Thank you for considering presenting your work as a poster at Aptamers 2023.
Flash-talks: Poster presenters will have the (optional) opportunity to introduce their poster in a short, 90 second flash presentation using 1-2 PowerPoint slides during their allocated break.
If you are planning to give a flash-talk, please submit 1-2 PowerPoint slides, appropriately named to match your digital poster file (e.g., <your surname>-APT23-slides.pptx), along with your digital poster. The session chair will share your slides on your behalf during your presentation.
Please check the website for your poster presentation schedule. Practice beforehand so that you can finish your presentation within your allocated time slot.
The poster PDF files, whether the presenter is attending virtually or in-person, will be made available via the secure conference documents page to the conference participants before the meeting. The JPG/PNG files will be available via @AptamerSociety.
Hardcopy posters: If attending in-person, you may bring along a printed copy of your (maximum A1 size, portrait), to be displayed during the conference. Only A1-sized posters will be displayed. Your poster may be displayed on Day 1 or Day 2 only due to space constraints.
There will be two ways to interact with the poster presenters:
Before uploading your poster and flash-talk slides, you must make sure that you follow ALL of the instructions above!
(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.
Aptameric biosensors for amoxicillin detection
Wafaa Al Borhani, Amani Chrouda, Mohammed Zourob
AlFaisal Department of Medicine, AlFaisal University, Al Zahrawi St., Riyadh, Saudi Arabia
Antibiotic resistance has increased rapidly in recent years becoming a major issue worldwide. Antibiotics can easily enter the environment reaching our food and water sources leading to health problems. A sensitive and rapid screening method for antibiotics has become essential in order to protect the sensitive consumers from life-threatening reactions. The current detection methods for antibiotics are mostly based on immunoassays which are costly and time consuming. In this work, we developed an aptamer/gold-based electrochemical biosensor for sensitive and low-cost detection of Amoxicillin, one of the top ten most used generic antibiotics. The selection of DNA aptamers against Amoxicillin was successfully carried out using systemic evolution of ligands by exponential enrichment (SELEX) method. Among the selected aptamers, Amx3 aptamer sequence has shown high specificity and affinity to Amoxicillin with a dissociation constant (Kd) of 112.9 nM. The aptamer was then integrated in a voltametric biosensor utilizing gold-modified screen-printed electrodes. The method has a high sensitivity, with a detection limit as low as 5.5 × 10−14 M. Our biosensor did not show any significant cross-reactivity with other similar antibiotics such as Ciproflex and Azithromycin, indicating that the sensor had high specificity to Amoxicillin.
Role of the Calcium Ion in the Ochratoxin A-binding aptamer
Zachary R Churcher1, Meghan Osborne1, Cameron Mackreth2, Richard A Manderville3, Philip E Johnson1
1Department of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, Ontario, Canada
2Institut Européen de Chimie et Biologie, Université de Bordeaux, Bordeaux, France
3Departments of Chemistry and Toxicology, University of Guelph, Guelph, Ontario, Canada
The interaction of the ochratoxin A-binding aptamer (OTA-1) with its ligand ochratoxin A, and calcium are being studied by using a combinatorial NMR spectroscopy, Isothermal Titration Calorimetry (ITC), and Differential Scanning Calorimetry (DSC) approach to investigate the structure and function of the aptamer. OTA-1 is a monomolecular anti-parallel G-Quadruplex centred around a two-tetrad core, while 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 the brain and cause oxidative damage to DNA. We have used our multifaceted approach to study the OTA-1 aptamer using calcium as the ion to help facilitate binding between ochratoxin A and the aptamer. Our results show tight binding between the OTA-1 aptamer and ochratoxin A in the presence of calcium. We also find that the aptamer structure is highly stable in the presence of calcium and ochratoxin A. We investigated the role the calcium ion plays in the formation of the aptamer structure as well as how it helps facilitate the interaction between OTA-1 and ochratoxin A. A calcium or magnesium ion is needed for binding between the aptamer and ochratoxin A. Of those two, calcium allows for the strongest interaction between the OTA-1 aptamer and ochratoxin A. Not only is this cation needed for binding, but it also helps to structure the OTA-1 aptamer when ochratoxin A is not present. We have used our NMR data to help construct a 3D model of the OTA-1 aptamer with the ochratoxin A and calcium ion included. Overall, our results show the complexity of aptamer-ligand systems and how choices such as salt selection in buffers can impact the structure and function of an aptamer system.
Structure-affinity relationship of the dopamine-binding aptamer with dopamine derivatives
Hoi Pui Chao1, Yunus K. Kaiyum1, Cameron Mackereth2 and Philip E. Johnson1
1Department of Chemistry, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3
2 Institut Européen de Chimie et Biologie, 2 Rue Robert Escarpit-33607, Pessac, France
The dopamine binding aptamer, DA-Mut3 has been utilized to investigate the binding relationship of aptamer-dopamine ligand interactions because of its high specificity and selectivity. Dopamine is a neurotransmitter that controls emotion and movements in brains and deficiency of dopamine has been linked to Parkinson disease. It is a small ligand with one amine group attached via an ethyl chain and a catechol structure which is a benzene ring with two hydroxyl side groups. A series of ITC binding studies were performed to identify the changes in binding affinity with respect to modifications made to dopamine. The data suggests that the terminal primary amine group was a key factor in the recognition of the ligand for DA-Mut3, as modifications made to this site caused loss of binding. Additionally, the two phenolic groups as well as the aliphatic chain length appear to play a role in the interaction between DA-Mut3 and dopamine. The modifications that do not result in a loss of binding are limited indicating a high specificity of this aptamer for dopamine. Previous research done has shown that this aptamer exhibits structure switching characteristic with binding which along with its specificity for dopamine. A series of truncation studies of DA-Mut 3 helps to simplify the NMR data analysis. Interestingly, the removal of several bases at the 5’ end of were suspected to be adjacent to the binding site led to an increase in binding strength indicated by lower Kd values. The goal of this research aims to gain insight into how dopamine aptamer interacts with its ligand and this could help better designing biosensors.
The development of aptamers against human red blood cells for forensic detection
Hayley Costanzo, James Gooch, Nunzianda Frascione
King’s College London, Department of Analytical, Environmental & Forensic Sciences, London, SE1 9NH, UK
Blood is one of the most commonly found biological fluids at crime scenes, with the detection and identification of blood holding a high degree of evidential value. It can provide not only information about the nature of the crime but can also lead to identification via DNA profiling. Presumptive tests for blood are usually sensitive, but not specific, so small amounts of the substrate can be detected but false-positive results are often encountered which can be misleading. Novel methods for the detection of red blood cells based on aptamer-target interactions may be able to overcome these issues. Aptamers are single stranded DNA or RNA sequences capable of undergoing selective antigen association due to three-dimensional structure formation. The incorporation of a target specific sensing moiety into an optical biosensor means that limited cross reactivity can be achieved, which is a hinderance to current testing methods. Within this study, a modified Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process is used to generate aptamers against whole red blood cells via the cyclical target-incubation and enrichment of randomized oligonucleotide libraries. Obtained aptamer pools from Round 6, 8 and 10 were analysed via Massively Parallel Sequencing to identify viable sequences that demonstrate a high affinity for red blood cells. Through the use of bioinformatics platforms such as AptaSuite and Galaxy, 5 aptamer candidates were identified through their enrichment profiles. Binding characterisation of generated aptamers was also conducted via Enzyme-Linked Oligonucleotide Assay (ELONA) and Microscale Thermophoresis to quantify binding to the target. The calculated KD values have been estimated within the nanomolar range for all aptamer candidates (KD = 0.23 – 1.36 nM). Future work is focused on the incorporation of developed aptamers into a ‘turn-on’ optical biosensing platform that can be used at crime scenes for the real-time detection of human blood.
