Thank you for considering presenting your work as a poster at Aptamers 2021 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 09th April 2021. Late posters may not be included in the symposium programme. Please DO NOT send your poster files by email.
Please ensure you send us the very final version of your poster (as well as your poster abstract), as once published, it cannot be replaced.
Naming your poster files: Name your poster files as follows: <your surname>-APT21V-Poster.pdf | <your surname>-APT21V-Poster.png | <your surname>-APT21V-Poster.jpg, etc. For example, for David Jones, name your file as Jones-APT21V-Poster.pdf. DO NOT name your poster files as, e.g., Oxford-poster, Aptamers2021v, Oxford-aptamers-poster. Such files will be automatically rejected.
Poster presenters will also have the (optional) opportunity to introduce their poster in a short, 60 second presentation, using a single PowerPoint slide, during their allocated break each day. Please check the website for your poster presentation day. If you have any questions, please get in touch with your session coordinator- Dr Maureen McKeague (firstname.lastname@example.org) for presentations on 14th April, or Dr Sarah Shigdar (email@example.com) for presentations on 15th April.
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.
(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.
The Aggregation and Long-Term Preservation of Aptamer Research in a Publicly Available Database
Hailey C Ferrell 1 and Ali Askari 1, Sumedha Kota 1, Kayla Goodman 1, Daniela Hernandez 1, Taylor Oliphant 1, Brandon Ta 3, Isaac Weislow 1, Gwendolyn M Stovall 1,2
1 Texas Institute for Discovery Education in Science Freshman Research Initiative, University of Texas at Austin, Austin, Texas 78712, United States
2 Texas Institute for Discovery Education in Science High School Research Initiative, The University of Texas at Austin, Austin, Texas 78712, United States
Aptamer research has been growing at an unprecedented rate and has piqued the interest of many researchers around the world; therefore, a comprehensive publicly available aptamer dataset is needed to standardize aptamer reporting, aggregate data, and expand on current research in the aptamer field (Wang and Tao et al, 2019). There have been several attempts to solve this problem via an aptamer database; however, the progress of most of these databases has been hindered due to various limitations, leaving the majority nonfunctional (Darmostuk, 2015). Our approach aims to create a continuously updated dataset where aptamer information is preserved while offering an interactive and searchable experience in the form of a publicly available database. This project seeks to obtain all the published aptamers since 1990. The information stored about each aptamer includes the year of publication, journal DOI, citation, genetic material, name of aptamer, target, sequence, affinity, binding/selection buffer, the molecular weight of the target, possible application, pool type, post-SELEX modifications to the aptamer, additional relevant information, and serial number. The information is routinely peer-reviewed to ensure accuracy and consistency across all entries, and as of March 30th, 2021, there are 1027 completed peer-reviewed entries. Keeping in mind the limitations of previous databases, we have implemented sustaining mechanisms that include submission forms to submit published aptamers, data stored separately from the database platform (Caspio), training protocols, and a continuously growing team of researchers committed to updating the database. Upon the publication of this ongoing project, we will have created the largest known functional aptamer database in the world, thus achieving the aggregation and long-term preservation of aptamer research and knowledge as well as creating an easily accessible, user-friendly platform for this data.
An investigation of fluorescent probe design for the Ochratoxin A aptamer
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
Ochratoxin A (OTA) is a mycotoxin that can contaminate a variety of foodstuffs, including fruits, grains, and drinks such as wine and coffee. OTA concentration in food supplies is typically measured using time-consuming methods that require skilled personnel, such as HPLC, LC-MS, and ELISA assays. Aptamers, which are single-stranded nucleic acids, may be useful in the development of rapid biosensors to OTA. Since the first Ochratoxin A DNA aptamer was developed in 2008, many detection methods have been developed for OTA. A large proportion of these methods involve strand displacement, in which a DNA “probe” sequence in a duplex with the aptamer is displaced upon aptamer binding to target. In previously proposed detection methods, conditions necessary for probe displacement vary. Some methods require that the OTA be added before the probe, rather than after, while others require high heat or additional reaction elements. This is likely in part due to the difference in design of the probe strand used in each application. Using a simple fluorescent system, we performed a systematic investigation of optimal probe design for the OTA aptamer. Here we present an analysis of the optimal probe characteristics, including length, placement, and mismatches within the sequence.
Using NMR to investigate the interaction of levamisole with the cocaine-binding aptamer
Zachary R. Churcher, Aron A. Shoara, Sladjana Slavkovic, Philip E. Johnson
Department of Chemistry, York University, Toronto, ON, Canada.
