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(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.
Cotranscriptional RNA strand invasion mediates ligand sensing and gene regulation in multiple riboswitches
#KBerman #AptOx22
Katherine Berman 1, Laura M Hertz 1, Angela M Yu 2, Russell Steans 3 and Julius Lucks 4
1 Interdisciplinary Biological Sciences Graduate Program, Northwestern University, Evanston, IL 60208, USA
2 Department of Electrical and Computer Engineering, University of Washington, Seattle, WA 98195, USA
3 Weinberg College of Arts and Sciences, Northwestern University, Evanston, IL 60208, USA
4 Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208, USA
RNA folds immediately as it is transcribed by RNA polymerase, thereby creating RNA structures faster than new nucleotides being added to an emerging RNA chain. Thus, cotranscriptionally folded RNA structures can differ greatly from equilibrium folded structures. Riboswitches are structured RNA elements that respond to a variety of small molecules to regulate transcription, translation, splicing, and mRNA degradation. Riboswitches use cotranscriptional RNA folding pathways to regulate gene expression by altering the folding pathway depending on the ligand binding. Here, we investigate the hypothesis that the E. coli thiB TPP riboswitch functions through an RNA strand invasion mechanism by which TPP binding blocks a key rearrangement event that hides the ribosome binding site, preventing translation. To examine this hypothesis, we used cotranscriptional SHAPE-seq, a technique which combines chemical probing, precise polymerase arrest and NGS, to study intermediate structures in the folding pathway. We then used secondary structure modeling informed by SHAPE data to reconstruct a riboswitch folding pathway as well as compare equilibrium refolded structures to dynamic, cotranscriptionally folded structures. These studies revealed a central hairpin that is essential for exposing the ribosome binding site. Functional gene expression studies further revealed details in the role of the strand displacement in TPP sensing and regulation. Moreover, we have found strand displacement utilized by several riboswitches, including the fluoride transcriptional riboswitch. We functionally show that a single wobble base pair within the strand displacement sequence regulates the riboswitch transcriptional function at 37 °C. Thus, this combinatorial work along with recent publications on the Adenine riboswitch, ZTP riboswitch and the signal recognition particle (SRP) RNA have demonstrated the prevalence of strand invasion in RNA cotranscriptional folding pathways.
Designing a positive cooperative binding of bifunctional aptamer
#HPChao #AptOx22
Hoi Pui Chao, Sladjana Slavkovic and Philip E Johnson
Department of Chemistry, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3
Aptamers are short single-stranded nucleic acid molecules that bind ligands and are widely used in biotechnology as biosensors. In our lab, we study cocaine-binding aptamers using quinine as the ligand as we’ve shown it to binds 50 fold tighter to this ligand than cocaine. I wish to design a bifunctional aptamer that displays positive cooperativity. We believe that an aptamer with positive binding cooperativity will be more sensitive in biosensor applications. Binding experiments are performed using Isothermal Titration Calorimetry (ITC) to quantify the strength of the interaction between quinine and aptamers. ITC provides useful information such as the thermodynamics and affinity of binding. In my work, a variant of the cocaine-binding aptamer (MN25) is being used in the experiments because as it has an extension at the 5’end that is complementary to another MN25 molecule. It has two base pairs in stem one and should be unfolded in the free state. Upon quinine binding at one site, a second MN25 molecule should associate, form an inter-aptamer helix and the formation of this helix should structure the second MN25 molecule and a second quinine molecule can come in and bind the second MN25 tighter than the first quinine. A goal of this project is not only to fabricate a more sensitive biosensor for cocaine, but is to see how aptamers can be manipulated in general in order to better design their functions and be able to apply what we learn to other aptamer systems.
Identification of clickmers that bind to prostate cancer cells with multiple modifications
#MChoukeife #AptOx22
Moujab Choukeife, Günter Mayer
University of Bonn, LIMES Institute, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany
In terms of therapeutics and diagnostics, aptamers possess a great deal of potential. There is, however, limited chemical diversity of nucleobases in nature, thereby limiting the interaction with the target. It may be possible to overcome this obstacle by adding functional groups to the nucleobases in order to increase chemical diversity in aptamers. Split-combine SELEX has been developed in order to screen multiple modifications to the target of interest in one selection. By using this method, we have been able to enrich a variety of clickmers with different modifications in one selection or the most suitable modification to the selected target. In this study, we applied this technology to prostate cancer cells. We identified a new class of clickmer that is capable of recognizing prostate cancer cells modified with different modifications. Each modification affects the binding to prostate cancer cells. In addition, these clickmers exhibited different binding properties when a variety of modifications were applied to them. Consequently, S1 aptamer can also recognize MCF-7 cells when modified with one modification, but not with the other modification.
Reduction in Dynamics of Base Pair Opening upon Ligand Binding by the Cocaine-Binding Aptamer
#ZChurcher #AptOx22
Zachary R. Churcher, David M. Garaev, Howard N. Hunter, Philip Johnson
Department of Chemistry, York University, Toronto, ON, Canada.
The effect of ligand binding on the base pair dynamics in the cocaine-binding aptamer was investigated using NMR spectroscopy. The cocaine-binding aptamer consists of three stems attached via a three-way junction with a tandem A-G mismatch and a dinucleotide bulge. The cocaine-binding aptamer shows off-target ligand binding with quinine and binds quinine ~50x times tighter than its selected ligand, cocaine. The base pair dynamics of the aptamer was investigated by measuring the imino proton exchange rate constant (kex) using magnetization transfer as a function of temperature in the free, cocaine-bound, and quinine-bound aptamers. The imino proton resonances were selectively magnetized and the intensity of the peak was measured as a function of time. The decay rate of these peaks was measured and used to calculate the kex values for each resonance at a specific temperature. This was done for two variants of the cocaine-binding aptamer, one with a longer stem 1 (MN4), and one with a shorter stem 1 (MN19). Because of the difference in the length of stem 1, MN4 is more structured in the free state, while free MN19 is more loosely folded and dynamic. Our study showed a reduction in both kex values and a decrease in the rate of how kex values increased with temperature at the ligand binding site. We saw little change elsewhere in the aptamer with ligand binding. It was also seen that MN19 is more dynamic even in the presence of either ligand. Using these kex values we were able to calculate the ΔH, ΔS, and ΔG of base pair opening. We saw an increase in the ΔG of base pairs at the ligand binding site in the presence of either ligand, or a slight decrease away from the binding site. This increase in ΔG shows us that there is an increase in the stability of the base pairs near the binding site. For bases that were visible in both the cocaine-bound and quinine-bound forms, there was little difference in the change in ΔG between the two bound states.