SELEX with fully-modified serum stable NA analogues
John RD Hervey1, Niklas Freund2, Gillian Houlihan2, Gurpreet Dhaliwal1, Philipp Holliger2 and Alexander I Taylor1
1Cambridge Institute of Therapeutic Immunology & Infectious Disease (CITIID), University of Cambridge, Cambridge, UK
2Medical Research Council Laboratory of Molecular Biology, Cambridge, UK
The in vitro evolution of functional nucleic acids (aptamers and NAzymes) by SELEX has typically been limited to natural DNA, RNA or partially modified polymer chemistries. The replacement of all four nucleotides with analogues has been challenging, limiting the structure space explored during SELEX and, for clinical applications, necessitates further engineering of resulting functional sequences by trial-and-error medicinal chemistry to reduce their degradation by serum nucleases, which can reduce or abrogate activity. By screening blends of engineered polymerases, we have established six synthetic genetic systems capable of efficient DNA-templated synthesis and reverse transcription of fully modified diverse-sequence (N40) libraries composed of serum nuclease-resistant nucleic acid chemistries with track records in development of oligonucleotide therapeutics: locked nucleic acid (LNA), 2′-O-methyl-RNA (2′OMe-RNA), as well as mixtures of the two. We demonstrate that, unlike previous SELEX setups, these systems enable preparation of libraries with resistance to human serum nucleases (stable in 90% human serum after five days at 37°C). We find that libraries can be prepared and replicated (via cDNA) with yields of synthesis and reverse transcription comparable to RNA systems. By deep sequencing N40 as well as defined sequences following complete cycles of replication and amplification, we find no measurable loss of diversity and aggregate error rates within suitable ranges for SELEX, including a pure LNA system (7.05 x 10-3) with fidelity comparable to a DNA only SELEX system (5.74 x 10-3). As proof-of-concept, we demonstrate discovery of novel serum-stable RNA-cleaving catalysts in the all-2’OMe-RNA system. Such ‘X-SELEX’ systems will expedite the development of biostable functional nucleic acids for biomedical and therapeutic applications.
Development of a label-free electrochemical cortisol aptasensor and automated plug-and-play sensor preparation strategy based on consumer-grade materials and processes
Alexandra Dobrea1,2, Rami Ghannam1, Damion K. Corrigan*2, Melanie Jimenez*2,3
1James Watt School of Engineering, University of Glasgow, UK
2Biomedical Engineering Department, University of Strathclyde, Glasgow, UK
3Institute of Infection, Immunity and Inflammation, University of Glasgow, UK
Cortisol is a steroid hormone that has gathered significant attention in the biosensing world due to its myriad physiological roles in health and disease. Because of its circadian rhythmicity and fast responsiveness to common sampling procedures (e.g., blood collection) and everyday stressors, multiple samples must be collected over the time period of interest (weeks or months) to gain a true understanding of its secretion patterns. However, state-of-the art cortisol quantification techniques such as mass spectroscopy are expensive and time consuming posing a roadblock to performing large-scale, long-term cortisol investigations with sufficiently high temporal resolution in a cost-effective manner. This work presents a proof-of-concept label-free low-cost electrochemical aptasensor based on a duplex dissociation principle, using an aptamer first reported by Yang et al. (2017). With a linear range of 625 – 10,000 pg/mL, the limit of detection was 625 pg/mL in spiked buffer; for reference salivary cortisol levels for healthy adults are 10.2–27.3 ng/mL (morning) and 2.2–4.1 ng/mL (night). Although these sensors hold great promise for reducing the costs of biomarker investigations, sensor preparation is often performed manually which increases the risk of sensor variability and lowers reliability. To address this issue, a low-cost fluidic cell was developed using laser-cut acrylic sheets and off-the-shelf stick-and-play tape to allow for precise delivery of reagents to the sensor surface. The flow cell design can be easily modified to fit any planar electrode format and was shown to significantly decrease SAM variability during sensor functionalisation (CoV dropped from 47% to 6%). Laser cutting allows hundreds of flow cells to be made within a short time frame, offering the possibility to upscale production. The fluidic design could also potentially be used for screening newly discovered aptamer sequences to rapidly assess their feasibility for biosensing applications.
High-throughput selection and characterisation of aptamers on optical sequencers
Alissa Drees, Christian Ahlers, Markus Fischer
Hamburg School of Food Science, Department of Chemistry, University of Hamburg, Germany
High-Throughput Sequencing-Fluorescent Ligand Interaction Profiling (HiTS-FLIP) is the first experiment to enable the quantitative measurement of millions of DNA-protein interactions in parallel. For this, it utilizes the capability of optical sequencers to perform fluorescence-based assays on the immobilized DNA-clusters subsequent to sequencing. Using different protocols between sequencing and screening of binding, HiTS-FLIP can be used to study a wide range of interactions of fluorescent molecules with ssDNA, dsDNA, RNA and even peptides. We successfully modified the common sequencer “Illumina MiSeq” to automatically perform custom high-throughput binding screenings. Only minor hardware modifications, i.e. additional tubing and an additional external valve, were required, making HiTS-FLIP for the first time available to anyone with access to a MiSeq. To demonstrate the potential of HiTS-FLIP, we exploited its’ capacity to enable high-throughput selection as well as characterisation of high-affinity aptamers for different medical-relevant target proteins within a period of a few days. Compared to the conventional selection of aptamers by Systematic Evolution of Ligands by Exponential Enrichment (SELEX), the required effort was significantly reduced while the information content about the binding interactions was drastically increased, providing potential data for machine learning approaches.
Targeting a hairpin containing G-quadruplex in HIV-1 RNA U3 Region with L-RNA Aptamer
Hengxin Feng1, Chun Kit Kwok1,2
1Department of Chemistry and State Key Laboratory of Marine Pollution, City University of Hong Kong, Kowloon Tong, Hong Kong SAR, China
2Shenzhen Research Institute of City University of Hong Kong, Shenzhen, China
G-quadruplexes (G4s) are special nucleic acid structures playing important roles in gene expression and becoming potential therapeutic targets. Recently, a stable rG4 with hairpin structure in loop position in human immunodeficiency virus 1 (HIV-1) genomic RNA U3 region has been reported, named LTR-III. Part of LTR-III is involved in the HIV-1 U3 region complementary to tRNA3Lys, which initiates the viral reverse transcription and promotes minus strand transfer in infected cells subsequently. Therefore, LTR-III is a potential target for HIV-1 diagnosis and interrupting minus strand transfer. However, targeting on specific G4 remains challenging. We performed in vitro selection to obtain L-RNA aptamer targeting on LTR-III, in which APP 3’ UTR rG4 is used in negative selection to reduce non-specific binding to other rG4s. During in vitro selection, the LTR-III and control oligos are converted into L-form, while the RNA pool remains D-form for amplification, subsequently converted back to D-LTR-III and L-aptamer for binding and selectivity test. The binding affinity is analysed by EMSA and MST, validating much stronger binding between the L-aptamer and D-LTR-III than other tested rG4s, confirming the selectivity of the L-aptamer. This LTR-III targeting L-RNA aptamer is a potential HIV-1 reverse transcription inhibitor, which can be further applied in HIV-1 diagnosis and therapeutics.