Levamisole is an anti-parasitic worm medicine that is no longer prescribed in North America to humans but is often prescribed to livestock. It can have several side effects including dizziness, headache, vomiting, and abdominal pain. Besides its intended purpose, levamisole is also often used as an adulterant in street cocaine samples. Approximately 90% of street cocaine samples contain an adulterant, while 65% of those samples contain levamisole. These adulterants can interfere with the ability of a scientist to accurately analyse a street cocaine sample due to signal suppression or overlap in electrochemical methods. The cocaine-binding aptamer is a 36-nucleotide aptamer consisting of three stems centred around a three-way junction containing a tandem A-G mismatch. The cocaine-binding aptamer was originally selected in 2000 by Stojanovic et. al. to find an aptamer that would bind to cocaine, but not the common cocaine metabolites benzoyl ecgonine and ecgonine methyl ester. Though it was selected to not bind to cocaine metabolites, the cocaine-binding aptamer has shown the ability to bind to a wide variety of ligands it was not selected for. Though off target ligand-binding is not uncommon, the cocaine-binding aptamer is able to bind to certain ligands such as quinine and amodiaquine tighter than it binds to cocaine. If the cocaine-binding aptamer shows a wide array of off-target ligand binding, could it interact with levamisole? To study the interaction of the cocaine-binding aptamer and levamisole two aptamer constructs were used. Initial ITC and UV-Vis spectroscopy studies did not show any interaction between the cocaine-binding aptamer and levamisole. NMR studies however showed the cocaine-binding aptamer binds to levamisole, albeit about 100x weaker than it does to cocaine. NMR spectroscopy was also used to investigate whether cocaine and levamisole share the same binding site, and how much of an effect pH has on the binding of levamisole.
Extracting Kinetic parameters of the cocaine-binding aptamer by Isothermal Titration Calorimetry
Nusaibah Dawood & Philip E. Johnson
Department of Chemistry, York University, Toronto, Ontario, Canada M3J1P3
The cocaine-binding aptamer is one of the most well-studied aptamers used in developing biosensors and aptamer-based technologies. There have been many studies conducted on the thermodynamics of binding for the cocaine-binding aptamer, however, one feature that has not been reported yet is the kinetics. By employing kinITC methods, it is possible to retrieve kinetic information from Isothermal Titration Calorimetry (ITC) experiments. Here, we compare the kinetics of two aptamer variants of the cocaine-binding aptamer, MN4 and MN19 binding to quinine at different temperatures. The secondary structure of the MN4 aptamer is pre-formed in the absence of quinine and is retained with ligand binding. The length of stem one in the cocaine-binding aptamer controls whether the structure-switching binding mechanism in the aptamer occurs or not. When the length is shortened to three base pairs, such as for MN19, the aptamer remains loosely folded in its unbound state and undergoes structural switching when bound to quinine. An unusual feature of the cocaine-binding aptamer is that it binds off-target ligands like quinine up to 30 folds tighter than what it was initially selected for, therefore all our experiments were performed using quinine as the ligand. Using ITC, we compare the binding kinetics of MN4 and MN19 to determine the activation energy of free binding since MN19 exhibits conformational changes upon ligand binding.
Aptamer selections for Islet Amyloid Polypeptide (IAPP)
Madison Ferguson, Maria DeRosa
Department of Chemistry, Carleton University, 1125 colonel By Drive, Ottawa, Ontario, K1S 5B6, Canada
Diabetes is one of the most predominate diseases across the world.1 Islet amyloid polypeptide (IAPP) is typically found in monomeric form within a heathy individual, although will form fibrils in diabetic patients.1 The nanostructural conformation changes taking place within this peptide and how these are influenced by inhibitors or activators is very key to understanding this system. We are interested in selecting aptamers specific for the monomeric form of this peptide but given its rapid aggregation kinetics, we first had to find conditions that would support the monomeric form. Previous studies on amyloid polypeptide (APP), an aggregating peptide implicated in Alzheimer’s, were tested against IAPP.1,2 We found that all APP aggregation inhibitors including ammonium hydroxide, hydrochloric acid, PBS solution pH 7.5, hexafluroisopropanol (HFIP), and sodium chloride all induced fibrilization of IAPP within one day and these fibrils grew in size for day two.1,2 Zinc chloride 2mM, on the other hand, showed inhibition of the aggregation events, with the onset of fibrilization not observed until day 6. Zinc(II) has been reported to interact with His18 preventing the fibrilization occurring from Ser20 – Ser 29, but cannot prevent or inhibit aggregation across a longer time period.3 This was then the basis of the conditions under which we performed the systematic evolution of ligands by exponential enrichment (SELEX). We will present our recent results from our SELEX experiment, where we noted that our enriched libraries can inhibit aggregation of the peptide for upwards of three weeks.
