Twitter: @AptamerSociety | @JAptamers | #AptaOx18
Email: AptamersOxford@gmail.com
Our 5th Oxford meeting – Aptamers 2018 – will address therapeutic, diagnostic, analytical and basic research applications of aptamers and invites oral and poster abstracts from academic and industrial researchers. We particularly encourage final year doctoral students and early/mid career postdoctoral researchers/junior group leaders to submit oral abstracts, as we believe this event will provide their research excellent exposure, critical analysis by attending senior researchers and new career opportunities.
The symposium will feature:
>High-impact talks, discussions & several hours of networking
>Oral presentations by an international faculty of senior & junior researchers from academia & industry
>Update on aptamer patents
>Posters, plus flash talks by selected poster presenters
>Trade exhibition
>Excellent networking opportunities, & a relaxed and friendly environment
Aptamers 2018 will be co-hosted with Oligo 2018 (10th April) – 4th symposium on antisense & therapeutic oligonucleotides (separate registration required).
We look forward to welcoming you to the symposium.
Symposium Chair
Dr Julian A Tanner
Associate Professor
The School of Biomedical Sciences
The University of Hong Kong
Hong Kong, PR China
0745 – 0830: Registration and welcome coffee (JdP foyer)
0830: Housekeeping
0835: Aptamers updates by Dr Muhammad Sohail
0840: INSOAP updates by Dr Sarah Shigdar
0850: Welcome by Dr Julian Tanner
0900: Professor Yoshikazu Nakamura, Ribomic Inc and the University of Tokyo, Japan
Anti-chymase RNA aptamer improved cardiac function in experimental heart failure
0930: Professor Adrian C Williams, University of Reading, UK
Passive diffusion of an RNA-based aptamer though intact human skin
1000: Miss Ke Zhang, University of Manchester, UK
Development of therapeutic aptamers as kinase inhibitors by Closed-loop Array Based Directed Evolution (CLADE)
1020: Refreshments, networking, exhibition and posters (JdP foyer)
1100: Dr Nebojsa Janjic, SomaLogic, Inc, USA
Base modifications profoundly influence the pharmacology of DNA aptamers
1130: Dr Marcel Hollenstein, Institut Pasteur, France
Selection of base-modified aptamers
1200: Miss Dehui Kong, University of Georgia, USA
Expanding aptamer chemical diversity by LOOPER
1220: Lifetime Achievement Award: 2018 Awardee – Emeritus Professor Uli Hahn
1230: Professor Uli Hahn, University of Hamburg, Germany
A life without nucleic acids – unimaginable
1250: Group photo (JdP Lawns)
1300: Lunch, networking, exhibition and posters (JdP foyer)
1350: Mrs Helen Lavender, Centauri Therapeutics, UK
The Development of Aptamers as Targeting Agents for Immunotherapeutics
1420: Professor Maria DeRosa, Carleton University, Canada
Aptamers in the central nervous system: Dopamine and Alpha-Synuclein
1450: Dr Sarah Shigdar, Deakin University, Australia
Aptamers for targeted therapeutic delivery
1520: Refreshments, networking, exhibition and posters (JdP foyer)
1600 – 1700: Presenters include: Atheer Alotaibi, Adrien Boussebayle, Stella Givanoudi, Muslum Ilgu, Jeannine Jäger, Przemyslaw Jurek, Peter (Shiwei) Li, Amir Nasiri, Marit Nilsen-Hamilton, Ayesha Siddiqua, Zoltán Tolnai
1700: Poster viewing and evaluation (JdP foyer)
1745: INSOAP AGM (open to all attendees)
1815: Aptamers Editorial Board meeting (Aptamers editorial board members only)
1915: Networking dinner (by prior booking or invitation only)
0900: Professor Darryl Bornhop, Vanderbilt University, USA
Compensated Interferometry Enables Nanovolume, Label-free OPNA and Opiate Screening in Urine and Serum at the Picomolar Level
0930: Dr Marcus Menger, Fraunhofer Inst for Cell Therapy and Immunology, Germany
Binding affinity analysis of DNA aptamers for therapeutic anthracyclines
0950: Dr Laura Cerchia, Istituto per l’Endocrinologia e l’Oncologia Sperimentale “G. Salvatore”, Italy
Theranostic anti-PDGFRβ aptamer for imaging and suppression of triple-negative breast cancer metastases
1010: Dr Guozhen Liu, The University of New South Wales, Australia
Graphene Quantum Dots based aptamer nanosensors for monitoring intracellular interferon-gamma (IFN-γ)
1030: Refreshments, networking, exhibition and posters (JdP foyer)
1100: Dr Julian Tanner, University of Hong Kong, PR China
Are aptamers better? Learning from malaria diagnosis
1130: Dr Marimuthu Citartan, Universiti Sains Malaysia, Penang, Malaysia
Stabilized RNA aptamers against Dengue virus 2 NS1 glycoprotein towards RNAaptanostics
1150: Professor Dr Beatrix Suess, Technical University of Darmstadt, Germany
A TetR-binding aptamer as versatile regulatory element
1220: Dr Anna Maria Weber, Life & Medical Science Institute Bonn, Bonn, Germany
Development of a light dependent aptamer-photoreceptor system
1240: Dr Shalen Kumar, Victoria University of Wellington, Wellington, New Zealand
Selection, characterization and application of DNA aptamers capable of binding a plethora of small organic molecules
1300: Lunch, networking, exhibition and posters (JdP foyer)
1350: Professor Philip Johnson, York University, Canada
Aptawire: Ligand-induced self-assembly of an aptamer-based DNA suprastructure
1420: Mr Beñat Olave, POLYMAT, University of the Basque Country (UPV/EHU), Spain