Unveilling novel triple negative breast cancer-associated biomarkers using an aptamer-based proteomic pipeline
Debora Ferreira1,2, Laura Figueiredo1,2, Catia Silva3,4, Hugo Osório3,4,5, Sergio F Sousa6,7 and Ligia R Rodrigues1,2
1CEB – Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal
2LABBELS – Associate Laboratory, Guimarães, Braga, Portugal
3i3S—Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal
4Ipatimup—Institute of Molecular Pathology and Immunology of the University of Porto, University of Porto, 4200-135 Porto, Portugal
5Department of Pathology, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal
6Associate Laboratory i4HB – Institute for Health and Bioeconomy, Faculty of Medicine, University of Porto, Alameda Professor Hernâni Monteiro, 4200-319 Porto, Portugal
7UCIBIO – Applied Molecular Biosciences Unit, BioSIM – Department of Biomedicine, Faculty of Medicine, University of Porto, Alameda Professor Hernâni Monteiro, 4200-319 Porto, Portugal
Breast cancer is a major public health problem worldwide. Particularly, triple negative breast cancer (TNBC), accounts for approximately 10-20% of all breast cancers and has a more aggressive phenotype and poorer prognosis. These factors, allied with the typical heterogeneity of breast cancer, make the search for new therapies essential. Therefore, the exploitation of novel and specific ligands, as aptamers, for use in TNBC-targeted therapies is of utmost importance. In this study, we combined cell-SELEX with high-throughput sequencing for the identification of novel ssDNA aptamer sequences that specifically recognize the metastatic TNBC cell line MDA-MB-231. After 8 rounds of evolved enrichment, the aptamer pool was sequenced. Two aptamer candidates, Apt1 and Apt2, were characterized considering their binding affinity to the target cells, with kd values at the nanomolar range. The potential translation to clinical applications was proven for Apt2 by immunofluorescence staining of tissue sections. To provide a proof-of-concept for the use of Apt2, this aptamer was conjugated with carboxylate-modified nanoparticles (NPs), creating NP-Apt2 conjugates. It was observed through fluorescence and flow cytometry that the NP-Apt2 conjugates led to an incredibly high fluorescence intensity compared to unconjugated NPs. Specifically, Apt2 seemed to be associated with cell membrane epitopes only present in the target cells. Aptamer-mediated pull-down coupled with proteomics gave us some hints about the possible biomarker to which this Apt2 binds to. After generating the 3D structures for Apt2, Apt2-biomarker docking studies were performed. Molecular dynamic simulations of the docked complexes and free binding energy calculations by molecular mechanics-generalized born surface area (MM-GBSA) allowed the identification of the potential biomarker to which Apt2 binds to. Ultimately, this novel ligand could be a truly helpful tool for targeted delivery in TNBC tumours.
The therapeutic potential of a novel aptamer-drug conjugate against Glioblastoma
Breanna L Giles1,2, Rasika M Samarasinghe1,2, Sarah L Shigdar1,2
1School of Medicine, Deakin University, Geelong VIC 3220, Australia
2Institute for Mental and Physical Health and Clinical Translation, School of Medicine, Deakin University, Geelong VIC 3220, Australia
The limited success of current treatment options for glioblastoma, an aggressive brain cancer with poor survivability can be attributed to the blood-brain barrier (BBB). This barrier is known to prevent entry of most chemotherapeutic drugs to treat brain cancer thus, a novel targeted therapeutic capable of crossing the BBB to deliver drugs is essential. Aptamers, or chemical antibodies, are small single-stranded nucleotide sequences that can bind specifically and selectively to desired targets on the cell surface. Most importantly, aptamers can be modified as a drug delivery vehicle for therapeutic purposes. In this instance, we previously generated a bifunctional aptamer by combining a transferrin receptor and epithelial cell adhesion molecule (EpCAM) aptamer together and intercalated the chemotherapeutic doxorubicin (DOX) for treatment of brain metastases. We were able to demonstrate that the bifunctional aptamer-drug conjugate, termed TEPP-DOX, was successfully able to deliver drug payloads across the BBB to EpCAM positive brain metastases both in vitro and in vivo, and reduce metastases spread and tumourigenicity. In this study, for the first time, this bifunctional aptamer-drug conjugate is being tested for treating the primary brain cancer, glioblastoma. To examine the potential of treating glioblastoma, the binding affinity of two bifunctional aptamers to glioblastoma was determined by flow cytometry, including with DOX conjugation, where a strong binding affinity towards the transferrin receptor on glioblastoma cells was observed. Next, the internalization and drug retention of aptamer-conjugates was visualized over a 96-hour period through confocal microscopy. With aptamer-DOX internalisation and drug retention visualised, the results of this study show that this conjugate would make an ideal therapeutic candidate for future studies.
Selection of aptamers for detection of blood coagulation status
Melis G Girbas1, Can Dincer2,3, Stefan Partel4, Meltem Avci-Adali1
1Department of Thoracic and Cardiovascular Surgery, University Hospital Tübingen, Calwerstr. 7/1, 72076 Tübingen, Germany
2Department of Microsystems Engineering (IMTEK), University of Freiburg, Georges-Koehler Allee 103, 79110 Freiburg, Germany
3FIT Freiburg Centre for Interactive Materials and Bioinspired Technology, University of Freiburg, Georges-Koehler Allee 105, 79110 Freiburg, Germany
4Research Centre for Microtechnology, Vorarlberg University of Applied Sciences, Hochschulstraße 1, 6850 Dornbirn, Austria
In clinical coagulation diagnostics, there is an urgent need for reliable testing devices to determine the coagulation status, especially during or after extracorporeal procedures. Infrequent and inaccurate measurements can lead to life-threatening complications, such as bleeding or thrombosis. Conventional coagulation tests require long turnaround times and costly equipment. Thus, they are inappropriate for on-site use. For an accurate diagnosis, it is essential to measure multiple biomarkers simultaneously. This project aims to implement a microfluidic sensor using aptamers for the assessment of blood coagulation status in real time. For this purpose, aptamers against proteins involved in blood coagulation, beta-thromboglobulin, plasminogen, and fibrinogen are selected. To select aptamers, systematic evolution of ligands by exponential enrichment is used. Every round, protein-DNA complex is bound to mixed cellulose ester membrane and aptamer candidates are amplified by polymerase chain reaction (PCR). The double-stranded DNA is digested with lambda exonucleases to receive single-stranded DNA. Before proceeding with the next round, the quality of the products is ensured by polyacrylamide gel electrophoresis. After multiple rounds, binding assays are performed and demonstrated the accumulation of putative aptamers against corresponding targets via real-time PCR. Selected aptamer pools are analyzed by Next-Generation-Sequencing and next, clustered aptamer sequences will be further tested regarding their binding affinity. The successful selection of beta-thromboglobulin, plasminogen, and fibrinogen-specific aptamers and their incorporation into biosensors could enable the real-time monitoring of blood coagulation.
Characterization of an aptamer inhibitor for hot start RTX, a novel reverse transcriptase
Jon C Graswich, Gwen Stovall
Department of Molecular Biosciences, UT Austin, USA
RNA amplification is critical in modern biochemistry, and it is very important that the reactions used to amplify RNA are accurate. However, current RNA amplification generally relies on reverse transcription (RT) to create cDNA. RT in its current form is an inaccurate process, as most reverse transcriptases lack an exonuclease domain to proofread base pairing. This process is commonly paired with polymerase chain reaction (amplification with exonuclease proofreading) to improve accuracy and yield. Consequently, a single enzyme reaction would be highly desirable. Reverse transcription xenopolymerase (RTX) is a novel reverse transcriptase evolved from a KOD DNA polymerase. RTX has a proofreading domain and is capable of performing nucleic acid amplification with RNA and DNA templates. RT reactions performed by RTX show evidence of reduced errors compared to traditional reverse transcriptases, however further improvements can be made by using an aptamer for a hot start application. Aptamers have previously been shown to have high potential for polymerase inhibition, as evidenced by AptaTaq, an aptamer inhibitor for Taq polymerase. Binding affinities for these applications are often on the same order of antibodies. It is also important that aptamers are resistant to degradation and relatively inexpensive to produce, making them both efficient and accessible for this purpose. In the presence of an inhibitory aptamer, RTX will be unable to bind targets at room temperature, consequently lowering mispriming incidents. Following ten rounds of aptamer selection, recurring sequences have been found in the pool via Sanger sequencing. Samples are currently being prepared for NGS, and an inhibition assay will be run on promising candidates identified via NGS. Additional testing will be done to examine the difference in transcriptional errors between RTX and the hot start RTX-aptamer complex once acceptable candidates have been identified and characterized.