Generation of ssDNA via Denaturing HPLC for use in SELEX
#PCoombes #AptOx22
Paul E Coombes and Mark J Dickman
Department of Chemical and Biological Engineering. University of Sheffield, Sheffield, United Kingdom
A key factor in the low success rate of SELEX protocols is problems associated with the regeneration of single-stranded DNA following the PCR amplification stage. Current methods for the generation of ssDNA are limited by several common issues, including low yields, time-consuming methods and contamination. These issues lead to poor enrichment and the accumulation of parasitic PCR by-products with increasing rounds. We are currently developing a new method for rapid regeneration of ssDNA using denaturing HPLC. Utilizing hydrophobic 5’ modifications on the reverse primer we can shift the retention time of the unwanted complementary strand enabling the simple purification of the ssDNA under denaturing conditions during HPLC. Initially, a non-random 80 base pair template was used so that mass-spec analysis could confirm the purity of the isolated ssDNA, then, once the chromatography was optimized the experiment was successfully repeated with PCR product from the amplification of an 80 base pair SELEX library. Following isolation of the ssDNA, the volatile nature of the ion-pair reagent tri-ethyl ammonium acetate makes de-salting trivial.
Offline functionalized LSPR-based aptasensor for the detection of SARS-CoV-2 S1 protein
#EGiroux #AptOx22
Erin Giroux 1, Tyra Lewis 2 and Sanela Martic 1,2
1 Department of Forensic Science, Trent University, Peterborough, Ontario, Canada, K9L 0G2
2 Department of Environmental and Life Sciences, Trent University, Peterborough, Ontario, Canada, K9L 0G2
Biosensors with the capacity to detect SARS-CoV-2 proteins rapidly and selectively are of great importance for the development of diagnostic methods, therapeutic treatments, and more. Aptamers offer the high degree of selectivity required of probes for these sensors. In this work, we designed and developed a rapid, selective, and sensitive aptasensor for the detection SARS-CoV-2 S1 protein. A portable two-channel Localized Surface Plasmon Resonance instrument was equipped with an AuNP-biotin sensor chip, and biotinylated aptamers were immobilized to the surface using biotin-streptavidin interaction. The effectiveness of offline chip modification was compared to the traditional, online, in-instrument method. The repeatability and shelf-life stability of the offline prepared sensors were also tested and determined to remain stable for up to 24 days. Additionally, the aptasensor preferentially detected the S1 protein of SARS-CoV-2 compared to SARS-CoV. This optimized sensor fabrication and detection method may be modified to be selective for other targets by utilizing different aptamer.
Tunable Aptamer Probes for Molecular Thrombi Imaging
#BPGray #AptOx22
Bethany Powell Gray1, Linsley Kelly 2, Kady-Ann Steen-Burrell 2 and Bruce A. Sullenger 2
1 Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
2 Department of Surgery, Duke University School of Medicine, Durham, North Carolina, USA
Thrombosis, formation of a blood clot within an artery or vein, is a major driver for cardiovascular morbidity worldwide and a major post-surgical complication. Early detection and treatment of thrombi improve outcomes for patients. As the serine protease thrombin plays a central role in thrombogenesis, targeting and imaging clot-bound thrombin may provide a way of identifying thrombi. High target affinity and short circulating half-lives make aptamers ideal for imaging applications. Additionally, aptamer function can be turned off or modulated using ‘antidote’ oligonucleotides complementary to a portion of the aptamer sequence; these antidotes bind to the aptamer, disrupting its structure and function. Thus, we sought to develop a tunable imaging method using an aptamer-antidote pair as a rapid binding-rapid reversal probe for the identification and imaging of thrombi by conjugating the thrombin-binding RNA aptamer Tog25t to the near-infrared dye Alexa Fluor 680 (AF680). In murine thrombosis models, circulating AF680-Tog25t bound to thrombin, allowing for specific imaging of jugular clot formation, as evidenced by whole animal near-infrared imaging. Antidote treatment rapidly reversed clot-bound AF680-Tog25t, demonstrating that an aptamer-antidote pair can be used to turn on and off clot imaging. Importantly, both whole body near-infrared imaging of jugular clots in mice and intravital microscopy of femoral vein clots in mice demonstrated that AF680-Tog25t also targets and images preformed clots. Subsequent antidote treatment sped up the detection of the clots by removing AF680-Tog25t from circulation and allowing for distinct, clear imaging of clot-bound AF680-Tog25t. Thus, we used an aptamer-antidote pair to create a novel tunable imaging technique. Our results also demonstrate that thrombin is a suitable target for imaging newly formed clots and that aptamer-antidote pairs serve as rapid binding-rapid reversal probes.