Function of aptamers in non-conventional ionic solvents
1440: Dr Philip Webber, UK and European Patent Attorney, Dehn Patent and Trade Mark Attorneys, Oxford, UK
Patenting of aptamers
1500: Poster prize (sponsored by MDPI Pharmaceuticals)
1520: Discussion and close
In Vitro Selection of High Affinity DNA Aptamer for Detection of sepiapterin, a Sepiapterin Reductase Deficiency (SRD) Biomarker
Shahd Alkhaldi, Mohammed Zourob
College of Science, Alfaisal University, Riyadh, Saudi Arabia
Sepiapterin reductase deficiency (SRD) patients have depletion in biogenic amines in brain, intellectual disability etc. Sepiapterin (SP)is accumulated in caribospinal fluid ( CSF) of the SRD patiants. Due to insufficient sepiapterin reductase, the ptrein metabolic pathway is for the biosynthesis of tetradydrobiopterin (BH4)is inhibited. BH4 is an important cofactor for several enzymatic reactions. Low level of BH4 in CSF will leads to mental retardation, convulsions, hypersalivation, swallowing difficulties, and temperature instability and oculogyric crises. There for sensitive and specific detection of sepiapetrin in important to identify the SRD patients. The current method of testing is by semi quantitative colorimetric assay to assess. The normal range to have the results time is about two weeks. The objective of this research to develop rapid screening test for SRD using high affinity and SP specific aptamer to have faster results. SP specific aptamer is selected from 1015 random sequence of DNA library by SELEX method…
Development of truncated aptameric biosensor for the detection of microcystin-LR
Razan AlZabn, Raja Chinnappan, Mohammed Zourob
College of Science, Department of Chemistry, Alfaisal University, Riyadh 11533, Saudi Arabia
Cyanobacteria is one of the algae that exist in marine ecosystems and produce toxins, which produce microcystin toxins. Microcystins are the most common cyanotoxin and can cause harm to liver, kidney and lungs due to intensive exposure to contaminated water through using lakes and rivers for recreational purposes and consumption of drinking water. The common methods to detect Microcystin are laborious, expensive and time-consuming. Development of aptamer-based biosensors are promising techniques that can overcome the antibody’s disadvantages. In this study, we truncate the parent aptamer to get high affinity short aptamer for the development of sensitive detection of Microcystin-LR (Mc-LR). We used molecular beacon assay and fluorescence based competitive displacement assay. In the competitive displacement assay, the fluorescein and a quencher labelled complementary aptamer sequences are hybridized with aptamer and form quencher-fluorescein pair. The duplex of the aptamer dissociates and quencher-fluorescein pair is separated in the presence of the MC-LR. Therefore, increase in the fluorescence intensity…
‘Aptahistochemistry’ to detect co-localised surface stemness markers in colorectal cancer xenografts and identify putative cancer stem cells
Michelle L Bauer, Alister C Ward, Sarah L Shigdar
Centre for Molecular and Medical Research, School of Medicine, Deakin University, Geelong, VIC 3128, Australia
Immunohistochemistry (IHC) is an indispensable method for the diagnosis of malignancy when alterations in tissue morphology remain deceptively subtle. IHC can provide prognostic indications and guide personalised treatment decisions by the detection of clinically relevant markers of tumourigenicity and invasiveness. For more than forty years, antibodies have demonstrated efficacy in chromogenic IHC. However, antibodies continue to harbour issues of batch-to-batch variability and cross-reactivity that are of detriment to the accuracy and precision of clinical histopathology. In contrast, aptamers are sequence-defined and synthesised entirely in vitro – affording them ease of functionalisation and high batch-to-batch fidelity. Aptamers are also around 10-20 times smaller than antibodies, which may prove beneficial in detecting cells which co-express multiple surface markers of tumourigenicity. Currently, as a result of the large size of the antibody-chromogen complex, and the resulting potential for steric hindrance…
Development of synthetic riboswitches using an optimized selection strategy to select structure-switching aptamers
Adrien Boussebayle, Daniel Torka, Florian Groher, Cristina Bofill-Bosch, Simon Fürbacher, Johannes Braun, Beatrix Suess
Synthetic Genetic Circuits, Department of Biology, TU Darmstadt, Darmstadt, Germany
Engineered riboswitches are of high interest in Synthetic Biology. In our group, we are adapting SELEX procedures to enrich RNA libraries against small molecule ligands and then screen these libraries in vivo in order to identify new synthetic riboswitches. The major drawback with traditional SELEX approaches is the need to immobilize the target on a matrix. This challenge is even harder with small molecules such as metabolites or antibiotics. For this reason, we developed an optimized selection strategy based on the refolding of the RNA upon ligand binding where the ligand is free in solution. In this new and improved protocol, the RNA is retained on magnetic beads and the aptamers are removed upon ligand addition and binding to the ligand…