A novel synthetic caffeine-binding riboswitch
Vincent J Gunawan, Leon Kraus, Beatrix Suess
Department of Biology, Technical University Darmstadt, Schnittspahnstraße 10, Darmstadt, Germany
The ligand binding capacity of aptamers can be utilized in a wide variety of ways. One of the evolutionary oldest ones might be the ability of RNA aptamers to recognize a ligand and thereby activating a functional RNA element. In this work, a synthetic RNA aptamer for caffeine with regulatory activity was identified and optimized. A caffeine binding aptamer-pool was enriched after 10 rounds of Capture-SELEX. Two candidates with regulatory activity were then identified in a subsequent screen of 1260 individual transformants of S. cerevisiae. There, the aptamers were inserted within the 5’UTR of GFP encoding mRNA and fluorescence measured in the presence and absence of 250 µM caffeine by flow-cytometry. The structure of an identified riboswitch was characterized by inline-probing and mutational studies to optimize its regulatory activity were performed. The optimized riboswitch exhibited a kD of 8,6 +/-3,3 µM and a 4-fold reduction of GFP expression in the presence of 250 µM of caffeine while retaining about 40% of the vector’s expression in the ligand free state. Afterwards, a caffeine-dependant hammerhead aptazyme was developed by replacing the P3 stem of the extended hammerhead ribozyme with the identified binding domain of the caffeine-riboswitch. The aptazyme construct was inserted into the 3’UTR of GFP encoding mRNA and transformants of S. cerevisiae were measured by flow cytometry. The aptazyme exhibited a 2.3 fold decrease in fluorescence in the presence of 250 µM caffeine while retaining about 60% of the fluorescence of an inactive A to G mutant in its ligand free state. The caffeine-binding domain was also fused to the DHBI binding domain of the iSpinach fluorogenic aptamer and fluorescence measured in vitro. The designed biosensor exhibited an increase in fluorescence between 5 and 2500 µM of caffeine where it reached about 80% of the fluorescence of the iSpinach aptamer.
Targeting highly homologous Flavivirus NS1 proteins with supremely specific Clickmers
Maren Hamann, Johanna Kauppila, Elisa Merklinger, Nora Karnowski
Clickmer Systems GmbH, part of APIS Assay Technologies Ltd., Marie-Curie-Str. 1, 52259 Rheinbach, Germany
Flaviviruses are arthropod-borne pathogenic ssRNA viruses. The genus includes the prominent members Yellow Fever Virus (YFV), West Nile Virus (WNV), Tick-borne Encephalitis Virus (TBEV), Dengue virus (DENV) and Zika Virus (ZIKV) which may cause meningitis, encephalitis, haemorrhagic fever and neurologic diseases. Their RNA genome encodes a polyprotein which is cleaved by host and viral proteases into 3 structural and 7 non-structural proteins. The non-structural protein 1 (NS1) is highly conserved across flavivirus species. Hexameric NS1 glycoprotein is secreted from infected cells, modulating the hosts immune response by interacting with the complement system and contributing to immune evasion as well as vascular leakage. It is therefore not only a promising diagnostic biomarker for infection but also a potential candidate for a therapeutic approach. NS1 can be detected in serum of infected individuals with NS1 concentrations ranging from as little as 30 ng/ml (ZIKV) up to 50 µg/ml (DENV). We performed Click-SELEX to discover Clickmers able to differentiate the highly homologous NS1 proteins of YFV, WNV, TBEC, DENV and ZIKV. The Clickmers show supreme specificity and can be used to detect NS1 from human serum in clinically relevant concentrations. With their outstanding specificity the NS1 Clickmers could aid in differential diagnosis of Flavivirus infections. Clickmers can bind to NS1 in presence of serum as well as full blood and show, in contrast to many NS1 Antibodies, no cross-reactivity towards Thrombin and Plasminogen. These properties can be advantageous in therapeutic approaches targeting NS1. With an in silico algorithm prototype we are now performing docking simulations and k-mer analysis to understand the role of DNA sequence plus modifications in target binding. We aim for a machine learning assisted fast and efficient in silico optimization of Clickmers through identification and modulation of binding sequence motifs and the required modifications.
Selection of naringenin aptamers using Capture-SELEX for sensor development
Bente K Hansen, Adrien Boussebayle, Ebbe Sloth Andersen
Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, Denmark
Flavonoids are natural polyphenolic compounds found in plants. One of these flavonoids is naringenin which have been shown to possess numerous beneficial effects on human health including antioxidant and anti-inflammatory properties. Here, we present a set of naringenin binding aptamers developed using Capture-SELEX. With this method structure-switching aptamers against the free molecule are selected and we used next-generation sequencing (NGS) analysis to identify the aptamers after 10 cycles of selection. The affinities of the aptamers were determined using microscale thermophoresis (MST) and we investigated the secondary structures of the aptamers using SHAPE probing. Building on the Capture-SELEX principle, we developed a strand displacement assay to measure naringenin concentrations in vitro. In this assay, the binding of naringenin to the aptamer displaces the Cy3-labelled capture-oligonucleotide (CO) and allows it to hybridize with a Cy5-labelled anti-CO, creating a FRET signal between the oligonucleotides. As a result. we have established a correlation between the FRET signal and naringenin concentration. Naringenin can be produced through the metabolic engineering of microorganisms using tyrosine as a precursor. To optimize the microbial production, we are working on using our naringenin aptamers to develop a Twister-based aptazyme for in vivo sensing of naringenin bioproduction.
Development of Inhibitory RNA Aptamers against Metallo–lactamases
Janis Hoetzel1,*, Claire Husser2,*, Michael Ryckelynck2, Beatrix Suess1,3
1Department of Biology, Technical University of Darmstadt, Schnittspahnstrasse 10, 64287, Darmstadt, Germany
2Université de Strasbourg, CNRS, Architecture et Réactivité de l’ARN, UPR 9002, Strasbourg, France
3Center for Synthetic Biology, Technical University of Darmstadt
The increasing occurrence of antibiotic-resistant bacteria is a growing threat for the public health worldwide. Combined with big pharmaceutical companies leaving the field of antibiotic compound development, bacterial infection could become the number one cause of death by 2050. To address this growing threat, we designed a high-throughput pipeline for the development of inhibitory RNA aptamers against proteins (DIRA). The pipeline consists of two parts: an initial screening for binding affinity via systematic evolution of ligands by exponential enrichment (SELEX) and a subsequent microfluidic screening for enzymatic activity which selects for inhibitory capabilities of the RNA sequences. The combination of these methods allows for a fast and effective development of inhibitory RNA aptamers against any given protein. Since bacteria expressing metallo–lactamases are particularly difficult to treat, we selected the Sao Paolo metallo–lactamase 1 (SPM-1) as the first target for our pipeline. First, we started with a randomized RNA pool and obtained an enriched pool of target-binding sequences. Second, we further selected the pre-selected pool for inhibition of the target protein activity via the microfluidic pipeline. After the first complete run through the pipeline, we obtained a highly enriched sequence that proved to bind SPM-1 with high affinity (89,8 ± 10,4 pM) and effectively inhibited the protein activity. The sequence was further characterized and reduced to a minimal binding motif that was only 37 nucleotides long and still served as an effective inhibitor. In the next step this minimal aptamer will be linked to a bacterial siderophore and tested for its in-vivo efficiency. With the functional minimal aptamer, we have proven the effectiveness of our pipeline and are theoretically capable of quickly developing inhibitory RNA aptamers against any given protein.