Investigating Interaction Mechanisms of Dopamine and Serotonin-Binding Aptamers
Yunus A Kaiyum, Zachary R Churcher, Aron A Shoara, Sladjana Slavkovic, Philip E Johnson
Department of Chemistry, York University, Toronto, Ontario, Canada M3J1P3
Aptamers represent a step forward in the development of robust and selective biosensors that are cost-effective and have high efficacy. Uncovering the mechanisms by which aptamers perform their functions can open an entire avenue of biosensors, diagnostic tools, and therapeutics. The dopamine and serotonin binding aptamers have shown strong and selective binding properties with their respective ligands. Despite the presence of species with similar structures, the short DNA strands undergo structural changes only when complexed to the ligands for which the aptamers were selected for. By investigating the specific structural mechanisms that allow the aptamers to bind their small molecule ligands, the data can then be used to fine-tune the aptamers to optimize binding properties and increase biocompatibility. Using a combination of 1D and 2D NMR studies, as well as ITC and fluorescence experiments, we have begun to characterize the behaviours of these aptamers free and ligand-bound. Imino proton resonances of the aptamer were assigned using a 2D NOESY, and those resonances were observed during a titration with their respective ligands to determine which imino proton resonances changed with ligand binding.
Controlling Gene Expression With DNA-Integrated Aptamers
Michael Y. Malca 1, Maureen H. McKeague 1,2
1 Department of Chemistry, McGill University, Montreal, Quebec, H3A 0B8, Canada
2 Depatment of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, H3G 1Y6, Canada
Controlling gene expression has become a powerful metabolic engineering tool for optimizing cell-based systems. Aptazyme-based gene control has emerged as a means towards better controlling gene expression output. While being powerful tools, these constructs typically necessitate mM concentrations of aptamer ligand and are often limited by the range of expressional output they can control. Indeed, high-affinity aptamers reported can often have nM dissociation constants but require mM concentration to see a significant switching effect in vivo. This discrepancy can be rationalized as being due to the fact that one gene can generate thousands of mRNA transcripts that are substrates to target to achieve a significant change in expression profile. It is well understood that DNA architecture largely influences gene expression output. A notable advancement in this field was the discovery of how G-Quadruplex forming sequences (G4) near or within promoters can affect the expression output of their downstream gene. For instance, it’s been shown that the G4 in the cMYC promoter can control up to 90% of MYC protein expression when stabilized in its folded state. This suggests that controlling gene expression at the DNA-level control could provide a lot more sensitivity and larger dynamic range when compared to controlling its mRNA counterpart. Given this, we investigate the applicability of DNA-level control by integrating G4 sequences within an SV40 promoter that regulates luciferase protein expression. Particularly, we focus on how integrating different synthetic G4s can influence the basal expressional output, dynamic range, and expression output in response to different concentrations of various G4 ligands. We compare these G4 systems to previously reported high activity aptazymes to determine their practical use for in vivo studies. In the future, we plan to replace these G4 structure with high-stability DNA aptamers to achieve DNA-level control of gene expression.
Developing a ligand displacement assay using the fluorescence of antimalarial drugs in a competitive affinity mechanism to the cocaine-binding aptamer
Aron A. Shoara, Logan W. Donaldson, Philip E. Johnson
Department of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, ON M3J 1P3, CANADA
Two main properties are often viewed in aptamer development studies: signal transduction of aptamer sensors in the ligand-induced folding mechanism and the competitive ligand displacement binding mechanism. The structure-switching binding mechanism of an aptamer is more favourable in biosensing technology than the aptamer’s binding affinity. The ligand displacement binding mechanism can be utilized to determine sequence selectivity of aptamers for ligands with similar chemical structures. We used the intrinsic fluorescence of chloroquine and cocaine in both steady state and polarized fluorescence analyses to demonstrate a competitive binding mechanism with the cocaine-binding DNA aptamer. Using the intrinsic fluorescence of chloroquine and cocaine, we have observed quenching of ligand fluorescence upon binding of the aptamer. We have also observed an increase in the fluorescence anisotropy of the ligands when titrated with the aptamer. Quantification of the fluorescence quenching and anisotropy provide a sensitive method to measure the displacement constant with great accuracy and precision. Our steady-state quenching, coupled with the fluorescence anisotropy results, shows that chloroquine binds to the cocaine-binding aptamer with higher affinity than cocaine, and chloroquine is able to displace cocaine from aptamer•cocaine complex. These results verify the effectiveness of our fluorescence-based ligand displacement assay in aptamer development studies.
Binding of the antimalarial agent artemisinin to the cocaine-binding aptamer
Sladjana Slavkovic, Zach Churcher, A. Aron Shoara and Philip E Johnson
Department of Chemistry, York University, Toronto, ON, Canada
Aptamers have generated great interest as biosensors as their selection process allows them to bind a wide variety of ligands often with high affinity and specificity. An aptamer that is an exception to this rule is the cocaine-binding aptamer as one of the unique features of this aptamer is its ligand-binding promiscuity. Despite being selected for cocaine, the cocaine-binding aptamer binds quinine as well as quinine-based antimalarial compounds such as amodiaquine, chloroquine and mefloquine with much higher affinity than cocaine. Previously, we showed that the quinine-based antimalarial compounds bind to the cocaine-binding aptamer to both sites under high salt conditions. Using isothermal titration calorimetry, we show that two molecules of artemisinin bind to the cocaine-binding aptamer tighter than quinine under the same set of conditions. NMR spectroscopy and UV melt experiments were used to confirm the binding. Competition experiments indicate that artemisinin does not compete for the same binding site with quinine. In this work, we also explore the interaction of artemisinin with different DNA structures to see if artemisinin is generic DNA binder or specific for the cocaine-binding aptamer.