Generation of chemically modified aptamers to capture human C5 complement component in myasthenia gravis treatment
#JGuzdek #AptOx22
Joanna Guzdek, Joanna Majewska, Marika Piskorz, Filipa V. Pires, Paulina Dobosz, David Carter, Karolina Krzaczek, Tomasz Bąkowski, Agnieszka Sok-Grochowska
Pure Biologics S.A., Research & Development Department, Wrocław, Poland
Myasthenia gravis (MG) is an autoimmune disease caused by disrupted neurotransmission at neuromuscular junctions, leading to weakness and fatigue of muscles, with a risk of respiratory failure. A new class of drugs approved for treatment of MG targets the complement cascade with an aim to prevent complement activation, which is believed to contribute to the damaging autoimmune response. Our goal is to develop a novel aptamer-based therapeutic medical device, which will selectively remove the C5 complement component from patients’ plasma while leaving other blood components intact, thereby supporting currently available treatment, especially in myasthenic crisis cases. To do so, an aptamer selection campaign was carried out using Pure Biologics’ proprietary selection platform PureApta™. As a result, ten chemically modified DNA sequences were identified. All ten aptamers showed strong and specific binding towards the purified C5 protein and eight were also demonstrated to effectively bind native C5 protein captured directly from human plasma. Binding was confirmed with Surface Plasmon Resonance (SPR) with KD values in the picomolar range. Molecules with the best parameters will undergo an optimization process to obtain truncated aptamers which retain strong and specific binding to C5, as well as improved stability in human plasma. Leading molecules will then be subjected to functional tests with an aim to develop optimal conditions of capturing the C5 protein from serum via aptamers immobilized to a resin.
Binding and NMR Based Structural Studies on a Dopamine Aptamer -Identification of Guanines involved in Tetrad Formation
#YKaiyum #AptOx22
Yunus A Kaiyum, Philip E Johnson
Department of Chemistry, York University, Toronto, ON, Canada
Uncovering the mechanisms by which aptamers bind their ligands can open avenues into the development of biosensors, diagnostic tools, and therapeutics. In previous research, a DNA aptamer was selected to specifically bind to the neurotransmitter dopamine with a micromolar-scaled affinity in a counter selection process against similarly structured molecules. Thought to undergo ligand-induced structural change and folds upon binding dopamine, the exact mechanics of which are loosely explained. Prior research suggests that this aptamer forms a two-tetrad G-quadruplex to facilitate binding. Investigation of each of the 10 guanine bases suspected to participate in G-quadruplex formation, was performed through a series of site-specific mutations, by which 8 guanines were identified by Isothermal Titration Calorimetry to be essential to ligand binding which suggests their role in G-quadruplex formation. A series of mutations involving base-pair deletions were also investigated to determine the minimal binding aptamer that retains a strong affinity and is well structured. Aptamer-variants were catalogued and compared against one another by means of isothermal titration calorimetry and nuclear magnetic resonance experiments to determine whether they were able to bind dopamine as well as any changes in the observed affinities and structure. Assignment of the NMR spectra of the aptamer will allow for structural determination and defining a secondary structure.
Aptamer-based detection system for disease-associated glycosylation patterns
#YKerler #AptOx22
Yannick Kerler 1, Sophia Rosencrantz 2, Nico Dreymann 1, Denise Czepluch 1, Anja Möller 1, Wiebke Sabrowski 1, Marcus Menger 1
1 Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (IZI-BB), Functional Nucleic Acids – Aptamers, Am Mühlenberg 13, 14476 Potsdam, Germany
2 Fraunhofer Institute for Applied Polymer Research (IAP), Glycobiotechnology, Geiselbergstraße 69, 14476 Potsdam, Germany
For the early diagnosis of the autoimmune disease rheumatoid arthritis (RA), multiple studies suggest serum immunoglobulin G (IgG) glycosylation as a biomarker for the monitoring of the progression of RA before symptoms develop. In patients of RA, the N-glycans on the Fc-region of serum IgG show a loss of terminal galactose and sialic acid. As this underglycosylation leads to pro-inflammatory effects and autoimmunity, it is associated with RA. This underlines the analysis importance of the glycosylation pattern variations for an early diagnosis. While high- and ultra-performance liquid chromatography (HPLC/UPLC) as well as mass spectrometry (MS) are commonly used for this, these methods are also time and cost intensive and require well-trained personnel. Our aim is to develop a detection system based on aptamers specific for binding degalactosylated N-glycans with a simple measurement signal output. For the detection system, we tested the proof of principle with several established aptamers. We designed fluorophore and quencher linked adapter-oligo-pairs complementary to the aptamer and monitored the dissociation of the adapters during aptamer-target-complex formation. After dissociating, the adapter-pair should form a hetero-duplex, which leads to fluorophore and quencher being in spatial proximity for a measurable quenching effect on the fluorescence intensity. In parallel, we generate aptamers specific against N-glycans by a semi-automated in vitro selection procedure (SELEX). Here we tested the classic SELEX approach with the target immobilized on magnetic beads as well as the Capture-SELEX with the target in solution and the DNA library immobilized on beads with the help of adapter-primers. After characterization and modification of the aptamers, they will be integrated into the established detection system to develop a fast, simple and highly sensitive analytical tool for the early diagnosis of RA.
Improvements of SELEX protocol by comparing the two most used single-stranded DNA generation methods
#LLDortz #AptOx22
Lisa Lucie Le Dortz, Clotilde Rouxel, Henri-Jean Boulouis, Nadia Haddad, Anne-Claire Lagrée, Pierre Lucien Deshuillers
Anses, INRAe, Ecole Nationale Vétérinaire d’Alfort, UMR BIPAR, Laboratoire de Santé Animale, Maisons-Alfort, F-94700, France
Despite the potential of aptamers, these innovative oligonucleotides remain underdeveloped by their complex and time-consuming selection method (Systematic Evolution of Ligands by EXponential Enrichment, SELEX), associated with a high failure rate of up to 70%. The most critical step of SELEX is the generation of single-stranded DNA (ssDNA), as it directly affects the enrichment and the selection of potentially binding sequences. Several ssDNA generation methods have been reported for SELEX, including asymmetric PCR, strand separation under denaturing conditions, magnetic separation with streptavidin-coated beads and lambda exonuclease digestion. Before starting the selection of aptamers against a desired target, researchers must choose the best-suited method to produce ssDNA free of impurities and in sufficient quantity. However, very few studies have compared these methods and the yield data were determined without taking into account the purification step, making the advised selection of a ssDNA generation method difficult. In addition, several methods are currently used for the quantification of the ssDNA generated. However, their performance had not yet been compared on a standardized basis. To overcome these gaps of knowledge, our aim was double: 1/ to compare the quality and the quantity of the ssDNA generated by the two most used methods, the capture on streptavidin-coated beads and lambda exonuclease digestion, before and after purification step; 2/ to compare the performances of three different techniques (Qubit, Gel quantification and Nanodrop) for quantifying the ssDNA generated. The results obtained on a rigorous basis allowing comparisons demonstrated both the accuracy of the gel-based quantification and the superiority of lambda exonuclease digestion compared to the capture on streptavidin-coated beads, in terms of quantity and quality of ssDNA. Our in-depth study will provide the many scientists working on aptamers with a solid ground for implementing essential but often overlooked steps for successful SELEX: ssDNA generation, purification and quantification. The protocol developed is currently used in our laboratory for the selection of aptamers against Anaplasma phagocytophilum, a strict intracellular, zoonotic and tick-borne bacterium. The first results are promising as a rapid and specific enrichment of potential sequences was observed by qPCR and sequencing.