CCL22 inhibition by aptamers inhibit immune cell chemotaxis in vivo
Anna Jonczyk1, Marlene Gottschalk2, Matthew Mangan4, Eicke Latz4, Irmgard Förster2, Günter Mayer1,3
1Chemical Biology and Chemical Genetics, Life and Medical Sciences (LIMES) Institute, University of Bonn, 53121 Bonn, Germany
2Immunology and Environment, Life and Medical Sciences (LIMES) Institute, University of Bonn, 53115 Bonn, Germany
3Center of Aptamer Research & Development, University of Bonn, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany
4Institute of Innate Immunity, University Hospital Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany
The chemokines CCL22 and CCL17 are both ligands of the receptor CCR4 and were shown to promote allergic and inflammatory diseases. CCL17 mainly induces chemotaxis towards T cells and thus promotes inflammation. We previously reported 2’F-RNA aptamers that bind CCL17 and significantly improved symptoms in a contact hypersensitivity (CHS) mouse model. In contrast, to the pro-inflammatory action of CCL17, CCL22 is mainly associated with the recruitment of regulatory T cells (Treg). To investigate whether the inhibition of CCL22 promotes or reduces the inflammatory response, we developed a 29-nt DNA aptamer, named AJ2, to impair CCL22-mediated chemotaxis. AJ2 specifically binds to murine CCL22 and effectively inhibits the migration of CCR4+ T cells in a transwell-migration assay. To investigate how CCL22 inhibition affects allergic reactions, we studied AJ2 in vivo using a CHS mouse model. To protect AJ2 from rapid renal clearance and nuclease degradation we generated a modified analog, dubbed AJ2m, with a 20 kDa 5’-PEG tail and 3’-dT cap. AJ2m does not induce innate immune response, measured as TNF-α secretion of macrophages upon AJ2m treatment. The half-life of AJ2m was determined being 2 hours in mouse serum. The systemic application (i.p.) of AJ2m significantly improved the CHS symptoms measured by the ear-swelling response. To further investigate the therapeutic potential of AJ2 we applied a skin cream containing fluorescently labeled AJ2 ex vivo on mouse skin and visualized penetration of the aptamer by confocal microscopy. AJ2 was observed to penetrate the ear skin and was localized in the epidermis and dermis. We are currently investigating the topical application of AJ2 in a CHS mouse model. Our study validates CCL22 as therapeutic target using an aptamer-based approach. The CCL22-binding aptamer AJ2 inhibits chemokine-mediated chemotaxis and, thus, represents a promising tool for the treatment of allergic and inflammatory diseases.
Prediction of 30-days mortality in Danish Covid19 cohort by translation of unique blood protein signatures using the novel APTASHAPE technology
Asger Givskov Jørgensen1, Daniel Miotto Dupont1, Søren Fjelstrup1, Claus Bus1, Thomas Benfield3, Peter Garred4, Maria Møller3, Simone Israelsen3, Håkon Sandholdt3, Cecilie Hansen4, Peter Heegaard5, and Jørgen Kjems1,2.
1Interdisciplinary Nanoscience Center (iNANO) Aarhus University, 2Department of Molecular Biology and Genetics Aarhus University, 3Department of Infectious Diseases, Amager and Hvidovre Hospital, 4Rigshospitalet Copenhagen, 5Health Technology Technical University of Denmark
Coronavirus disease (COVID-19), caused by infection with SARS-CoV-2 virus continues to impact our economy and healthcare systems negatively. More than 673 million people have been registered as infected with SARS-COV-2, challenging the capacity of our healthcare systems due to the broad spectrum of clinical presentation, i.e., asymptomatic to death. Demanding the development of novel strategies to stratify patient populations to predict prognosis, guide treatment management, and focus resources. The molecular composition of blood reflects our physiology, health state, lifestyle etc., in many ways, an “ocean” of biological information challenging to access. We developed a novel technology, APTASHAPE, to translate this “difficult-to-read” language into readable digital information. The APTASHAPE technology utilises 2’-fluoro-protected RNA aptamer pools to decipher fluctuations in the protein compositions of blood, generating imprints of the protein composition that are translated to unique sample-specific patterns through the application of NGS and machine learning algorithms. Fjelstrup et al., demonstrated the APTASHAPE technology’s potential to characterise patient samples concerning non-cancerous vs bladder cancer and early-stage vs late-stage bladder cancer. We hypothesised that the technology could identify disease-specific patterns for COVID-19, allowing diagnosis and prediction of disease severity by measuring fluctuations in the plasma proteome. We performed plasma profiling of two Danish COVID-19 cohorts to identify aptamers discriminating healthy controls and COVID-19 patients and demonstrated that the 88 discriminatory aptamers identified could predict 30-day mortality in the validation cohort (n= 168). The developed assay predicts the 30-day mortality in females with high sensitivity and specificity with an AUC of 0.914. The technology possesses the potential to serve as a health-state measuring and prediction tool for treatment management.
Tertiary structure determination of a dopamine aptamer – structure assignment by NOESY and proton exchange rate NMR techniques
Yunus A Kaiyum1, Hoi Pui Chao1, Cameron Mackereth2, Philip E Johnson1
1Department of Chemistry, York University, Toronto, ON, Canada
2Institut Européen de Chimie et Biologie, Université de Bordeaux, Bordeaux, France
Aptamer function, specificity and selectivity are widely a consequence of the 3-dimensional structures they adopt when binding a ligand and understanding the specific mechanisms by which structure and function are related within aptamers can open avenues for specific biosensor and therapeutic design. Previous research had selected an aptamer to bind the neurotransmitter dopamine with a sub-micromolar-scale affinity in a counter selection process against similar analogues. Initially thought to undergo ligand-induced structural change to form a G-quadruplex however, recent 2D NMR studies contraindicate a G-quadruplex. Several mutations were made to a DA Mut3 Del7 variant of the aptamer to probe this alternate structure and interestingly it appeared that the removal of several bases that are thought to partially occupy the binding site for dopamine, resulted in a 25-fold stronger binding. A subsequent 2D NOESY study was performed on this modified variant, denoted as RKEC1, to determine if the optimization of this sequence lead to an overall more rigid structure. Proton exchange rate experiments were performed to determine the rate of imino proton exchange with surrounding solvent to determine the degree by which a base may be protected or buried within the greater aptamer structure in the bound state. This can further lend support to the proposed tertiary structure for this aptamer and further explain observations made regarding the binding studies of these variants of the dopamine-binding aptamer.
Development of novel, synthetic riboswitches with RNA capture-SELEX
Leon Kraus1,2, Elke Duchardt-Ferner3, Beatrix Suess1,2
1Department of Biology, Technical University of Darmstadt, Schnittspahnstrasse 10, 64287 Darmstadt, Germany
2Centre for Synthetic Biology, Technical University of Darmstadt, 64287 Darmstadt, Germany
3Institut für Molekulare Biowissenschaften und Zentrum für Biomolekulare Magnetische Resonanz (BMRZ) Goethe-Universität Frankfurt, Max-von-Laue Str. 9, 60438 Frankfurt, Germany
One of the most exciting areas of synthetic biology is to control cellular behaviour using engineered genetic circuits. The expression levels of the corresponding genes need to be regulated and fine-tuned to avoid unbalanced gene expression and the accumulation of toxic intermediates. Synthetic RNA-based systems have increasingly been used for the regulation of gene expression and can be employed for the control of translation initiation, pre-mRNA splicing, RNA self-cleavage as well as fluorogenic aptamers. Due to their structural properties, riboswitches provide a convenient basis for the development of ligand-dependent, controllable systems. Here we present the selection and engineering of a synthetic tobramycin riboswitch with the use of RNA capture-SELEX. We were able to achieve a 17.7-fold regulation and could identify two separate binding sites for tobramycin using isothermal titration calorimetry (ITC) and nuclear magnetic resonance spectroscopy (NMR). Finally, we were able to engineer a minimal regulating sequence with retained switching behaviour.
