptamer, meaning these bases are either required for binding or required for proper folding of the aptamer.
Twitter: #AptaOx20V, #SCunningham
Stephen Cunningham1,2, Lokesh Joshi1
1Advanced Glycoscience Research Cluster (AGRC), Biomedical Sciences, National University of Ireland Galway, Galway, Ireland. 2Centre for Research in Medical Devices (CÚRAM), National University of Ireland Galway, Galway, Ireland
Simple or complex carbohydrates – known as Glycans – play key roles as recognition determinants and modulators of wide ranging physiological and pathological processes. Their direct engagement and control of such biological processes presents them as ideal candidates for greater exploitation and utilisation across biotechnological, diagnostic and therapeutic fields. Though the roles of lectin-glycan interaction has long been established and studied, there remains a great need for molecular recognition molecules, which interact and bind glycans with high specificity and affinity. We present DNA aptamers, which have been evolved to selectively bind and discriminate members of the Lewis antigen system (Lea, Leb, Lex and Ley). Focusing initially on the Lewis B (Leb), Fuca1-2Galb1-3(Fuca1-4)GlcNAcb1-R, we report exclusive specificity, showing direct comparison with established anti-Leb commercial monoclonal antibodies. Favourable specificity and affinity of the top ranked aptamers after 12 rounds of evolution was observed (plate assay format). In vitro detection and quantification of cell surface presented Leb performed by competitive enzyme linked oligonucleotide assay (ELONA) and real-time quantitative PCR (qPCR) have been standardised. Currently, aptamers against LeB are undergoing assessment for the competitive inhibition and displacement of Helicobacter pylori, the main pathogenic bacterium involved in chronic gastritis and peptic ulcer and a class 1 carcinogen in gastric cancer. H. pylori interacts and binds through adhesins, with the principal one of note being the blood group antigen-binding adhesion (BabA) which binds to the Leb antigen present on red blood cells and gastrointestinal mucosa epithelial cells. Leading to Type IV secretion system (TFSS)-dependent host cell signalling leading to inflammation, intestinal metaplasia development, and associated precancerous transformation.
Twitter: #AptaOx20V, #FAEbanks
Fiona A Ebanks, Dr. Maria C DeRosa
Department of Chemistry, Carleton University, Ottawa, Ontario K1S 5B6
An aptamer-based colorimetric assay was developed for the detection of aflatoxin B1 (AFB1) a carcinogenic mycotoxin that is a persistent agricultural contaminant. In this study a solution-based aptamer-gold mediated assay was performed. This utilized the adsorption-desorption approach in which the aptamer is adsorbed unto the surface of the gold nanoparticle. Upon the addition of AFB1, the aptamer interacts with AFB1 leaving the surface of the gold nanoparticle. When of NaCl is added, aggregation of the gold nanoparticles is induced and there is a colour change from red to blue. Using this method, a limit of detection of 4 nM was achieved for AFB1.
Twitter: #AptaOx20V, #SHirka
Serhii Hirka 1, Donatien De Rochambeau 1, Daniel Saliba 1, Violeta Toader 1, Michael Dore 1, Hanadi Sleiman 1, Maureen McKeague 1, 2
1 Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, Quebec, H3A 0B8, Canada
2 Department of Pharmacology and Therapeutics, McGill University, 3655 Prom. Sir-William-Osler, Montreal, Quebec, H3G 1Y6, Canada
Protein with hydrophobic domains and hydrophobic small molecules form an array of biomolecules found in diverse environments, in which they may function as structural elements or biomarkers of various conditions. Levels of those biomarkers can be indicative of certain pathologies. Yet, their hydrophobicity makes them elusive targets for hydrophilic, negatively charged aptamers. Limited chemical functionality of natural nucleic acids is one of the major factors that hampers their development and makes the task of evolving a canonical nucleobase aptamer to hydrophobic motifs rather difficult. We present two approaches – direct synthesis library with subsequent screening and magnetic beads click-SELEX to impart hydrophobic functionality onto the aptamers and evolve new modified aptamers to hydrophobic targets. We show that both these approaches are useful strategies to improve binding of aptamers and allow to screen and select for aptamers with greater specificity and affinity.