High Throughput Sequencing (HTS) analysis of multiple branched selections to detect specific aptamers
Alix Bouvier-Müller 123, Romain Schmitt123, Cynthia Forier123, Adrien Henriques123, Benoit Lelandais12345, Clément Bouvier123 and Frédéric Ducongé123
1 CEA, Fundamental Research Division (DRF), Institut of biology François Jacob (Jacob), Molecular Imaging Research Center (MIRCen), 18 route du panorama, 92265 Fontenay-aux-Roses, France
2 Neurodegenerative Diseases Laboratory, CNRS CEA URA 2210, Fontenay-aux-Roses, France
3 Paris-Saclay University, Fontenay-aux-Roses, France
4 Present Address: Unité Imagerie et Modélisation, Institut Pasteur, 25 rue du docteur roux, 75015, Paris, France
5 Present Address: UMR 3691, CNRS; C3BI, USR 3756, IP CNRS, Paris, France
Aptamers are short synthetic oligonucleotides able to bind a target with high specificity and affinity. These molecules are identified through a process of directed evolution named SELEX. Although counter-selection steps could be used during SELEX to improve the specificity of aptamers, it is sometimes difficult to select aptamers that are able to discriminate very similar molecules. Here we used High Throughput Sequencing (HTS) to identify sequences with very high specificity. This method was used to find candidates specific to four proteins A, B, C and D that are very similar. In a previous study, we performed several SELEX with counter-selection steps, but no aptamers were able to discriminate the four proteins. To identify aptamers possessing such specificities that may be present at very low…
Aptamer selection using exotic nucleic acid for targeting Plasmodium falciparum lactate dehydrogenase
Yee-Wai Cheung1, Alvin Wai-Chung Wong1, Pascal Röthlisberger2, Marcel Hollenstein2 and Julian A. Tanner 1
1School of Biomedical Sciences, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong
2Institut Pasteur, Department of Structural Biology and Chemistry, Laboratory for Bioorganic Chemistry of Nucleic Acids, CNRS UMR3523, 28, rue du Docteur Roux, 75724 Paris CEDEX 15, France
Nucleic acid aptamers have characteristics including ease of manipulation and their stability which benefits diagnostic application. The incorporation of our previous identified DNA aptamer to different platforms for malaria diagnosis demonstrates the flexibility of aptamer application. However, the diagnosis of low Plasmodium parasitaemia infection mainly rely on microscopic and nucleic acid amplification approaches that only available in laboratory, whereas the point-of-care rapid diagnosis test (RDT) still remains a challenge. Modified nucleic acids with different functional groups provide opportunities for obtaining aptamer with higher affinity. As nucleic acids with highly hydrophobic cubane residues may enhance the affinity of aptamer, herein, we selected aptamer by a conventional SELEX process using a cubane modified deoxyuridine triphosphate (dUcTP) instead of using of deoxythymidine triphosphate (dTTP)…
Ligand-dependent base pair stability in the cocaine-binding aptamer
Zachary R Churcher, Philip E Johnson
Department of Chemistry, York University, 4700 Keele Street, Toronto, Ontario, Canada, M3J 1P3
Using NMR spectroscopy, the effect of temperature on the base pair stability of of the cocaine-binding aptamer has been investigated by studying the imino proton exchange rates of the base pairs in their free and ligand bound states. MN4 is a variant of the cocaine-binding aptamer that binds to cocaine tighter than the originally selected sequence. It consists of a three stems centered on a three-way junction with a dinucleotide bulge and 2 A-G mismatches. What make this aptamer exciting however is that it binds to quinine tighter than the ligand it was selected for. Off target ligand binding is not uncommon with aptamers, but off target binding that is tighter the intended ligand is rarely seen. The MN4 aptamer was studied using magnetization transfer NMR. This is done by selectively magnetizing the potion of the NMR spectrum where the imino resonances are found, and then seeing how long it take for the peaks…
Making sense of SELEX data
Tristan Cragnolini12, Alex Martin3, Helen Lavender3, Lucy Colwell12
1 Aptamex Ltd, 6 Arlington St, London, SW1A 1RE
2 Department of Chemistry, Lensfield Road, Cambridge, CB2 1EW
3 Centauri Therapeutics, Discovery Park, Sandwich, CT13 9ND, Kent, UK
SELEX has shown considerable success as a method to discover RNA sequences with specific binding properties, yet identification of the best aptamers from a library that contains numerous sequences remains a significant challenge.To address this challenge, we have developed an interactive computational workbench that streamlines SELEX data analysis, providing tools that identify candidate aptamers, and facilitate optimisation of their sequence and structure.In this poster we use simulated SELEX datasets to demonstrate the role played by these tools in the aptamer discovery process, through identification of highly amplified sequences over multiple SELEX rounds, visualisations that identify noteworthy change in the pool composition between rounds, and structure refinement using embedded automatic prediction tools.