Novel Early Diagnostics for Oesophageal Cancer Using Novel Aptamers and Epigenetic Panel
Louise R Lawless, Niamh Gilmartin, Therese Murphy
School of Biological and Health Sciences, TUDublin, Grangegorman, 191 North Circular Road, D07 EWV4, Ireland
Oesophageal cancer (OC) is currently the 8th most common form of cancer worldwide with the 5-year survival rate being around 10-15%. The high mortality rates are mostly linked to the absence of early clinical symptoms and that the cancer is usually detected in later stages. Due to this lack of early clinical symptoms, there is a great need for early diagnostic and prognostic biomarkers. Early biomarkers would also aid in treatment options and detection of reoccurrence. Patients have a much lower mortality rate if OC is caught in the first stages with a five-year survival rate of approximately 41-45%. Once the cancer has metastasised into other organs or to surrounding lymph nodes, this excludes surgery as a treatment option and further treatments are far less effective. Endoscopy and biopsy are currently considered the gold standard for diagnostics in OC but both are invasive and are often carried out at later stages of cancer. Barrett’s Oesophagus (BO) is a pre-cancerous condition that occurs when the normal squamous epithelium in the oesophagus is transitioned to columnar lined cells, and is the only known precursor to OC and may also have aptamer and epigenetic diagnostic potential. There is a general lack of knowledge surround the biology in OC and therefore aptamers and epigenetics has been a major area of research in terms of biomarker discovery. There is currently a lack of aptamers targeting early OC specifically and a potential novel aptamer will be used in conjunction with an epigenetic panel to produce a novel, non-invasive method for OC diagnostics. Initial rounds of cell-SELEX have been used to produce an aptamer and has been amplified using PCR. Flow cytometry has also been used to determine the binding potential of current OC associated aptamers (e.g., SYL3C) and, in the future, of the potential novel aptamer. This work shows that an aptamer can be generated using these methods and its diagnostic potential will continue to be investigated.
Selection and development of aptamers against Anaplasma phagocytophilum, a tick-borne, zoonotic bacterium
Lisa Lucie Le Dortz1, Clotilde Rouxel1, Quentin Leroy1, Frédéric Ducongé2, Henri-Jean Boulouis1, Nadia Haddad1, Pierre Deshuillers1 and Anne-Claire Lagrée1
1Anses, INRAe, Ecole Nationale Vétérinaire d’Alfort, UMR BIPAR, Laboratoire de Santé Animale, Maisons-Alfort, F-94700, France
2CEA, Fundamental Research Division (DRF), Institute of Biology François Jacob (Jacob), Molecular Imaging Research Center, CNRS UMR9199, Paris-Saclay University, 92265 Fontenay-aux-Roses, France
Anaplasma phagocytophilum is a tick-borne, zoonotic bacterium of great interest in animal and human health. It is the agent of granulocytic anaplasmosis, reported in USA, Europe and Asia. Many questions remain, regarding the variability and links between strains infecting different animal species and/or humans, their epidemiological cycles and the mechanisms of host/pathogen interactions. This lack of knowledge results from the difficulty to isolate and cultivate A. phagocytophilum, due to its strict intracellular nature and short life span in samples (granulocyte neutrophils) as well as the paucity of research tools (few or no monoclonal antibodies). Aptamers are innovative and powerful tools in infectiology employed as valuable substitutes to antibodies. These oligonucleotides are able to bind with high affinity and specificity to a wide variety of targets (e.g. proteins). Thereby, our team is working on the selection of aptamers directed against A. phagocytophilum and/or molecules expressed during infection. The first step consisted in selecting aptamers against infected cell lysates by performing 12 rounds of SELEX. The progress of selection was monitored by quantitative PCR and NGS sequencing and demonstrated an enrichment of sequences from round 6. Three different oligonucleotides were chosen for further binding and specificity studies. The first results are promising as they suggest that certain sequences could be specific to proteins expressed during infection or to A. phagocytophilum. Aptamers specific to molecules expressed during the infection will help to better understand the interaction between A. phagocytophilum and its hosts, by identifying the target of aptamers and by studying their function. Aptamers specific to A. phagocytophilum are a real opportunity to develop a capture method of bacteria directly from blood samples for in-depth genomic studies and for diagnostic purposes in animals and humans. In this context, several objectives will be pursued: to better explore epidemiological cycles, to understand the mechanisms involved in virulence and species barriers and to identify new potential vaccine and therapeutic targets in the longer term.
Selection of sialic acid-modified aptamers targeting influenza A virus
Kevin Neis1,4, Julián Valero1,2,3, Jørgen Kjems1,2,3,4
1Interdisciplinary Nanoscience Center, Aarhus University, DK-8000 Aarhus, Denmark
2Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus, Denmark
3Centre for Cellular Signal Patterns (CellPAT), Aarhus University, Aarhus DK-8000, Denmark
4Center for Multifunctional Biomolecular Drug Design (CEMBID), Aarhus University, Aarhus DK-8000, Denmark
Traditional influenza therapies face challenges due to the constantly evolving nature of the virus, which can lead to drug resistance and reduced recognition by the immune system. In this context, aptamers show great promise as new alternatives for neutralizing influenza viruses. However, reported aptamers targeting influenza hemagglutinin are limited by the restricted chemical space of their canonical nucleobases. To address this issue, this study introduces a modified SELEX method using sialic acid for RNA modification, with the aim of improving selection of high affinity aptamers to viral hemagglutinin, and potentially other influenza virus proteins, for virus neutralization. The results consistently show high transcription yields using sialic acid modifications and preservation of modified nucleotides after multiple SELEX rounds. Sialic acid-modified aptamers with low nanomolar affinities to influenza A H1N1 virus hemagglutinin have been selected, and further research will examine the effect of these modifications on RNA-target interaction. This study highlights the potential of utilizing natural influenza ligands to broaden the chemical space of RNA aptamers targeting influenza.
The analysis of the affinity and stability of modifications of the glucose-binding and cocaine-binding aptamers
Meghan T Osborne1, Zach Churcher2, Philip E Johnson2
1Department of Biology, York University, Toronto, Ontario, Canada
2Department of Chemistry, York University, Toronto, Ontario, Canada
Aptamers are selected to bind to their ligands, usually with high affinity and selectivity for their targets. Due to this aptamers can be used as sensing molecules in several different biosensor applications. Previously published research by the Stojanovic group selected an aptamer to bind glucose, but not any structurally related carbohydrates. The Kd of this aptamer-ligand interaction is only 10mM, which is much lower than the nM affinities seen in other aptamer-ligand systems. This is of physiological relevance as the blood glucose concentration usually falls between 4 mM to 11 mM, meaning it can be used in possible in vivo applications. Glucose is a difficult molecule to select an aptamer for as there are no strong epitopes present of glucose for DNA bases to interact with. NMR spectroscopy is suited to study weaker aptamer-ligand interactions such as these due to the greater concentrations required for NMR relative to other methods. 1H NMR spectroscopy was used to investigate the affinity, specificity, and stability of the wild-type glucose aptamer, and to try and engineer an aptamer variant with a stronger affinity for glucose than the wild-type aptamer that could be useful for future biosensing applications. The stability of high affinity glucose binding modifications will be studied using differential scanning calorimetry (DSC) similar to an analysis performed on the cocaine-binding aptamer. The cocaine-binding aptamer is a DNA aptamer selected to bind cocaine and is composed of three stems constructed around a three-way junction with 2 A-G mismatches at the centre. DSC is being used to investigate the thermostability of the cocaine-binding aptamer as a function of how many base pairs are in stem one. The length of stem one can change how tightly the aptamer binds a ligand, and how well defined the free state of the aptamer is. This was done with the hope of determining which stem one variant is the most thermodynamically stable.