3D printing and retooling simple devices for aptamer-target detection
Nancy J Ruiz-Pérez, Jaime Sánchez-Navarrete and Julia D Toscano-Garibay
Dirección de Investigación, Hospital Juárez de México, Magdalena de las Salinas, Gustavo A. Madero, Mexico
Additive manufacturing (AM) or 3D printing originated from 1980s rapid prototyping and is currently used for the generation of three-dimensional objects through computer-aid design and layer-by-layer heated material deposition (extrusion). AM is widely used for manufacturing temporal industrial prototypes but it is also utilized on small-scale production and tooling applications, with the advantage of creating almost unlimited shapes for personalized equipment and devices. Although there are other principles for 3D printing, extrusion is by far the commonest, cheapest and readily available technology nowadays. In here, we designed and 3D-printed a vacuum-filtering box and retooled a xy-plataform to easily visualize aptamer-target complexes. We used a biotinylated version of aptamer IGA3 isolated for insuline recognition for testing the device. Vacuum was provided by adapting the box to a dental thermoforming apparatus allowing rapid separation of aptamer-target complexes and free aptamer on a double membrane array. After filtration, retained aptamer was incubated with streptavidin-conjugated HRP and revealed using ECL reagents. Signal was detected with a CMOS camera attached to the xy-plataform for surface scanning. The use of AM for designing and retooling lab equipment is an accessible alternative to reduce research costs, ultimately leading to the fabrication of customized equipment for local health applications.
Top Cancer Biomarkers for Future Aptamer Selections
Brittany Uebbing1, Doru Gucer1, Sheetal Mohanty1, Nicole Nnadi1, Nhu Nguyen1, Gwendolyn M Stovall1,2
1Texas Institute for Discovery Education in Science Freshman Research Initiative, University of Texas at Austin, Austin, Texas 78712, United States
2Texas Institute for Discovery Education in Science High School Research Initiative, The University of Texas at Austin, Austin, Texas 78712, United States
Choosing a biomarker with a high potential for future clinical translation is an important first step during aptamer selections with diagnostic applications. Although the in-vitro nature of aptamer selection affords a host of benefits when compared to their in-vivo counterparts, selecting for aptamers can still be a time-consuming and resource intensive process. Cancer diagnostics are often dependent on the reliable detection of biomarkers with low concentrations. Due to their high specificity and sensitivity, aptamers are well suited for functionalization in prognostic and diagnostic tools for cancer biomarkers. For this project a three tier method will be employed to create a list of the most promising cancer biomarkers for future aptamer selections. The initial tier will involve mass biomarker collection from cancer review and primary research articles within the past 2 years. The second tier will identify and remove biomarkers that already have aptamers selected against them. Lastly, the final tier will utilize a combination of sensitivity, specificity, and predictive values as well as receiver operating characteristic (ROC) curves to generate a final list of biomarkers. Due to their small size and high variation in potential targets, aptamers are well suited for multiplexing; thus recommendations will also be made for combining biomarkers into panels. Currently, 150 biomarker entries have been collected with the goal of reaching 1000 before the end of tier one. Ideally this biomarker list will be used by aptamer researchers globally to guide the next generation of aptamer selections against cancer biomarkers.
(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.
Analysing criteria affecting the functionality of GQ-based DNA aptazymes as colorimetric biosensors and development of quinine-binding aptazymes
Yasaman Ahmadi, Regina Soldo, Krista Rathammer, Laura Eibler, Ivan Barisic
Molecular Diagnostics, Center for Health and Bioresources, AIT Austrian Institute of Technology GmbH, Giefinggasse 4, 1210 Vienna, Austria
A DNA aptazyme consists of an aptamer domain and a DNAzyme module, in which the DNAzyme activity can be regulated by the aptamer-target interaction. The complex of G-quadruplex (GQ) and hemin mimics the catalytic activity of HRP enzyme and catalyses the peroxidase reaction of ABTS/H2O2 generating a colorimetric signal. The development of GQ-based aptazymes is of high interest as they can be used as label-free biosensors for the real-time detection of pathogens. Intrigued by this concept, we rationally designed ca. 200 GQ-based aptazyme candidates and evaluated the suitability of 14 aptamers targeting quinine, ATP, Protein A and Staphylococcus Enterotoxin B for this detection concept. As a result, six novel aptazymes were developed for the specific detection of quinine based on two quinine-binding aptamers. The rest of designed probes, however, hardly showed significant functionality. False-positive signal, due to the inherent activity of DNAzyme, is generally considered as the main obstacle for obtaining active aptazymes. Our results, however, showed that the increase in the reporter activity of the GQ/hemin complex in the presence of targets is a significant problem. Moreover, ELONA (Enzyme-Linked Oligonucleotide Assay) results showed that once conjugated to the GQ sequence or upon integration into the aptazyme probe, the target affinity of the integrated aptamer may be irreversibly reduced or even blocked. The lack of target-induced structure-switching functionality in the parent aptamer and the presence of GQ configuration in many SELEX-derived aptamers are among other factors, which limit designing functional GQ-based DNA aptazymes.