Comparison of pharmacological effects of HD1 and HD22 thrombin binding aptamers on human platelets, thrombin activity and thrombus formation
Katarzyna Derszniak1,2, Kamil Przyborowski 1, Karolina Matyjaszczyk 1,3, Maria Nowakowska 2, Stefan Chlopicki 1, 4
1 Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Krakow, Poland
2 Faculty of Chemistry, Jagiellonian University, Krakow, Poland
3 Chair and Department of Toxicology, Jagiellonian University, Krakow, Poland
4 Chair of Pharmacology, Jagiellonian University, Medical College, Krakow, Poland
HD1 and HD22 anti-thrombin aptamer binding thrombin exosite I and exosite II, respectively are intensively studied. However, their pharmacological effects are still not well-characterized. The aim of this work was to compare pharmacological effects of HD1 and HD22 aptamers on thrombin-induced platelets aggregation in platelet rich plasma (PRP) and human washed platelets (WP) by optical aggregometry, thrombin generation in human plasma by Calibrated Automated Thrombography (CAT) as well as thrombi formation in microchip-based flow chamber system in whole human blood (Total Thrombus Formation Analysis System, T-TAS). Dissociation constants (Kd) of HD1 and HD22 with thrombin were determined in Tris-HCl buffer (pH=7.4), in artificial plasma and in artificial plasma with K+, Mg2+, Ca2+ ions addition by Capillary Electrophoresis (CE)…
Deciphering the binding characteristics of a DNA aptamer (FELIAP) on Factor XIa
David A Donkor 1,2, Varsha Bhakta 1, Amy Nouanesengsy 2 and William P Sheffield 1,2
1 Centre for Innovation, Canadian Blood Services, Hamilton, Ontario, Canada
2 Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada
Coagulation factor XI (FXI) is activated to FXIa either by FXIIa of the contact pathway, or by thrombin; FXIa then activates FIX. Both humans and mice deficient in FXI appear to be protected from thrombosis, with only a mild to moderate bleeding tendency. FXIa is thus an attractive target for antithrombotic agent development. Using a combinatorial DNA library and in vitro selection, we isolated a 78 nucleotide long Factor ELeven Inhibitory Aptamer (FELIAP) that bound FXIa with high affinity (mean KD 1.8 nM) but with a relatively high inhibitory constant (mean Ki 29 µM) (Donkor DA et al., Sci Rep 2017). FELIAP exhibits competitive inhibition of FXIa, suggestive of either allostery or binding near the active site without full active site blockade. Biotin conjugated FELIAP in complex with avidin (FELIAP_BIOT_AVIDIN) significantly increased the inhibitory potency of FELIAP by 4-fold versus FELIAP alone; the correlation of expanded FELIAP size…
Selection of high-affinity fully modified 1,5-anhydrohexitol aptamers against rat vascular endothelial growth factor 164
Elena Eremeeva 1, Lia Margamuljana 1, Mikhail Abramov 1, Elisabetta Groaz 1, Piotr Leonczak 1, Sam Noppen 2, Peter Carmeliet 3, and Piet Herdewijn 1
1 Medicinal Chemistry, Rega Institute for Medicinal Research, KU Leuven, Herestraat 49, 1041, 3000 Leuven, Belgium
2 Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology, Rega Institute for Medicinal Research, KU Leuven, Herestraat 49, 1043, 3000 Leuven, Belgium
3 Laboratory of Angiogenesis and Vascular Metabolism, VIB Vesalius Research Centre, VIB, Herestraat 49, 912, Leuven 3000, Belgium
Aptamers are unique nucleic acid molecules that hold a promising potential for various applications in therapy and biotechnology. Fully modified 1,5-anhydrohexitol (HNA)–2’-OMe-RNA hybrid aptamers targeting the rat vascular endothelial growth factor 164 (rVEGF164) have been selected using an in vitro evolution procedure. Three sequences were found to interact with the target protein with affinities in the low-nanomolar to picomolar range. The evolution of such chemically altered aptamers was accomplished starting from a completely sugar-modified library containing a 20 2’-OMe-ribonucleotide sequence at the 5’-end followed by distinct 47 mer HNA regions. The resulting aptamers exhibited a remarkable resistance to nuclease degradation even after 24 h of incubation with DNase I. The high affinity of the selected aptamers was…
Development of target-specific DNA aptamers for histamine, cadaverine and putrescine
Stella Givanoudi 1,3, Marc Heyndrickx 3, Patrick Wagner 1, Johan Robbens 2
1Soft-Matter Physics and Biophysics Section ZMB, KU Leuven, Celestijnenlaan 200 D, Leuven, Belgium
2Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Aquatic Environment and Quality, Ankerstraat 1, Oostende, Belgium
3Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Food Safety, Brusselsesteenweg 370, Melle, Belgium
Histamine is a monoamine naturally occurring in the human body. Dietary intake and accumulation of increased levels of histamine is known to cause foodborne poisoning. Outbreaks of histamine poisoning are reported every year and they are mostly associated with consumption of spoiled fish-related products and Swiss type cheese. Moreover, histamine is an indicator of seafood freshness as its presence signifies food spoilage by microbial contamination. For that reason, the levels of histamine in commercial seafood is officially regulated and monitored. Two other amines, cadaverine and putrescine are known to enhance the toxicity of histamine. The objective of the present research is to develop aptamers as bio-recognition elements that can bind specifically to three important biogenic amines found in food: histamine, cadaverine and putrescine. For this purpose, we conducted magnetic beads-based SELEX…