Twitter: #AptaOx20V, #AAShoara
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. We use size-exclusion chromatography, fluorescence spectrophotometry, and UV thermal melts to show that the quinine-bound oligos form a larger assembly of aptachain units than in the absence of ligand. When split into two overlapping DNA strands, the aptamer remains functional. Size-exclusion chromatography shows that quinine-bound oligos form a larger assembly of aptamer units than in the absence of ligand. Finally, the oligomer forming ability of the aptachain oligos in a biosensor application for quinine that brings gold nanoparticles closer together resulting in a shift in plasmonic resonance to a longer wavelength and an observed colour shift. We propose that splitting aptamers into overlapping strands that form oligomers in the presence of a ligand will generally be applicable to aptamers and prove useful in a variety of biotechnology applications.
Twitter: #AptaOx20V, #SSlavkovic
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. Adding a full turn of the B-form helix between two binding sites results in an ATP-binding aptamer variant that binds both adenosine and ATP in an independent manner.
Twitter: #AptaOx20V, #MRequena
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. To demonstrate the utility of this method, the epidermal growth factor receptor (EGFR)-binding antagonistic aptamer E07 was immobilized on magnetic beads and used to isolate a purified population of EGFR(+) cells from a mixture of EGFR(-) and EGFR(+) cells. An antisense oligonucleotide antidote complementary to a region of E07 reverses aptamer binding thereby simultaneously detaching the cells and restoring receptor function. Quantitative western blot analysis of cell lysates after E07-FACS showed no statistically significant difference in EGFR autophosphorylation following incubation with epidermal growth factor compared to unsorted cells. We also used E07-MACS to recover labeled EGFR(+) cells from whole blood added at concentrations equivalent to 5% the white blood cell count. These results suggest aptamers and reversal antidotes can be used to isolate rare cell types in their native state for a wide variety of sensitive applications such as CAR-T cell therapy or mechanistic receptor signaling studies.
Twitter: #AptaOx20V, #HSPereira
Higor Sette Pereira1, Ananda Pereira Aguilar1, Alexandre Haruo Miyamoto Vieira1, Maria Eduarda Almeida Pinto1, Lívia Reis Valente1, Maria Clara Starling2, Camila Costa de Amorim2, Andréa de Oliveira Barros Ribon1, Tiago Antônio de Oliveira Mendes1
1Department of Biochemistry and Molecular Biology, Universidade Federal de Viçosa, Viçosa, Brazil
2Department of Sanitary Engineering, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
Excessive release of non-metabolized antibiotics or their residues in the environment has raised concern since it facilitates the development of resistance in bacteria. Thereafter, develop strategies to detect those antibiotics and their resistance factors that have been thrown into the environment and human samples such as blood is gaining importance. Aptamer-based sensors could be coupled to a wide diversity of reporter molecules without modifying their activity. So, it’s become an attractive path to detect environmental contaminants by linking aptamers with colorimetric reporters. In this work, we aim to build a DNA-based gold-nanoparticle (AuNPs) aptasensors to detect ampicillin, kanamycin, and related resistance factors expressed by blaTEM, blaCTX, and blaSHV genes. The aptamers Ky2 and AMP4 that recognize kanamycin and ampicillin, respectively, were recovery on literature. Aptamers against resistance factors are being selected via nitrocellulose membrane SELEX immobilizing the enzymes codified by target genes. We determine DNA amounts that prevent aggregation of gold-nanoparticle in NaCl salt solution. Antibiotic detections were performed by adding 2 nM AuNPs, 50 mM NaCl and 200 nM of aptamer with different antibiotic concentrations. The AuNPs response were quantified by spectrophotometry measuring absorbance at 620 and 520 nm. We noted that ampicillin aggregates at a final concentration of 50 mg.mL-1 and kanamycin in 0.06 μg.mL-1. Compared with other colorimetric assays, our findings suggest an excellent sensitivity in kanamycin detection. We are already improving limit detection of ampicillin system and selecting aptamers probes that could recognize those antibiotic resistance factors in natural effluents and human samples. Our aptamer-based AuNP is a promising test due to its reliability in detect antibiotics and to be cheap, easy-to-use and fast approach.