Cryptoaptamers: Covalent Bi-Modular Parallel and Antiparallel G-Quadruplex DNA Nanocostructs Reduce Viability of Patient Glioma Primary Cell Cultures
Anastasia A. Bizyaeva 1, Valeria A. Legatova 1, Nadezhda S. Samoylenkova 2, Alexander M. Arutyunyan 3, Vadim N. Tashlitsky 3, Dmitry Y. Usachev 2, Galina V. Pavlova 2,4,5 and Alexey M. Kopylov 1
1 Chemistry Department, Lomonosov Moscow State University, Russia
2 Burdenko Neurosurgical Center, Russia
3 Belozersky Research Institute of Physical Chemical Biology, Lomonosov Moscow State University, Russia
4 Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, Russia
5 Department of Medical Genetics, Sechenov First Moscow State Medical University, Russia.
G-quadruplex oligonucleotides (GQs) exhibit specific anti-proliferative activity in human cancer cell lines, and they can selectively inhibit the viability and/or proliferation of cancer cell lines vs. noncancer ones. Because the exact target for every GQ is not known yet, GQ could be coined as cryptoaptamer. GQ inhibiting ability could be translated into a cancer treatment for glioblastoma multiform (GBM), which currently has a poor prognosis and low-efficiency therapeutic treatments. A novel bi-modular GQ, bi-(AID-1-T), a twin of the previously described three-quartet AID-1-T, was designed and studied in terms of both its structure and function. Structural methods included circular dichroism spectroscopy and size-exclusion HPLC; and MTT assay was applied as a functional method. A covalent conjugation of two AID-1-T modules via three thymidine link, TTT, did not interfere with its initial GQ structure and presumably led to another dimerization way. A comparison of bi-(AID-1-T) with its mono-modular AID-1-T and its derivative bi-(AID-1-C), mono-modular two-quartet HD1, and bi-modular bi-HD1, as well as conventional two-quartet AS1411, was made. Among the GQs studied, bi-(AID-1-T) had the highest anti-proliferative activity − for the neural cancer cell line U87, while not affecting the control cell line, human embryonic fibroblasts. GQs, for the first time, were tested on several primary glioma cultures from patient surgical samples. MTT assay data on the cell viability of six patient primary glioma cell cultures after bi-GQ treatment demonstrated that the sensitivity of the patient primary glioma cultures toward GQs varied, with an apparent IC50 of less than 1 µM for bi-(AID-1-T) toward the most sensitive G11 cell culture (glioma, Grade III). This research was funded by the Ministry of Education and Science of Russia Foundation, grant number № 075-15-2020-809 (13.1902.21.0030) and by RFBR according to the research project № 18-29-01047.
Chemically-modified aptamers generation to recognize relevant bacterial antigens and antibiotic resistance proteins
Monika Czarnecka 1, Magda Puchała 1, Joanna Guzdek 1, Marta Radzińska 1, David Carter 1, Zbigniew Darżynkiewicz 1, Kamilla Sołtys 1, Aleksandra Adamowicz-Skrzypkowska 1, Yasaman Ahmadi 2, Ivan Barisic 2, Agnieszka Sok-Grochowska 1
1 Pure Biologics S.A., Research & Development Department, Wrocław, Poland
2 AIT Austrian Institute of Technology GmbH, Vienna, Austria
Targeted treatment of bacterial infectious diseases and the prevention of the spread of multi-drug resistant pathogens remains a clinical challenge. Our goal was to develop a panel of aptamers with chemically modified side chains designed to specifically recognize and bind clinically relevant bacterial surface markers and antibiotic-resistance enzymes. The aptamer selection campaigns were carried out using Pure Biologics’ patent-covered PureApta™ proprietary selection platform based on the SELEX procedure. These selections resulted in the identification of a dozen of highly specific aptamers against five antigens, including β-lactamase enzymes: KPC-2, OXA-48, and IMP-1, responsible for the most prominent antibiotic resistance mechanisms in opportunistic bacteria, and against protein G and protein A, surface proteins on Streptococcal and Staphylococcal bacterial cells, respectively. Aptamers were further optimized for their length relative to the strength of binding to their molecular targets. The strongest and the shortest aptamers were identified for the proteins: OXA-48, KPC-2 and protein A, where at the length of 46-, 63- and 61- nucleotides respectively, their Kd parameters were in sub-nanomolar range. Additionally, for protein A aptamer, a bacterial cells binding assay was successfully performed, thus demonstrating its potential utility in the development of new tools in diagnostic tests.