Automated de-novo selection of Broccoli based sensors
#TLegen #AptOx22
Tjasa Legen and Günter Mayer
The Centre of Aptamer Research and Development (CARD), Life and Medical Sciences Institute (LIMES), Bonn, Germany
Sensing of intracellular metabolites is fundamental to the understanding of metabolic pathways native to cells. Thus, sensor molecules are required that transduce variations in metabolite concentrations into a suitable output signal. In this regard, RNA light-up sensors are promising molecular tools as they fluoresce upon binding to another molecule. They consist of a fluorophore binding domain and a target sensing domain. However, to date only RNA light-up sensors are available, whose target sensing domain is based on naturally occurring riboswitches. Here we show a new selection strategy of Broccoli light-up sensors adjustable to the development of sensors for any type of targets. Selection protocol was based on Capture-SELEX approach and was adapted in a way where it can be performed on a customized robotic platform. We designed a special library, which holds a constant region complementary to Broccoli and Red broccoli sequence. As a proof of principle, we chose Thiamine pyrophosphate as a model target, which led to a selection of a sensor with fluorescence increase of 2-fold upon addition of the target molecule measured in vitro. This new design lays the foundation for selection of sensors detecting any desired types of intracellular metabolites.
Selective Aptasensor for Detection of SARS-CoV-2 Spike Protein
#TLewis #AptOx22
Tyra Lewis, Erin Giroux and Sanela Martic
Department of Forensic Science, Environmental and Life Sciences, Trent University, Peterborough, Ontario, Canada, K9L 0G2
During the current COVID-19 pandemic, there has been a high demand on testing of suspected cases which has demonstrated the need for efficient, accurate and reliable solutions for viral detection. Conventional methods such as polymerase chain reaction (PCR) techniques, which rely on nucleic acid detection, and serological tests based on antibody detection are routine detection strategies which have been heavily relied upon in current times. However, these methods can be time consuming, costly and have demonstrated concern for high false-negative and -positive rates. Thus, alternative detection methods are needed that offer faster response times, and overall improved reliability and accuracy in results. Viral proteins, such as the SARS-CoV-2 spike protein play a vital role in the function of the coronavirus and can be used as diagnostic biomarkers for viral detection. Compared to traditional methods based on antibody detection, approaches that target viral proteins lead to higher sensitivity, and offers the potential for early diagnostic testing of viral infection. Within biosensing applications, aptamers have gained much attention as suitable high-affinity and cost-effective binding partners to targets, including viral proteins. Herein, a localized surface plasmon resonance (LSPR) assay was fabricated using ssDNA aptamers to monitor bioaffinity interactions with SARS-CoV-2 proteins. The sensor was developed using a biotin-streptavidin platform functionalized with a biotinylated S1 aptamer as the detection probe. The S1 aptamer was found to selectively bind to the S1 protein with high affinity (KD = 0.26 nM). Real-time, rapid detection of the S1 protein was achieved and ka, kd parameters were determined for the specific aptamer-protein interaction. Spiked saliva samples achieved >90% recovery with the S1 aptasensor and the sensor exhibited excellent shelf-life stability. Overall, using LSPR, the aptasensor is a promising detection tool for viral infections.
Aptamers for recognition of the viral protein RBD SARS-CoV-2 and H1N1 influenza virus to improve the diagnosis of respiratory diseases
#CMartinezLiu #AptOx22
Cynthia Martinez-Liu 1, Natalia Martinez-Acuña 1, Gabriela Leija-Montoya 2, Sonia Lozano-Sepulveda 1, Kame Galan-Huerta 1, Ana María Rivas-Estilla 1
1 Department of Biochemistry and Molecular Medicine, School of Medicine and Hospital Universitario “Dr. Jose E. Gonzalez”, Autonomous University of Nuevo León, Monterrey, N.L. México
2 School of Medicine, Universidad Autónoma de Baja California, Mexicali, Baja California, México
The novel coronavirus named SARS-CoV-2, which causes the disease COVID-19, has spread from China to the whole world causing a pandemic. COVID-19 is currently considered a global health problem because to its contagious potential, the constant emergence of new variants that modify its transmissibility and symptomatology, and additionally because there is still no treatment to stop the infection. The need to implement sensitive methods for the identification of individuals with COVID-19 and treatments for prevention and progression of the virus have led to the accelerated development of different diagnosis strategies. Aptamers have emerged as an excellent therapeutic alternative for the identification and treatment of various important viruses. Due to its advantages over other molecules, such as high specificity towards its target molecule, low immunogenicity, small size, chemical synthesis, and feasibility to coupling to other molecules. In this work, we present the design and production of new DNA aptamers that differentiate between the RBD protein of SARS-CoV-2 and Hemagglutinin 1 protein of the influenza virus H1N1 for use in point-of-care detection systems in order to differentiate between respiratory diseases caused by these viruses. To obtain single-stranded DNA aptamers, we performed a specific SELEX selection method using recombinant proteins from both viruses (RBD and H1N1) immobilized on a membrane and a random library, which consists of a sequence of 40 random nucleotides flanked for a sequence of 18 nucleotides. Further, the oligonucleotides obtained were amplified by PCR and the ssDNA was recovered by asymmetric PCR for the next cycle of selection. Five cycles of positive selection and three cycles of negative selection were carried out to obtain specific aptamers for the recognition of each virus. Until now, 3 pools of enriched aptamers have been isolated, two specifics for the RBD protein and one specific for H1 protein, which were cloned in the TOPO vector and sequenced for their subsequent characterization.