Optoribogenetics: Light dependent control of cellular processes through PAL-aptamer platform
Tejal Patwari1, Günter Mayer1,2
1Life and Medical Sciences Institute (LIMES), University of Bonn, Bonn, Germany
2Center of Aptamer Research & Development Sensory, University of Bonn, Bonn, Germany
photoreceptors nurture light-dependent adaptations in nature and enable the optogenetic control of organismal behaviour and physiology. One such photoreceptor is PAL which was identified in the a gram-positive actinobacterium Nakamurella multipartite. The acronym PAL is derived from its unique architecture, comprising of Per-ARNT-Sim (PAS), AmiR and NasR transcription antitermination regulators (ANTAR) and light-oxygen-voltage (LOV) domains. A crystal structure reconciles the unusual receptor architecture of PAL with C-terminal LOV photosensor and N-terminal effector units. Using Opto-SELEX, a short RNA hairpin aptamer, named 53.19, was selected for PAL protein as a target. 53.19 binds to the light state of the protein with around 20nM affinity and weaker than 1µM in darkness. The PAL-RNA aptamer complex was exploited to regulate the translation of target mRNAs in a light-switchable manner in mammalian cells. Herein, aptamer 53.19 was embedded in the 5’ untranslated regions (UTRs) of the Luciferase gene. Upon light irradiation, PAL binds to the aptamer thus blocking the translation and reduction of Luciferase expression in the cells. Additionally, a novel method for controlling gene expression by light was developed using the PAL-aptamer system (CRISPRa-PAL). We used a CRISPR/dCas9 system (CRISPRa) that binds to a specific DNA sequence and recruits a transcriptional activator domain. The activator domain was fused to PAL and the RNA aptamer was embedded in sgRNA of the CRISPRa system. Here, PAL changes its conformation when exposed to blue light, which triggers the binding of the aptamer and activates gene expression of Blue Fluorescent Protein (BFP) in mammalian Cells. This system allows for reversible and tunable regulation of gene expression with minimal genetic engineering and low background activity in the dark. Furthermore, the CRISPRa/PAL system is being established in vivo to control the gene expression in Drosophila Melanogaster as a model organism. Successful development of such a system will add up to the existing gene expression tool box and provides a new approach for investigating complex cellular processes with spatiotemporal precision.
Selection of Single-Stranded DNA Molecular Recognition Elements Against Cyanotoxin L-BMAA
Xaimara Santiago-Maldonado1, José A. Rodríguez-Martínez2, Eduardo Nicolau-López1
1Department of Chemistry, University of Puerto Rico, Río Piedras Campus, San Juan, Puerto Rico, USA
2Department of Biology, University of Puerto Rico, Río Piedras Campus, San Juan, Puerto Rico, USA
-N-methylamino-L-alanine (L-BMAA) is a water-soluble non-protein amino acid that has been linked to neurodegenerative disorders such as Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex (ALS/PDC). Currently, the detection of L-BMAA is challenging and often unreliable, thus, the development of analytical techniques for its detection in aquatic environments is imperative. Molecular recognition elements (MREs), such as aptamers, provide the required analyte specificity for bioanalytical applications. Herein, we study the isolation of L-BMAA-specific MREs using the in vitro selection of single-stranded DNA (ssDNA) aptamers through the systematic evolution of ligands by exponential enrichment (SELEX). To achieve this, heterogenous and homogenous SELEX strategies were applied. A total of 18 selection rounds were performed using the Beads-SELEX (MB-SELEX) method and 13 rounds were completed using graphene oxide-assisted SELEX (GO-SELEX). Chosen rounds were sent for sequencing using the Illumina next-generation sequencing technology and analyzed using a combination of bioinformatic tools. The GO-SELEX method did not show significant changes in the pool composition or motif enrichment whereas the MB-SELEX method did show a significant shift in the base distribution, which was further supported by the presence of G-rich conserved motifs that were enriched across the selection. The prevalence of these motifs suggests that G-rich sequences might be relevant for L-BMAA binding. A total of 6 candidates were chosen to screen for binders. Preliminary results obtained with the SYBR Green (SG) fluorescent assay suggest the potential binding of two of these aptamers, with affinities within the low micromolar range. A full characterization of these L-BMAA-binding oligonucleotides will be performed using isothermal calorimetry (ITC). The successful identification of these sequences will set a precedent as the first selected aptamers against the cyanotoxin L-BMAA.
Structural insight in an aptamer-small molecule complex using NMR spectroscopy – the TESS.1_s_mod case
Sofie Schellinck1, Nataša Medved2, Dieter Buyst1,4, Annemieke Madder1, Karolien De Wael3, Janez Plavec2, José C. Martins1,4
1Department of Organic and Macromolecular Chemistry, Ghent University, Ghent, Belgium
2National Institute of Chemistry, Ljubljana, Slovenia
3Department of Bioscience Engineering, University of Antwerp, Antwerp, Belgium
4NMR Expertise Centre, Ghent University, Ghent, Belgium.
It is well accepted that detailed knowledge of aptamer-ligand structures would benefit our fundamental understanding of these systems and would benefit the design and optimisation of aptamers towards affinity and selectivity, thus contributing to the development of real-world applications. To contribute to filling the current knowledge gap, we are currently focussing on NMR spectroscopic investigations of the structure-switching TESS.1 aptamer, a testosterone binding aptamer proposed and extensively characterized by the Stojanovic group. NMR spectroscopy allows to acquire molecular level information about conformational changes and intermolecular interactions, making it a powerful technique for studying aptamer-ligand interactions. At this 51-nucleotide length, the original aptamer presents challenges both for synthesis and NMR spectroscopy. Therefore, the sequence has been truncated and further optimized to generate a more ‘NMR optimal’ 30-nucleotide long construct, labelled TESS.1_s_mod, which interacts with testosterone in a similar fashion as the originally sized aptamer TESS.1. We will present and discuss recent results that provide the first detailed molecular view on this aptamer and its interaction with the testosterone target.
Development of computational methods for aptamer design for biomedical applications
Sérgio F Sousa
UCIBIO@REQUIMTE – BioSIM, Dept Biomedicina, Faculdade de Medicina da Universidade do Porto, Portugal
Biomolecular simulations have long been an important part of the drug discovery and development process, with techniques such as docking, virtual screening, molecular dynamics and quantum mechanics being routinely used in the study of the interaction and selection of small molecular drugs with their target proteins or enzymes.More recently, the application of these techniques in aptamer selection and aptamer engineering has algo become a reality. Such methods can help to understand aptamer-target interaction and to rationally introduce modifications in selected aptamers to modulate their affinity, specificity or ability to carry other molecules. Here, we present a computational protocol developed by us for the selection of specific aptamers for protein recognition. The protocol combines protein-DNA docking, atomistic molecular dynamics simulations and free energy calculations, including the conformation variability of the protein and aptamer in the selection process. As a proof of concept, the protocol was applied to a set of 20 ssDNA aptamers comprised by 50 nucleotides each previously experimentally identified from the most prevalent sequences present in the pool obtained after 8 rounds of systematic evolution of ligands by EXponential enrichment (SELEX) for Cathepsin B, a protein overexpressed in prostate cancer cells. The computational protocol was able to identify the aptamer with the highest affinity towards Cathepsin B and to predict its interaction mode and dominant interactions with Cathepsin B with atomic level detail. In conclusion, this protocol can be used in the selection of aptamers for the recognition of specific protein targets and in rational customization of specific aptamers for improved protein affinity or specificity, working as a useful tools in understanding and engineering molecular recognition.