Riboswitching with ciprofloxacin – development and characterization of a novel RNA regulator
Florian Groher 1, Cristina Bofill-Bosch 1, Christopher Schneider 1, Johannes Braun 1, Sven Jager 2, Katharina Geißler 1, Kay Hamacher 2,3 and Beatrix Suess 1
1 Synthetic Genetic Circuits, Department of Biology, TU Darmstadt, Darmstadt, Germany
2 Computational Biology and Simulation, Dept. of Biology, TU Darmstadt, Darmstadt, Germany
3 Department of Physics, Dept. of Computer Science, TU Darmstadt, Darmstadt, Germany
RNA molecules play important and diverse regulatory roles in the cell. Inspired by this natural versatility, RNA devices are increasingly important for many synthetic biology applications, e.g. optimizing engineered metabolic pathways, gene therapeutics or building up complex logical units. A major advantage of RNA is the possibility of de novo design of RNA-based sensing domains via an in vitro selection process (SELEX). Here, we describe development of a novel ciprofloxacin-responsive riboswitch by in vitro selection and next-generation sequencing- guided cellular screening. The riboswitch recognizes the small molecule drug ciprofloxacin with a KD in the low nanomolar range and adopts a pseudoknot fold stabilized by ligand binding. It efficiently interferes with gene expression both in lower and higher eukaryotes. By controlling an auxotrophy marker and a resistance gene, respectively…
The impact of 2-aminopurine replacement on aptamer dynamics and ligand interaction
Muslum Ilgu,1,2,3,4 Shuting Yan, 1 Christopher L. Fritz,1 Sharif Anisuzzaman1, Monica H. Lamm,1 and Marit Nilsen-Hamilton1,2,3
1 Iowa State University, Ames, IA, USA
2 Ames National Laboratory, Ames IA, USA
3 Aptalogic Inc., Ames IA, USA
Present address: 4 Middle East Technical University, Ankara, Turkey
The use of 2-aminopurine (2AP) as a substitute for adenine in nucleic acids is a strategy that adds the benefit of its intrinsic fluoresecence properties to the molecule and has enabled detailed studies of nucleic acid structures and the activities of DNA interacting enzymes. 2AP replacement has been claimed to be an innocous substitution. However, our studies with RNA aptamers containing 2AP substitutions indicate that extra precaution should be taken when considering substituting an adenine with 2AP. Specifically, we found that 2AP replacements can affect the affinity of the aptamer for its ligand, most probably due to local structural changes. Molecular dynamics simulations show that two models of an RNA aptamer are compared with A or 2AP in particular position, the structural dymanics can be different. In this work, a variety of small molecule binding RNA aptamers with 2AP or A in specific locations will be compared for their ligand interactions. In summary…
A fluoroquinolone-binding aptamer as a biosensor
Jeannine Jaeger, Florian Groher, Cristina Bofill-Bosch, Johannes Braun, Beatrix Suess
Synthetic Genetic Circuits, Department of Biology, TU Darmstadt, Darmstadt, Germany
The massive use of antibiotics in the medical sector and animal fattening has led to a continuous increase in multidrug resistant bacteria (MDROs). In order to control and possibly contain the spread of antibiotics in food, it is necessary to detect them quickly, efficiently and cost-effectively. While today’s detection methods are based either on antibody-rapid-tests or require a great deal of technical effort, aptamer-based biosensors represent a fast and efficient analytical method. Nucleic acid-based aptamers (aptamers) consist of either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and can be characterized by their complex three-dimensional structure. This structure makes it possible to recognize any target molecule, like antibiotics, with high affinity and specificity. The aptamer-mutant R10K6_V11, which is able to bind ciprofloxacin (CFX) and other fluoroquinolones as a ligand, was used for an aptamer-based biosensor-design…
Modular platform for chemically modified aptamers
Przemysław M Jurek, Aleksandra Adamowicz, Agnieszka J Sok, Maciej P Mazurek
Pure Biologics Inc., Research & Development Department, Wrocław, Poland
In comparison with widely used antibodies, non-modified aptamers display only a limited range of chemical groups involved in the binding to the cognate targets. The narrow diversity of natural nucleobases is no match for the multifarious properties of the 20 proteinogenic aminoacids. In spite of that, many aptamers with excellent properties have been selected during the past 28 years that can rival antibodies in many aspects, and even outcompete them. This makes us believe that the true power of aptamers lies in the possibility of introducing new non-canonical (either natural or unnatural) modifications to the nucleobases that will allow even better binding properties, thanks to a significantly broadened chemical space coverage. To challenge this view, we have recently developed a modular platform termed PureApta™ for introduction of new chemical groups into oligonucleotides structure and its integration into the SELEX procedure…
Aptamers as Targeting Agents for Immunotherapeutics
Helen Lavender, Alex Martin, Carl Laxton, Emma Leire, Chris Pickford, Mike Westby
Centauri Therapeutics, Discovery Park, Sandwich, Kent, CT13 9ND