Characterization of aptamer families for biosensing
#NMitchell #AptOx22
Noelle M Mitchell1 and Anne M. Andrews1,2
1 Department of Chemistry and Biochemistry
2 Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, USA
Aptamers are short, single-stranded oligonucleotides that function as high specificity receptors for targets, including small molecules. Unlike antibodies and other macromolecular receptors, aptamers are identified in vitro and are chemically synthesized. Aptamers coupled with field effect transistors (FETs) for signal transduction enable direct monitoring of neurotransmitters and other small-molecule targets under physiological, high ionic-strength conditions. Target-induced aptamer reconfiguration produces a change in electric field close to semiconductor surfaces and thus, measurable changes in FET transconductance. Because structure underlies function, a comprehensive understanding of aptamer-target conformations will enable predictions of characteristics that improve biosensing. We used algorithms to predict the stem-loop structures of a family of serotonin aptamers identified from a single selection. These structures guided single-base mutations to test predictions about nucleotides involved in terminal and internal stems, and loops. We used circular dichroism to deduce target-induced G-plex formation and isothermal calorimetry to measure target binding affinities. We are using DNA cleavage protection mapping to identify target binding sites at single nucleotide resolution. Knowledge of aptamer structures, target binding sites, and target-induced aptamer rearrangements will enable selection of specific aptamer sequences having desired properties for various biosensing applications, e.g., FETs, electrochemical aptamers, FRET sensors, and for specific target concentration ranges. Establishing target recognition sites and loop rearrangements creates opportunities for predicting sites for graftable elements, e.g., for electrochemical reporters. By comparing structural and target-binding motifs across sequences, we can identify aptamer ‘prototypes’ or converging motifs for particular targets that can be intentionally tuned in terms of affinity and kinetic profiles.
Indole-functionalized clickmer interacts with and exhibits neutralizing potential against CoV-2 Spike variants
#NEsmaeili #AptOx22
Nima Moradzadeh-Esmaeili 1,2,3, Mehtap Bayin1,3, Anton Schmitz1,3, Michael Famulok1,2,3, Günter Mayer2,3
1 Max Planck Institute for Neurobiology of Behavior – Caesar (MPINB), Max Planck Fellow Chemical Biology, Ludwig-Erhard-Allee 2, 53175 Bonn, Germany
2 The Center of Aptamer Research and Development (CARD), University of Bonn, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany
3 Life and Medical Sciences Institute (LIMES), Gerhard-Domagk-Str. 1, 53121 Bonn, Germany
Rapid continental spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, has created a state of pandemic with detrimental global health and economic ramifications for over 2 years. Cell entry of SARS-CoV-2 is primarily mediated by Spike glycoprotein interaction with the human ACE2 receptor. The inhibition of this initial contact point of SARS-CoV-2 viral infection is a potential treatment strategy. Despite the speedy development pace of neutralizing antibodies, the appearance of SARS-CoV-2 immune escape variants shows that additional classes of affinity ligands with different binding modes and no antibody-dependent enhancement are needed. Aptamers are a class of affinity molecules that can be developed via in vitro process, SELEX. The chemical repertoire of unmodified oligonucleotides is limited and addition of various functional groups extends the chemical space by which an aptamer can interact with its target, in turn increasing the chance of successful SELEX. Here we implemented split-combine click-SELEX to introduce several functional groups using Copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry to a DNA library and screen for chemical moieties mediating best binding. We identified modified aptamer N2.5, clickmer, that interacts with CoV-2 Spike variants such as Mu, Delta and Omicron with high affinity (< 20 nM) upon indole functionalization solely. Cell culture experiments using SARS-CoV-2 spike-pseudotyped virus and live SARS-CoV-2 reveal neutralizing potential of the N2.5 against viral variants. Our results provide proof of principle for modified aptamers and the split-combine click-SELEX methodology applied against a medically relevant viral target.
Computational Modelling of Mass Transport and Distribution of Aptamer in Blood-Brain Barrier Domain for Tumour Therapy and Cancer Treatment
#MNakhjavani #AptOx22
Maryam Nakhjavani 1,2, Sarah Shigdar 1,2, Mohsen Sarafraz 3, Farid C. Christo 4, Bernard Rolfe 3
1 School of Medicine, Deakin University, Geelong, VIC 3220, Australia
2 Institute for Mental and Physical Health and Clinical Translation, School of Medicine, Deakin University, Geelong, VIC 3220, Australia
3 School of Engineering, Deakin University, Waurn Ponds, VIC 3216, Australia.4School of Engineering, Aerospace Engineering & Aviation, RMIT University, VIC 3082, Australia
Drug delivery to brain tumours is challenging due to the suppression of the mass transport across the blood-brain barrier (BBB). This is associated with the characteristics of the BBB that reduces the diffusion and convective mass transfer mechanisms. The BBB consists of several layers including endothelial cells (ECs), pericytes, basement membrane and astrocytes, which together, construct the neurovascular unit (NVU), and tightly regulate the transfer of materials to the brain. We previously showed that aptamers targeting transferrin receptors increased brain drug delivery. To get a better understanding of this phenomenon, a mathematical model based on the finite element method was developed accounting for the fluid flow and mass transport of the aptamer molecule inside an 8 µm capillary vessel across a 14 µm NVU. Coupled fluid flow and mass transport equations were solved to calculate the spatial blood velocity and aptamer concentration (Capt) profiles across the BBB. It was identified that the thickness of the astrocyte and EC layers were key parameters affecting the Capt delivered to the last neuron dendrites in the BBB. The modelling was performed for a blood velocity of 0.38 mm/s, which was independent of the inlet concentration of the aptamer. Predicted efficacy of the drug delivery of ~11% and ~14% was calculated at a porosity of 0.5 and 0.9, respectively. This low efficacy was attributed to the mass transfer resistance across the EC, astrocyte and pericyte layers, which decreased the Capt by ~7%. Furthermore, convective mass transport as the main transport mechanism of drug delivery in the capillary layer was switched to mixed convection mass transport in the porous layers and to pure diffusion once the aptamer reached the brain parenchyma. The results of this study provide first-hand data as a steppingstone for further studies in drug delivery and dose optimisation to brain tumours.