Binding affinity of structure-switching aptamers as a function of NaCl concentration
Kabisan Thavaseelan, Matthew Bowman, Philip E. Johnson
Department of Chemistry, York University, Toronto, ON, Canada
The goal of this research is to see how structure-switching aptamers behave towards ligand binding as a function of NaCl concentration. Many intermolecular forces contribute to the binding of the ligand to the aptamer such as Van der Waals, hydrogen bonding and electrostatic interactions. Since structure-switching aptamers are unfolded or loosely folded when in the free-state and only become structured when bound to their corresponding target ligand, the contribution of electrostatic forces is the primary interest for this research. Increasing NaCl concentrations are used because the Na+ ions produce a shielding effect on the negatively charged phosphate backbone on the DNA aptamer and therefore, disrupting the binding between the aptamer and ligand through electrostatic interactions. Non-structure-switching aptamers have been studied previously in the Johnson lab as a function of NaCl concentration and it was shown that aptamer affinity increased towards their ligand when NaCl concentration was decreased. Since structure-switching aptamers become more structured when binding to their target, we want to see if there is a parallel relationship with non-structure-switching aptamers under the same conditions. This will be investigated by analyzing the binding affinity (Ka) as a function of NaCl concentration through isothermal titration calorimetry (ITC) with various structure-switching aptamers. This includes MN19 and DAMut3, which are variants of the cocaine-binding aptamer and the dopamine-binding aptamer. Using Ka values obtained from ITC at different NaCl concentrations, the log of the Ka values can be plotted against the log of the NaCl concentrations. The slope of this data provides an indication of the electrostatic contribution for the affinity of the aptamer towards its ligand. Overall, we can see if structure-switching aptamers function similarly to non-structure-switching aptamers under the same conditions or if they can be differentiated from them.
A workflow for COVID-19 aptamer selection and characterisation
Sireethorn Tungsirisurp, Nunzianda Frascione
Department of Analytical, Environmental and Forensic Sciences, King’s College London, London, SE1 9NH, UK
DNA aptamers are short, single-stranded oligonucleotides developed with selective binding interactions for specific targets. Aptamers are selected through a laborious process (systematic evolution of ligands by exponential enrichment, SELEX) and their target binding properties are characterised using various techniques. In this study, a thorough literature research was initially performed to identify and select for developed DNA aptamers against SARS-CoV-2 virus. Subsequently, various biophysical techniques were used to experimentally characterise the aptamer binding properties against the receptor binding domain (RBD) protein. These techniques include enzymatically linked oligonucleotide assay (ELONA) and surface plasmon resonance (SPR) for binding affinity (dissociation constant; KD) and kinetics evaluation. The binding studies on an aptamer ‘1C’ using ELONA shows a comparable estimated KD to the literature at 3.05 ± 1.64 nM. However, a KD estimated using SPR is 10-fold higher (30.77 ± 2.11 nM) suggesting the variability of aptamer binding parameters depending on the techniques used. Moreover, circular dichroism (CD) was used for structural studies of the aptamer and aptamer-target complex. The CD spectra suggest that the tertiary structure of an aptamer ‘1C’ contains a parallel G-quadruplex which is enhanced in the presence of RBD protein. Different in silico simulations were also used to further characterise the DNA aptamers and their target interactions. Web-based servers such as MFold and QGRS Mapper were used for the aptamer secondary structure and G-quadruplex formation predictions, respectively. It is believed that this combinative workflow of experimental and simulations will allow a more in-depth assessment of aptamer-target interactions and appropriate aptamer-based biosensing design.
Development of multiple B-cell aptamers against CD19 and CD20 antigens using Multi-target Ligand-Guided Selection (LIGS)
Nicole B Williams1, Sana Batool2, Hasan Ekrem Zumrut3, Mohammad Jamal2, German Sosa2, Prabodhika Mallikaratchy1,2,3
1Ph.D. Program in Molecular, Cellular and Developmental Biology, CUNY Graduate Center, The City University of New York, 365 Fifth Avenue, New York, New York 10016, USA
2Department of Molecular, Cellular and Biomedical Sciences, CUNY School of Medicine, New York, NY 10031, USA
3Ph.D. Programs in Chemistry and Biochemistry, CUNY Graduate Center, The City University of New York, 365 Fifth Avenue, New York, New York 10016, USA
Nucleic Acid Aptamers (NAAs) are single-stranded DNA or RNA molecules which fold into complex functional three-dimensional structures. These three-dimensional structures can bind to their target molecules with high affinity and specificity. The method used to select Nucleic Acid Aptamers is an in vitro iterative process called Systematic Evolution of Ligands by Exponential enrichment (SELEX). We recently introduced Ligand-Guided Selection (LIGS), a variant of SELEX, to partition multiple aptamers against B cell antigens expressed in B cells, namely, CD19 and CD20, from an evolved cell-SELEX library. Here, multi-target LIGS takes advantage of the evolutionary step in cell-SELEX by introducing a high-affinity monoclonal antibody (mAb), such as anti-CD19 or CD20, to partition aptamers bound to pre-determined targets, CD19 or CD20, at 25°C. The evolved cell-SELEX and LIGS libraries were sequenced using the Illumina high-throughput (HT) DNA sequencing platform. The sequences were analyzed using FASTAptamer and Galaxy by designing a novel bioinformatics workflow. Two major sequence families consisting of 18 hit sequences for CD19 and 13 hit sequences for CD20 were identified based on defined fold-enrichment values. The potential hit sequences were screened against CD19/CD20 positive or negative cell lines to identify aptamers specific for each respective antigen (CD19 or CD20). Non-essential bases from CD19- and CD20 full-length aptamers were removed to minimize the aptamers to their smallest functional size. Their co-localization on the cell surface with the respective mAb was demonstrated using microscopy. This study establishes LIGS as a state-of-the-art screening technology that can partition highly specific aptamers against complex targets, such as cell surface receptors (proteins) expressed in their natural state.
Bispecific DNA aptamers constructed by MACE®-SELEX aptamers as analytical reagent and therapeutic drug
Keitaro Yoshimoto1, 2
1Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1, Komaba, Meguro-ku, Tokyo, Japan, 153-8902
2LinkBIO Co. Ltd., The ICI Center, 5270 Terada, Toride-shi, Ibaraki, Japan, 302-0021
We recently established the microbead-assisted capillary electrophoresis (MACE®)-SELEX method, which allowed accelerated identification of bioactive aptamers via specific enrichment under high exclusion pressure through capillary electrophoresis of aptamers that bind to proteins of interest. Although multiple human thrombin aptamers have been reported, M08 showed the highest affinity and in vitro anticoagulant activity with strong affinity (Mol. Ther. Nucleic Acids, 16, 348, 2019). An 87‐meric bispecific TBA aptamer containing M08 and rapid neutralizing activities of the bispecific aptamer with a 34‐meric short‐length antidote was successfully developed (Res. Pract. Thromb. Haemost., 5, e12503, 2021). As many as fifteen DNA aptamers with high-affinity against TNF-α were obtained and the selected Apt14 showed the highest binding affinity among previously reported monomeric aptamers. Homo-and hetero-dimerization through hybridization successfully improved not only the binding affinity but also the inhibitory activity of Apt14, which became a more potent inhibitor of the TNFα-TNFR1 interaction (ChemBioChem, 22, 3341, 2021). The methotrexate (MTX) detection system was established by linking the MTX-binding aptamer MSmt7 to hemin-G-quadruplex DNAzyme (PW17) and enhanced sensitivity to MTX was achieved by amplifying PW17 by RCA, which drastically lowered the LoD to 0.290 nM in 50% human serum (Anal. Chem., 94, 17255, 2022). LinkBIO Co. Ltd. in Japan started an aptamer discovery service using MACE®-SELEX and MACE® is a trademark of LinkBIO.