Aptamers have demonstrated potential as therapeutic agents. We are engaged in selecting aptamers as the targeting component of a novel immunotherapeutics platform, designated ‘alphamers’. Alphamers recruit natural immunity and can be designed to target specific pathogens or clinically important cell surface receptors. In vitro studies with a Group A Streptococcus alphamer have shown that pre-existing antibodies to the ‘aGal’ sugar epitope can be directed to promote an antibacterial immune response. Our current aptamer discovery programs focus on the identification of novel aptamers to ESKAPE pathogens and Oncology targets. SELEX methodologies are used to screen for new aptamer sequences with nuclease stable RNA compositions. Bioinformatic technologies are extensively utilised, to select sequences for biological characterisation, from the near infinite number of candidates within a SELEX pool. A typical bioinformatics workflow using opensource software will be described; reviewing different triage strategies…
Fluorescent-imaging assay for dissociation constant determination of triclosan specific aptamers
Shiwei Li 1,2, Shalen Kumar 1,2, Jeremy Jones 2, Mark Clarkson 3, Janet Pitman 1, Kenneth McNatty 1
1 School of Biological Sciences, Victoria University of Wellington, New Zealand
2 AuramerBio Limited, Callaghan Innovation Quarter, 69 Gracefield Road, Lower Hutt, New Zealand
3 Callaghan Innovation, 69 Gracefield Road, Lower Hutt, New Zealand
Aptamers are nucleic acid-based receptors that bind target molecules by adopting specific three-dimensional structures. The binding affinity and specificity of an aptamer and the dissociation constant (Kd) are important characteristics when assessing it’s utility for specific applications. However, there are no universally accepted methods or recommended guidelines for the measurement of aptamer Kd. Therefore, there is a need for new and effective techniques to determine the binding characteristics of aptamers. Herein, we report a novel fluorescent-imaging assay (FIA) for determining the binding characteristics of aptamers selected against the antibacterial agent, triclosan (TCS). FIA utilises fluorescently-labelled aptamers and, in this instance, TCS that had previously been conjugated to sepharose beads. Upon binding of the TCS-selected aptamer to the TCS-bead conjugate, the complex can be visualised and the image captured…
Automated electrochemical sensing system for monitoring luteinising hormone pulsatility
Shaolin Liang1,2,3, Andrew Kinghorn1, Waljit Dhillo2, Anthony E G Cass3 and Julian A Tanner1
1School of Biomedical Science, Faculty of Medicine, University of Hong Kong, 21 Sassoon Road, Hong Kong SAR, P.R.China.
2Department of Medicine, Imperial College London, South Kensington Campus, SW7 2AZ, UK
3Department of Chemistry, Imperial College London, South Kensington Campus, SW7 2AZ, UK
Normal fertility in human involves a highly orchestrated signal communication crossing the axis of hypothalamic-pituitary-gonadal (HPG). The pulsatile release of Luteinising Hormone (LH) from the pituitary gland is the key element in this “concerto” for the stimulation of sex steroid hormones synthesis and the production of mature eggs. Specific alternations in LH pulsatile pattern are linked to hypothalamic dysfunction in female patient with anovulatory infertility – by knowing the information of this pattern, clinicians can decide whether the patient needs treatment with pituitary hormones to recover the normal ovulational function. Here, we propose a novel platform using aptamer-based electrochemical sensor integrated with open-source liquid handling robot to assess LH pulsatility profile. A methylene blue (MB) modified LH specific aptamer was generated and adapted to a microwire electrode device designed for…
Binding affinity analysis of DNA aptamers for therapeutic anthracyclines
Stephan Sass1, Walter F. Stöcklein2, Anja Klevesath2, Jeanne Hurpin1, Marcus Menger2 & Carsten Hille1
1University of Potsdam, Institute of Chemistry, Physical Chemistry, 14476 Potsdam, Germany
2Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (IZI-BB), Functional Nucleic Acids – Aptamers, 14476 Potsdam, Germany
Aptamers are single-stranded RNA or DNA nucleotides, which are generated using an iterative in vitro selection procedure (SELEX) and bind to target molecules with high specificity and high affinity due to their specific tertiary structures. Thus, aptamers are preferable interaction molecules in many different analytical systems like biosensors or lateral flow assays. Anthracyclines such as daunorubicin (DRN) and doxorubicin (DOX) are key chemotherapeutic substances in cancer treatment since decades. However, their chronic administration can induce severe side effects. The development of new nanocarriers for targeted drug delivery could probably minimize these side effects, and aptamers seem to be favorable molecular targeting agents. Here, we present the binding properties of a single-stranded DNA aptamer, which has been previously generated against DRN. For this, we applied particularly two complementary methods, namely…
The Center of Aptamer Research and Development (CARD), an automated platform for aptamer selection
Amir H Nasiri, Stefan Künne, Günter Mayer
The Center of Aptamer Research and Development (CARD), Life and Medical Sciences Institute (LIMES), Bonn, Germany