The Analysis of the Stability and Change in Volume of Cocaine-Binding Aptamers with Ligand Binding
#MOsborne #AptOx22
Meghan T Osborne1, Philip E Johnson2
1Department of Biology, York University, Toronto, Ontario, Canada2Department of Chemistry, York University, Toronto, Ontario, Canada We are studying the stability of cocaine-binding aptamers both free and bound to its various ligands using Differential Scanning Calorimetry (DSC). DSC is a powerful tool which uses a global fitting analysis that accounts for thermolabile ligand conversion. This method works by measuring the heat flow into and out of an aptamer sample in the sample cell as a function of temperature and time while the reference cell containing buffer is put under the same conditions. Additionally, we are using pressure perturbation calorimetry to determine the volume change with ligand binding by the cocaine–binding aptamer. Cocaine-binding aptamers are used as a model system in electrochemical aptamer-based sensors. These sensors are used for real-time, feedback-controlled drug delivery and would provide new routes by which drugs with dangerously narrow therapeutic windows or complex optimal dosing regimens can be administered safely and efficiently. To achieve the most efficient sensor possible an appropriate aptamer must be chosen. We don’t really know how these aptamers work and we hypothesise that a change in volume is somehow related to how they function in electrochemical aptamer-based sensors. These experiments give the structurally sensitive thermodynamic parameters as well as a simultaneous measurement of heat capacity and expansibility for a given aptamer.
Enhancing aptamer resistance to nucleolytic degradation by post-SELEX chemical modification for preclinical studies
M. Puchała, K. Sołtys, B. Kucharska, M. Radzińska, D. Wilk, E. Zatorska, E. Liputa, S. Oleksy, D. Carter, A. Sok-Grochowska, T. Bąkowski*
Pure Biologics S.A., Research & Development Department, Wrocław, Poland
The use of chemical modifications to prevent degradation of nucleic acid aptamers by endogenous nucleases are valuable for expanding the development of aptamers as therapeutic molecules. Our research focuses on a non-systemic therapy for treatment of patients suffering from the neurological autoimmune disorder – Neuromyelitis Optica (NMO). The objective is to develop a biomolecular filter to be used during selective plasmapheresis, comprising an aptamer-functionalized adsorber that will precisely remove specific pathogenic autoantibodies from the patients’ blood. To pave the way for the clinical application of an aptamer-based device, a variety of aptamer modification strategies have been undertaken to improve aptamer affinity, specificity and stability. DNA aptamers were enhanced directly in the SELEX process by patented nucleobase modification, providing a lead molecule with strong molecular target binding ability. Subsequent stability development was carried out by post-SELEX chemical modification at various sites including aptamer termini, the sugar moiety and the phosphodiester backbone. All introduced alterations enabled preparation of an aptamer with sufficient stability in both human and animal serum, while maintaining high affinity towards the molecular target. This allows the molecule to undergo further development, including a safety and efficacy study in an animal model, followed by examination of the device in patients suffering from NMO.
DNA Aptamer Selection for SARS-CoV-2 Spike Glycoprotein Detection
#MARoque #AptOx22
Mateo A Martínez Roque 1, Pablo A Franco Urquijo 1, Víctor M García Velásquez 1, Moujab Choukeifeb 2, Günther Mayer 2, Sergio R Molina Ramírez 3 , Gabriela Figueroa-Miranda 3, Dirk Mayer 3, Luis M. Alvarez-Salasa 1
1 Laboratorio de Terapia Génica, Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del I.P.N., CDMX 07360, México
2 Life and Medical Sciences (LIMES) Institute, University of Bonn, 53121 Bonn, Germany
3 Institute of Biological Information Processing, Bioelectronics (IBI-3), Forschungszentrum Jülich GmbH, 52428 Jülich, Germany
The rapid spread of SARS-CoV-2 infection throughout the world led to a global public health and economic crisis triggering an urgent need for the development of low-cost vaccines, therapies and high-throughput detection assays. In this work, we used a combination of Ideal-Filter Capillary Electrophoresis SELEX (IFCE-SELEX), Next Generation Sequencing (NGS) and binding assays to isolate and validate single-stranded DNA aptamers that can specifically recognize the SARS-CoV-2 Spike glycoprotein. Two selected non-competing DNA aptamers, C7 and C9 were successfully used as sensitive and specific biological recognition elements for the development of electrochemical and fluorescent aptasensors for the SARS-CoV-2 Spike glycoprotein with detection limits of 0.07 fM and 41.87 nM, respectively.