ITC analysis of the arginine binding DNA aptamer
Hamish Dunham 1, Jennifer Soundy 2, Janet Pitman 1
1 School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand
2 AuramerBio 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 interaction sites for aptamer-target binding, as well as conferring smaller mass and energetic changes and usually smaller structural changes upon aptamer-ligand binding. This in turn makes observation of small-molecule aptamer interactions difficult, thus requiring methods sensitive enough for accurate detection of ligand binding. Isothermal titration calorimetry (ITC) is widely regarded as the gold standard to discern molecular kinetic data. Free amino acids deservedly fall into the category of small molecule targets, the argininamide-binding DNA aptamer, arg12-28, developed by Harada & Frankel (1995) was originally selected for binding arginine with a Kd of 2.5 mM. However, this aptamer was demonstrated to bind argininamide ~20-fold more tightly than arginine. This is due to the absence of a carboxylate group on argininamide compared to arginine. Consequently, this aptamer is now widely regarded as an argininamide aptamer and has been well investigated as a model system while further analysis of its affinity for arginine is paid less attention. Given the physiological and clinical implications of Arginine, which is a conditionally essential amino acid, where nutritional supplementation may be necessitated by health condition or life cycle status, the ability to detect and monitor arginine levels in a biological matrix is of practical utility. Here I present ITC analysis of the arg12-28 aptamer binding to arginine which I have not found in the literature. Interestingly, increasing ionic strength ablated the binding interaction decreasing the Kd, ΔH & ΔS, this has implications for the structural differences between arginine and argininamide, and may have justification given how this aptamer was originally selected. This information may provide useful insights for the future development of an effective aptamer system for arginine detection.
Synthesis of a vancomycin-modified uridine to be included in aptamers
Chiara Figazzolo 1,2,3, Fabienne Levi-Acobas 1, Marcel Hollenstein 1
1 Institut Pasteur, department of Structural Biology and Chemistry, Laboratory for Bioorganic Chemistry of Nucleic Acids, CNRS UMR 3523, 28 rue du Docteur Roux, 75015 Paris, France.
2 Université de Paris, 12 rue de l’École de Médecine, 75006 Paris, France.
3 Centre de Recherches Interdisciplinaires CRI, 8 Rue du Charles V, 75004 Paris, France
Antibiotic resistance (ABR) is one of the biggest public health challenges of our time and occurs when bacteria develop mechanisms that protect them from the effects of antibiotics. In this work, we explore the first steps towards combatting ABR by attaching antibiotics on the scaffolds of aptamers. The aim of this work was to synthesize a novel deoxyuridine nucleotide bearing a vancomycin moiety and assess its capability to be incorporated into DNA via enzymatic synthesis, and hence proving its potential application in SELEX experiments. Vancomycin’s resistance has been widely observed in S. aureus which can alter a peptidoglycan terminus, resulting in reduced vancomycin binding and failure to prevent bacterial wall synthesis. The identification of a vancomycin-bearing aptamer would potentially use the aptamer binding properties to mechanically force the vancomycin in the binding site and allow its function despite the presence of the aforementioned mutation. Initially, a suitably protected alkyne-modified deoxy-uridine was synthesized, triphosphorylated and then reacted with a vancomycin modified with an azide group. Subsequently, a biochemical characterization was performed to assess the substrate acceptance of the nucleotide by polymerases. The PCR showed that the nucleotide is accepted by the polymerases Deep Vent and Vent exo–. The primer extension (PEX) demonstrated the formation of full-length products with the polymerases Phusion, Q5, Therminator, Deep Vent and Vent exo–. PEX also showed the possibility to incorporate a series of vancomycin modified nucleotides in a row. Finally, we demonstrated that a template containing a vancomycin-modified uridine can be converted into unmodified DNA without mutations or misincorporations which is an important prerequisite for SELEX experiments. In conclusion, this work proves the possibility to enzymatically synthesize oligonucleotides containing a vancomycin-modified uridine and use them in SELEX experiments.
Towards the discovery of novel aptamerogenic biomarkers by on-blot SELEX
Tjaša Legen 1, Günter Mayer 1,2
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 using the resultant clickmers as staining reagents. Furthermore, NGS analysis of the libraries obtained by the selection process revealed individual clickmers specific for different proteins. Subsequently, protein targets of individual clickmers were assigned by LC-MS/MS analysis and obtained candidates will thus be confirmed by siRNA knockdown of specific gene in the next step. According to this study, clickmers may serve as alternative to antibodies as detection reagent in western blot analysis and may help to overcome the reproducibility crisis associated with antibody usage.