Twitter: #AptaOx20V, #JDOVilla
Ospina-Villa JD, Sánchez M
Instituto Colombiano de Medicina Tropical. Cra 43A #52 Sur – 99, Sabaneta, Antioquia, Colombia
Aptamers are a great alternative that have emerged in recent decades, to obtain novel therapeutic tools in different areas such as; infectious diseases, chronic and degenerative diseases, among others. Our research group focuses specifically on the application of the potential diagnostic uses of aptamers in different parasitosis that affect human health. In Colombia there are a great variety of parasitic diseases of public health interest, one of them is leishmaniasis caused by protozoal parasites of the genus Leishmania sp transmitted by the bite of the female phlebotomies sand fly. They can cause skin, mucous or visceral lesions. This disease mainly affects patients who live in rural areas without access to adequate diagnosis and / or treatment and produces chronic disability and even death. Therefore, as an alternative to conventional methods that use antibodies that are expensive and labile, our group intends to develop a diagnostic test to detect specific biomarkers of mucosal leishmaniasis caused by Leishmania panamensis. In our laboratory, we have previously identified through immunoproteomic tests with total protein extracts and sera from infected patients; four specific proteins for mucous leishmaniasis, which are not found in patients with Trypanosoma cruzi, and there are not in healthy patients. These proteins have been adequately characterized by means of mass spectrometry and emerge as potential biomarkers for the diagnosis of mucosal leishmaniasis in a precise and agile way. This work describes the methodology for the identification of specific aptamers against four potential biomarkers of mucosal leishmaniasis which could allow in the near future to develop a locally manufactured diagnostic test which meets the objective of being accessible to the vulnerable population.
Twitter: #AptaOx20V, #JDTGaribay
Juan C. Gutiérrez Santana 1, Nancy J Ruiz-Pérez 2, Luis M Álvarez-Salas 3 and Julia D Toscano-Garibay 2
1 Laboratorio de Bacteriología Experimental, Instituto Nacional Pediatría, Insurgentes Sur 3700, Letra C, Coyoacán, Mexico
2 Dirección de Investigación, Hospital Juárez de México, Av. Politécnico Nacional 5160,Magdalena de las Salinas, Gustavo A. Madero, Mexico
3 Laboratorio de Terapia Génica, Departamento de genética y Biología Molecular, CINVESTAV-IPN, Av. Instituto Politécnico Nacional 2508, San Pedro Zacatenco, Gustavo A. Madero, Mexico
Sporadic breast cancer (SBC) is one of the most frequent gynaecological pathologies worldwide, its aetiology has multifactorial nature, including alterations on the apoptosis regulatory pathways. Bcl-2 interacting killer (BIK) is a proapoptotic protein that has been detected as overexpressed on stage II and III biopsies of Mexican patients and has been proposed as an important biomarker for SBC. BIK belongs to the Bcl-2 associated-protein family and is classified as a BH3-only member along with Noxa and Puma; it is attached to the endoplasmic reticulum membrane, regulating the release of calcium and interacting with Bax to initiate the apoptotic cascade. In here, we used a 23-aa peptide of the N-terminal of BIK as target for a SELEX procedure by fixating it into PVDF, incubating the membranes with a N20 library and mechanically separating unbound molecules. After ten selection cycles, sequence C10.3 showed the most affinity. Then, we synthesized a biotinylated version of C10.3-RNA and used avidin-HRP to reveal BIK-aptamer interactions on cell extracts. Western-blots of MCF7 cells showed a single band with remarkably less background when using C10.3 as primary detection reagent as compared to two commercially available antibodies. Incubation after blotting was performed at room temperature and time was reduced from 4hrs to 30min. Additionally, we performed immunofluorescences on apoptotic tissues comparing the staining pattern of 5′-fluoresceinated-C10.3 to the one shown by the previous antibodies, C10.3 was able to render the same pattern both in localization and in intensity, probing its fitness to detect complete BIK in complex mixtures. Concluding remarks: C10.3 aptamer was easily adapted for use on immunodetection techniques by directly coupling it to signalling molecules (biotin-avidin-HRP and Fluorescein) and hence it is a suitable candidate for the design of diagnosis tests for SBC.