Influence of short complementary oligonucleotides on ligand-binding properties of G-quadruplex aptamer specific to ochratoxin A
#ASamokhvalov #AptOx22
Alexey V Samokhvalov, Anatoly V Zherdev, Boris B Dzantiev
A.N. Bach Institute of Biochemistry, Federal Centre of Biotechnology, Russian Acad. Sci., Moscow, Russia
Short complementary oligonucleotides are widely used in aptamer-based biosensors for the detection of various ligands. However, the relationship between specific characteristics of these oligonucleotides and their influence on the aptamer-ligand binding remain unclear. Therefore, we tested a set of short complementary oligonucleotides (ssDNAs) and identified their features that led to changes in structure and ligand binding ability of an aptamer. The chosen aptamer is a 36-nucleotide single-stranded DNA (5′-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA-3′) that is specific to a low molecular weight toxicant – ochratoxin A (OTA). The aptamer consists of 5′-tail (bases 1-5); antiparallel G-quadruplex (bases 6-24) and 3′-tail (bases 25-36). The set of oligonucleotides consist of 21 fully complementary ssDNAs and was used to demonstrate that the number of hydrogen bonds (H-bonds) formed between the ssDNAs and the aptamer affected the inactivation of the aptamer binding to OTA, and the binding position affected the inactivation kinetics. The aptamer–ssDNA interaction constants were measured by two methods: isothermal calorimetry and registration of labelled OTA fluorescence anisotropy. The results of these methods correlate well (R2 = 0.87). Constants in both sets increase as H-bonds increase. For H-bonds greater than 24, the obtained constants are higher than constants of the aptamer-OTA binding, 131±32 and 36±2 nM, respectively. For H-bonds less than 18-20, the ssDNAs do not affect the OTA binding. The location of the complementary region in G-quadruplex lead to the slow destruction (up to 40 min) of the quadruplex. In addition, the destruction caused the aptamer to lose its binding properties in complex with such ssDNA. This study was financially supported by the Russian Science Foundation (Project No. 20-74-00112).
Investigation by NMR spectroscopy of a structure-switching aptamer, the case of the testosterone binding TESS.1 aptamer
#SSchellinck #AptOx22
Sofie Schellinck1, Dieter Buyst2, Annemieke Madder3, Karolien De Wael4,5, José C. Martins1
1NMR and Structure Analysis Research Group, Department of Organic and Macromolecular Chemistry, Ghent University, Ghent 9000, Belgium
2Department of Organic and Macromolecular Chemistry, NMR Centre of Expertise, Ghent University, Ghent, Oost-Vlaanderen, 9000, Belgium
3Organic and Biomimetic Chemistry Research Group, Department of Organic and Macromolecular Chemistry, Ghent University, Ghent 9000, Belgium
4A-Sense Lab, Department of Bioscience Engineering, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium
5NANOlab Center of Excellence, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium.
The potential of DNA aptamers as small molecule bioreceptors in sensors is well recognized. However many challenges remain to be addressed for these to break-through in real world applications, as extensively reviewed by the Xiao group (Angew. Chem, 2021). One challenge concerns addressing the lack of insight at the molecular level of the factors determining affinity and selectivity, as well as going beyond the simplified, cartoon-like approach in describing the underlying sensing mechanism. We describe efforts to contribute towards this goal by developing NMR based strategies, using the structure-switching testosterone binding TESS.1 DNA aptamer developed and extensively characterized by the Stojanovic group as model system. While NMR spectroscopy is a uniquely suited technique to acquire molecular level information about conformational changes and intermolecular interactions, the TESS.1-testosterone model system provides two important complications. First, at 51 nucleotides, the impact of size and flexibility on spectral complexity and line-broadening effects prevents straightforward analysis using NMR, a problem generally encountered with most aptamers of interest. As used by others, trimming of the duplex stem and introduction of some nucleotide substitutions was used to generate a more ‘NMR optimal’ sequence that shows interaction with the target in a fashion similar to the original sequence, affording improved analysis and interpretation of the aptamer-target interaction. Second, the low sensitivity and concomitant need for sufficiently concentrated samples is at odds with the low solubility of steroids (~80 µM) such as testosterone. We devised a sample preparation protocol that could handle this challenge, and allowed to demonstrate the occurrence of conformational changes upon interaction with the target. Through both approaches, first molecular insights into the testosterone-TESS.1 complex as well as the structure-switching mechanism will be presented.
Analysis of the First Visible Fluorescent Light-up Probe for DNA Three-Way Junction Structures
#AShoara #AptOx22
Aron A Shoara, Abigail J Van Riesen, Sladjana Slavkovic, Zachary R Churcher, Richard A Manderville, Philip E Johnson
Department of Chemistry and Centre for Research on Biomolecular Interactions, York University, 4700 Keele St., Toronto, Ontario M3J 1P3, Canada
2Departments of Chemistry and Toxicology, University of Guelph, Guelph, Ontario N1G 2W1, Canada DNA three-way junctions (3WJs) consist of a Y-shaped hydrophobic branch point structure and form important building blocks for the construction of DNA nanostructures. The three double stranded stems serve as recognition elements for DNA aptasensors for a wide variety of diagnostic applications and are considered as specific druggable targets for cancer treatment. However, visible fluorescent light-up probes specifically for staining of DNA 3WJs are currently lacking. We report that a merocyanine containing the N-methyl benzothiazolium (Btz) acceptor vinyl linked to a 2-fluorophenolic (FPhO) donor (FPhOBtz) serves as a universal fluorescent turn-on dye for DNA 3WJs. Our evidence is based on a multifaceted approach using ultraviolet-visible spectrophotometry, fluorometry, isothermal titration calorimetry, and nuclear magnetic resonance spectroscopy techniques to define the specificity and affinity of FPhOBtz for 3WJ DNA aptamers: the cocaine-binding aptamer (MN4), the cholic acid binding aptamer (CABA), and four steroid aptamers (DOGS1, DISS1, BES1, DCAS1). FPhOBtz exhibits significant turn-on (up to 730-fold) fluorescence at 580 nm upon aptamer binding with low micromolar affinity. Direct FPhOBtz displacement from the 3WJ binding domain through competitive alkaloid and steroid ligand binding provides immediate fluorescent read-out for target detection strategies in human blood serum in the low micromolar scale. Our results present the first visible light-up fluorescent probe for DNA 3WJ detection strategies.