Development of molecular probes for surface markers on EBV-positive nasopharyngeal carcinoma cells using aptamer selected by combination of Cell-SELEX and in-vivo-SELEX
Alex Yongshu Li 1, Umar Mubarak Ishaq 2, Qian Wu1, Chun Kit Kwok 2, Julian Alexander Tanner 3, Kwok Wai Lo 1, George SW Tsao 3, Anna CM Tsang 1
1 The Chinese University of Hong Kong, Department of Anatomical and Cellular Pathology, Hong Kong, China
2 City University of Hong Kong, Department of Chemistry, Hong Kong, China
3 University of Hong Kong, School of Biomedical Sciences, Hong Kong, China
Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated cancer prevalent in Southeast Asia and Southern China. Our recent genomic sequencing revealed few druggable oncogenic mutations in this cancer, suggesting personalized and targeted therapies cannot be generally applicable to most NPC patients. Systemic chemotherapy remains as the major treatment modality in the management of primary, recurrent and metastatic NPC. Therefore, improvement in selective drug delivery is imperative for enhancing efficacy and lowering toxicity in treating NPC. Aptamers are single-stranded oligonucleotides with 3D folding and can bind to specific targets. They are known as ‘chemical antibody’ which can deliver toxic cargos against cell-surface proteins on tumor cells. The unique presence of EBV infection in NPC has enabled us to perform Cell-SELEX and In vivo-SELEX to enrich aptamers which can preferentially bind to the surface molecules on EBV-positive NPC cells, but not the EBV-negative counterparts. We have successfully discovered Apt-1194 binding to EBV-positive NPC cells. Importantly, this aptamer has shown preferential accumulation in EBV-positive NPC tumors in mice models. The binding partner of Apt-1194 was identified to be metadherin (MDTH) which may represent novel surface markers in EBV-associated NPC cells. To apply the newly discovered aptamer as a drug-delivery vehicle, several cytidines of Apt-1194 have been exchanged to gemcitabine which is a commonly used chemodrug in NPC treatment. The gemcitabine-incorporated Apt-1194 (Gi-Apt1194) displayed the same specificity and affinity to EBV-positive NPC tumor cells comparing with the drug-free Apt-1194. The cell toxicity of Gi-Apt-1194 in different NPC cell lines and its therapeutic effect in different NPC mouse models will be examined. This study indicates the feasibility of selecting tumor-targeting aptamers in EBV-associated NPC cells by a combination of Cell-SELEX and In vivo-SELEX.
Demonstrating aptasensing in whole saliva using the tobramycin aptamer
Yasmin Liu 1,2, Rebecca SJ Palmer 1,2, Tessa M Pilkington 1,2, Kelly A Campen 1,2, Jennifer P Soundy 1,2
1 AuramerBio Limited, Wellington, New Zealand
2 School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand
Aptasensing for point of care detection in complex biological fluids, such as saliva, has proved to be a significant challenge for the biosensing field. Here we have developed a disposable electrochemical aptasensor which is capable of detecting small molecule analytes in small volumes of untreated human saliva. The tobramycin DNA aptamer reported by Rowe et al 2010 was selected as a model system for this proof of concept study. Briefly, the dual labelled methylene blue and thiol aptamer was tethered onto a disposable laser ablated gold electrode followed by 6-mercapto-1-hexanol backfilling to increase the sensor’s antifouling properties. Introduction of the tobramycin target to the functionalised electrode induces a structure switch in the aptamer which was monitored by square wave voltammetry, and the results are reported as the signal change between no target and target. Three different matrices were studied for their impact on the tobramycin sensing capability of the electrochemical sensor – tobramycin binding buffer, simulated saliva buffer (SSB), and whole human saliva collected by the passive drool method. Surprisingly, the biosensor signal change in response to tobramycin was enhanced when measurements were performed in SSB compared to the reported tobramycin binding buffer, however, detection in whole saliva was less sensitive than in SSB. A preliminary study was performed to examine different saliva processing protocols and their influence on the biosensor signal. These results begin to demonstrate that 1) reliable sensing in complex unprocessed biological matrices is achievable using electrochemical aptasensors; and 2) the performance of structure switching-based aptasensors can be significantly influenced by the properties of the matrices.