The Center of Aptamer Research and Development (CARD) is located within the Life and Medical Sciences (LIMES)-Institute of the University of Bonn. CARD is centred on an automated aptamer development platform. This proprietary platform enables the fully automated generation of aptamers in a cost & time efficient manner. Our mission is to provide economic access to aptamers on a non-profit basis for researchers from Germany, Europe and worldwide. Based on our long-term experience in the aptamer field we have developed an automated selection protocol that facilitates the identification of RNA aptamers binding to protein targets. Based on this protocol we have successfully selected RNA aptamers, which specifically bind to different protein classes, e.g. kinases. These aptamers have the capacity to be a novel and promising class of protein kinase inhibitors. They are able to block domain specific functions…
DRAGINs: Aptamer-enabled uptake of small molecule ligands
Supipi Liyamali Auwardt 1,2,3 Yeon-Jung Seo 1, Muslum Ilgu 1,2,3,4, Judhajeet Ray 1,2,5, Robert R. Feldges 2, Shambhavi Shubham 1,2,6, Lee Bendickson 1,2, Howard A. Levine 1 and Marit Nilsen-Hamilton 1,2,3*
1 Iowa State University, Ames, IA, USA
2 Ames National Laboratory, Ames IA, USA
3 Aptalogic Inc., Ames IA, USA
4 Present address: Middle East Technical University, Ankara, Turkey
5 Cornell University, Ithaca, NY, USA
6 Currently at University of Iowa, Iowa City IA, USA
The relative ease of isolating aptamers that bind specific molecules suggests that molecular recognition may be common in the folds of natural RNAs. We show here that, when expressed in cells, aptamers can increase the intracellular concentrations of their small molecule ligands. We have named these aptamers as DRAGINs (Drug Binding Aptamers for Growing Intracellular Numbers). The DRAGIN property, assessed here by the ability to enhance the toxicity of their ligands, was found for some, but not all, aminoglycoside aptamers. One aptamer protected cells against killing by its ligand. Another aptamer promoted killing as a singlemer and protected against killing as a tandemer. Based on a mathematical model, cell protection vs. killing is proposed as governed by aptamer affinity and access to the inner surface of the cell membrane, with the latter being the most important determinant…
Development of Biosensor for the Detection of Antibiotics
Atheer M Al Otaibi, Raja Chinnappan, Mohammed Zourob
Al Faisal University, Riyadh, Saudi Arabia
Antibiotic is a medication that destroys or slows down the bacterial growth. Hence, they are used to treat different types of bacterial infection. However, the overuse of antibiotics causes antibiotic resistance which means that the bacteria is no longer affected by the antibiotics. This is a very serious problem that might be detrimental for the future generation. Furthermore, antibiotic resistance can be obtained through many ways other than direct ingestion. One of the ways is by sewage sludge that is recycled in the agricultural land that contains a significant amount of various antibiotics, and antibiotic resistant bacteria. The current detection methods like HPLC and GC require sophisticated instruments trained personals. Moreover, antibody based immune assays leads to false positive results due to thermal stability and cross reactivity of the antibodies…
Automated isolation of aptamers against non-modified small molecule targets for simplified assay development
Christine Reinemann, Edward T Barnes, David H J Bunka, Arron C Tolley
Aptamer Group, Suite 2.80 – 2.91, Bio Centre, Innovation Way, Heslington, York, YO10 5NY
There is an increasing need to reliably detect and monitor levels of small molecules in all areas of the life sciences. This need ranges from monitoring individual patient drug levels, to food or environmental contaminant, detection. In every case, more favourable outcomes are achieved when tests are performed ‘on-site’ or at ‘point-of-care’ as immediate action can be taken. Monitoring small molecules often relies on expensive laboratory based methods such as chromatography and mass spectrometry. Simple field based diagnostic devices, such as lateral flow devices, are widely employed as a first-pass assay to determine if more thorough analysis is required. These tests often utilise antibodies as the recognition element and frequently require a ‘sandwich-pair’ of antibodies, which is problematic for targets where antibodies are not available. The problem is exacerbated with small molecule antigens…
Utilizing the intrinsic fluorescence of cocaine-binding aptamer ligands: insights into the binding mechanism and development of a thermal shift assay
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
We used fluorescence spectroscopy to measure the binding affinity and provide new insights into the binding mechanism of cocaine and quinine with the cocaine-binding DNA aptamer. Using the intrinsic fluorescence of quinine and cocaine, we have observed quenching of ligand fluorescence upon binding of the aptamer. Quantification of this quenching provides an easy method to measure the binding constant using small amounts of sample. The observed quenching coupled with a red shift of the Stokes shift in the emission spectrum indicates that quinine and cocaine interact with the aptamer through stacking interactions.We then used the inherent fluorescence properties of the ligands in a thermal shift assay in two different projects. In the first, we developed a new cocaine-binding aptamer variant with a higher melting temperature when bound to a ligand than the currently used sequences without decreasing…