Engineering and functional characterization of anti-PSMAxCD3 bispecific aptamers
Anna Miodek, Anaïs Boutserin, Cécile Bauche, Renaud Vaillant and Frédéric Mourlane
Ixaka, Villejuif Bio Park, 1 Mail du Professeur Georges Mathé, 94800 Villejuif, France
Bispecific therapies redirecting cytotoxic effector T cells to malignant cells by simultaneous binding to CD3, the signalling component of the T-cell receptor, and tumour-associated antigens have demonstrated striking activity in patients across different cancers (blinatumomab and catumaxomab). Currently over 80 bispecific antibody fragments/derivatives are under active clinical developments in oncology, half of which involves CD3 recognition. Ixaka has been developing CD3-specific aptamers as new targeting agents to specifically deliver in vivo anti-cancer therapeutics to T lymphocytes. These G-quadruplex-based binders with nanomolar affinities recognize epitopes on the CD3 receptor that are different from OKT3 and other T-cell activating antibodies. They exhibit remarkable cross specificity with human, mouse and cynomolgus isoforms and extended serum stability. In order to further explore the therapeutic potential of these leads, we engineered PSMAxCD3 bispecific aptamers to develop new anticancer agents able to recruit cytotoxic T cells to induce killing of prostate-specific membrane antigen (PSMA)-positive tumour cells. Hexynyl-modified anti-CD3 moieties carrying hexaethylene glycol linkers at their 5’ends were dimerized by click chemistry with the model anti-PSMA RNA A10 aptamer functionalized with an azide group and TEG-biotin/Cy5 tag at its 3’ end. By combining biophysical and on-cell binding methods, we demonstrated that dimerization did not alter the specificity and affinity of PSMA and CD3 monomers due to steric hindrance. The serum stability of the PSMAxCD3 heterodimers was not any different from that of individual monomers despite the HEG linker and triazole inter-nucleotide dimerization. In vitro cytotoxicity assays revealed specific PSMA-positive cell killing with PSMAxCD3 aptamers when control monomer A10 lacking the CD3 binding moiety did not induce any cytotoxicity. Engineered bispecific aptamers are thus able to recruit effector T lymphocytes to target cells to redirect their cytolytic machinery and eliminate a particular cell population. Based on these promising properties, PSMAxCD3 aptamers have ben selected for further preclinical and clinical development as prostate cancer therapeutics.
Interactions of ochratoxin A-specific G-quadruplex aptamer with different short complementary oligonucleotides
Alexey V. Samokhvalov, Anatoly V. Zherdev, Boris B. Dzantiev
A.N. Bach Institute of Biochemistry, Federal Centre of Biotechnology, Russian Acad. Sci., Moscow, Russia
Aptamers are actively used for analytical purposes as receptor molecules. Their ligand-binding properties can be attenuated by adding short complementary strands and such interactions open new possibilities for aptamer-based assays. Among structural rearrangements of aptamers, special attention is drawn to changes in the frequently occurring G-quadruplex structure, which fixes conformation of aptamers and makes a significant contribution to their binding properties. However, dependences between structure of complementary strands and their effects of G-quadruplexes need detailed characterization and generalization.We have studied the interaction of G-quadruplex DNA aptamer and a row of partially complementary short ssDNA oligonucleotides with different length, structure and location of complementary sites. An aptamer specific to ochratoxin A (5´-GAT-CGG-GTG-TGG-GTG-GCG-TAA-AGG-GAG-CAT-CGG-ACA-3´) was taken for the experiments. Conditions of its binding with the target ligand were tested. The necessity of Mg2+ or Ca2+ to form the stabilized basket-like antiparallel G-quadruplex structure and to bind ochratoxin A was confirmed. Fifteen complementary strands were tested for their interaction with the quadruplex using isothermal titration calorimetry and competitive fluorescence anisotropy assay. The influence of the length and localization of their complementary regions on the affinity of the interaction and induced structural rearrangements were characterized. The measured equilibrium dissociation constants varied from 10-6 to 10-9 M. Factors influencing these differences are considered. This study was financially supported by the Russian Science Foundation (Project No. 20-74-00112).
The increasing issue of antibiotic resistance: selection of a new aptamer for a glycopeptide antibiotic
Geanina Ștefan 1,2, Oana Hosu 1, María Jesús Lobo Castañón 2, Noemí de-los-Santos-Álvarez 2, Cecilia Cristea 1
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, Av. Julián Clavería 8, 33006 Oviedo, Spain
Nowadays, antibiotic resistance (AR) is considered to be the root of the next global pandemic as it is one of the main causes of death due to the significant increase in the multidrug-resistant bacterial infections being estimated to account for 10 million deaths by 2050. When antibiotics do not act against bacteria, the treatment becomes very costly and much less likely to succeed. Human-animal-environmental cross-interaction increase the resistome transmission; therefore, resistance has emerged in all classes of antibiotics in agricultural and clinical use. Hence, accurate and timely detection and monitoring of antibiotics is critical to the global strategies to prevent and control AR. Vancomycin is a powerful glycopeptide antibiotic used in the treatment of infections caused by gram positive organisms; however, in high doses, it can be toxic to the renal and auditive systems, but also low doses can cause hypersensitivity reactions. Since the discovery of SELEX technology, new approaches of the basic method were designed to specifically enhance the selection process of nucleic acid sequences. Among these, magnetic-beads based SELEX is particularly valuable as it adds considerable enhancement in the efficiency of the selection process of DNA sequences. The incorporation of aptamers in electrochemical sensors enabled the in situ detection of various molecules with high affinity and selectivity. This poster presents our current progress in the selection of a new aptamer selected through magnetic beads-based SELEX technology. Chemical characterization of the magnetic beads functionalized with vancomycin by means of XPS and insights about the SELEX process based on negative and counter selections will be detailed. The selected aptamer will be further explored for its potential application in the detection of vancomycin from biological and environmental samples.