Selection Method for Generating Nucleic Acid Aptamers using electrochemical assay
Ayesha Siddiqua, Shimaa Eissa, Raja Chinnappan, Mohammed Zourob
Al Faisal University, Riyadh, Saudi Arabia
Aptamers are single stranded oligonucleotides that could be either DNA or RNA and they have the ability to specifically bind to the target analyte with high affinity. In addition to this property, they have several advantages which make them better candidates for various applications. Aptamers are selected by a process called SELEX which stands for systematic evolution of ligands by exponential enrichment. The conventional SELEX that is used today is tedious, time consuming and expensive as it usually utilizes beads as solid matrix for the immobilization of the target as well as fluorescently-labeled primers. In order to overcome these drawbacks, here, we employed gold electrodes as a solid matrix for the immobilization of the target. This makes the washing and the elution steps of the SELEX easier. Furthermore, the recovered DNA can be determined…
RNA aptamers recognizing murine CCL17 inhibit T cell chemotaxis and reduce contact hypersensitivity in vivo
Lorenz Fülle1,8, Nancy Steiner1,8, Markus Funke2,8, Fabian Gondorf1,8, Franziska Pfeiffer2, Julia Siegl2, Friederike V. Opitz1, Silvana Haßel2, Anna Belen Erazo1, Oliver Schanz1, H. James Stunden3, Michael Blank4, Carsten Gröber4, Kristian Händler5,6, Marc Beyer5,6,7, Heike Weighardt1, Eicke Latz3, Joachim L. Schultze5,6, Günter Mayer2,9 and Irmgard Förster1,9
1Immunology and Environment, Life and Medical Sciences (LIMES) Institute, University of Bonn, Carl-Troll-Straße 31, 53115 Bonn, Germany
2Chemical Biology and Chemical Genetics, Life and Medical Sciences (LIMES) Institute, University of Bonn, and Centre of Aptamer Research and Development, University of Bonn, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany
3Institute of Innate Immunity, University Hospital Bonn, Sigmund-Freud-Str. 25; 53127 Bonn, Germany
4AptaIT, Am Klopferspitz 19a, 82152 Planegg-Martinsried, Germany
5Genomics and Immunoregulation, Life and Medical Sciences (LIMES) Institute, University of Bonn, Carl-Troll-Straße 31, 53115 Bonn, Germany
6Platform for Single Cell Genomics and Epigenomics at the DZNE and the University of Bonn, Sigmund-Freud-Str. 27; 53127 Bonn, Germany
7Molecular Immunology in Neurodegeneration, German Center for Neurodegenerative Diseases (DZNE), Sigmund-Freud-Str. 27, 53127 Bonn, Germany
8these authors contributed equally
9joint senior authors
The chemokine CCL17, mainly produced by dendritic cells (DC) in the immune system, is involved in the pathogenesis of various inflammatory diseases. As a ligand of CCR4, CCL17 induces chemotaxis and facilitates T cell-DC interactions. We report the identification of two novel RNA aptamers that were validated in vitro and in vivo for their capability to neutralize CCL17. Both aptamers efficiently inhibited the direct migration of the CCR4+ lymphoma line BW5147.3 towards CCL17 in a dose-dependent manner. To study the effect of these aptamers in vivo, we used a murine model of contact hypersensitivity. Systemic application of the aptamers significantly prevent ear swelling and T cell inflation into the ears of sensitized mice after challenge with the contact sensitizer.
Insights into Ligand Binding by the Cocaine-Binding Aptamer and ATP Aptamer – an ITC Study
Sladjana Slavkovic, Zachary Churcher, Yanrui Zhu and Philip E Johnson
Department of Chemistry, York University, Toronto, ON, M3J 1P3, Canada
Both the cocaine-binding aptamer and the ATP aptamer are widely used as model systems in the development of a variety of biosensor applications. We have utilized isothermal titration calorimetry (ITC) methods to analyse the thermodynamics of how these aptamers interact with their ligands. Two unique features of the cocaine-binding aptamer are its ligand-binding promiscuity and salt-controlled two-site binding mechanism. Despite being selected for cocaine, the cocaine-binding aptamer binds quinine with higher affinity than cocaine. To further study the ability of this aptamer to bind different ligands, we looked at binding of quinine-based (amodiaquine, chloroquine, primaquine and mefloquine) and non-quinine based (pyrimethamine and dapsone) anti-malarial drugs to the cocaine-binding aptamer and compared results to both quinine and cocaine. Surprisingly, the quinine based anti-malarial drugs…
Selection of NT-proBNP specific aptamers
Zoltán Tolnai, Éva Nagyné Scholz, Zsuzsanna Szeitner, Ákos Harkai, Tamás Mészáros
Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary
Brain natriuretic peptide (BNP) are expressed and stored in myocardial tissue and plays role in regulating fluid and blood pressure. By heart failure patients the elevated serum level of BNP and of the inactive aminoterminal fragment of the BNP prohormone (NT-proBNP) are useful biomarkers in diagnosis of left-ventricular systolic dysfunction. Currently the measurements of serum BNP level are based on Immunoassay technics. Aptamers for the N terminal end of the NT-proBNP protein were selected previously in our laboratory. We aimed to select aptamers for another epitope of the NT-proBNP protein to aid the development of a proBNP specific sandwich assay. To obtain the suitable aptamers, peptides molecules from the C terminal end of proBNP and in vitro translated proBNP protein were alternately…