In-silico Screening and validation of high affinity ssDNA aptamer for Staphylococcal enterotoxin type A (SEA)
#SSingh #AptOx22
Smriti Singh and Seema Nara
Department of Biotechnology, Motilal Nehru National Institute of Technology Allahabad, Prayagraj, U.P., India
Now-a-days nucleic acid (NA) aptamers have attracted huge attention from scientific community as a diagnostics and therapeutics molecule. NA aptamers are screened using SELEX and Non-SELEX techniques against diversity of targets. However, In-vitro screening of aptamers is a time intensive, laborious, and costly process. Additionally, the protocol optimization needs careful analysis as it may lead to loss of high affinity sequences and could amplify non-specific sequences. As an alternative, instead of planning a SELEX experiment for every new target, in silico tools can be used to search a high affinity binder for a target from a pool/library of already reported aptamer sequences. Here, we attempt to screen a ssDNA aptamer sequence that binds with staphylococcus enterotoxin type A (SEA) with high affinity and specificity from a collection of already reported aptamers against SEA and SEB. A total of ten DNA aptamer (AptSEA1-AptSEA10) sequences reported to bind with SEA, SEB, and SEC1 (enterotoxins) were used as an initial DNA pool and analyzed using aptainformatic tools such as M-fold, RNA composer, discovery studio, Chimera, patch dock, and H-dock. The binding of aptamers with SEA was compared on the basis of their high binding score and docking score, and lesser RMSD and cross-reactivity with other enterotoxins. Best three sequences (AptSEA1, AptSEA2, and AptSEA8) were tested for their binding with SEA using Dot Blot assay. Out of these three, Aptamer AptSEA8 was originally screened against SEB, however our study it showed good binding with SEA as well in DOT blot experiments.
Single-round deoxyribozyme discovery
#TSramkova #AptOx22
Tereza Šrámková 1, 2, Jaroslav Kurfürst 1, 3, Edward A Curtis 1
1 Institute of Organic Chemistry and Biochemistry of the Czech academy of Sciences, Prague, 16000, Czech Republic
2 Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague, 16000, Czech Republic
3 Department of Informatics and Chemistry, University of Chemistry and Technology, Prague, 16000, Czech Republic
Artificial evolution experiments typically use libraries of ~1015 sequences and require multiple rounds of selection to identify rare variants with a desired activity. Based on the simple structures of some aptamers and nucleic acid enzymes, we hypothesized that functional motifs could be isolated from significantly smaller libraries in a single round of selection followed by high-throughput sequencing. Our selections yielded deoxyribozymes (and their sequence requirements) with activities 8- to 30-fold lower than those previously isolated under similar conditions from libraries containing 10^14 different sequences, indicating that the disadvantage of using a less diverse pool can be surprisingly small. Acknowledgement: This work was supported by the Chemical biology for drugging undraggable targets (ChemBioDrug) [CZ.02.1.01/0.0/0.0/16 019/0000729] from the European Regional development Fund (OP RDE).
Supernova: A deoxyribozyme that catalyzes a chemiluminescent reaction
#KSvehlova #AptOx22
Katerina Svehlova1,2, Ondřej Lukšan1, Martin Jakubec1,2, Karolína Pšenáková1,2, Kateřina Dušková1, Edward A. Curtis1
1Institute of Organic Chemistry and Biochemistry ASCR, Prague, Czech Republic
2Charles University in Prague, Faculty of Science, Prague, Czech Republic
Functional DNA molecules such as aptamers and deoxyribozymes are useful components in nanotechnology and synthetic biology. In many applications that utilize such building blocks, the ability to link a molecular input, such as the binding of a ligand, to an easily detectable signal is desirable. Although a wide range of binding domains have been developed in the past 30 years, few signalling components made of DNA have been described. To expand the toolkit of functional DNA parts, we used artificial evolution to identify a glowing deoxyribozyme called Supernova. This deoxyribozyme transfers a phosphate from a 1,2-dioxetane substrate called CDP-Star to its 5′ hydroxyl group, which triggers a chemiluminescent reaction and generates a flash of blue light. High-throughput sequencing and comparative sequence analysis revealed that the 46-nucleotide catalytic core of Supernova forms a triple helical structure in which almost every unpaired nucleotide is a purine. Under optimal conditions, the rate enhancement of light production catalyzed by Supernova is 6,500-fold, which far exceeds that of existing DNA-based systems. To further improve the efficiency of the reaction, a multiple turnover variant of Supernova was developed. An engineered version of Supernova can be programmed to only generate light in the presence of an oligonucleotide complementary to its 3′ end, demonstrating that signal production can be coupled to ligand binding. We anticipate that Supernova will be useful in a wide variety of applications, including as a signalling component in allosterically regulated sensors. Funding: This work was supported by an IOCB start-up grant awarded to E.A.C., a GAČR grant (19-20989S) awarded to E.A.C., a GAUK grant (102216) awarded to K.S, ELIXIR CZ research infrastructure project (MEYS Grant No. LM2018131), and the European Regional Development Fund; OP RDE; Project: ChemBioDrug (No. CZ.02.1.01/0.0/0.0/16_019/0000729).
In vitro selection of high affinity aptamers that detect hepatitis C virus core protein and inhibit virus production in cell culture
#BTorres #AptOx22
Beatriz Torres-Vázquez 1,2, Miguel Moreno 1, Carlos Briones 1,3
1 Department of Molecular Evolution, Centro de Astrobiología (CSIC-INTA), Torrejón de Ardoz, Madrid, Spain
2 Department of Medicine, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain
3 Centro de Investigación Biomédica en Red de enfermedades hepáticas y digestivas (CIBERehd), Spain
Hepatitis C virus (HCV) is the etiological agent of chronic hepatitis and a major cause of fibrosis, cirrhosis and hepatocellular carcinoma. It is estimated that around 71 million people have chronic HCV infection worldwide and up to 4 million new infections occur each year. Current diagnostic tests are based on serological assays that detect HCV-specific antibodies and antigenic assays that quantify HCV RNA or proteins in plasma samples. However, rapid, inexpensive and more sensitive analytical tools are still needed. HCV core is a highly conserved and multifunctional protein that forms the viral capsid, making it an attractive target for HCV detection and inhibition. In this work, RNA and ssDNA in vitro selection processes were carried out in parallel for six variants of HCV core protein belonging to genotypes 1 to 4 (which are responsible for about 95% of the reported HCV infections), using different protocols and counter-selection strategies. The aptamer populations obtained were analysed by means of Ultra-Deep Sequencing (UDS), the most abundant sequences were identified and a number of highly represented sequence motifs were unveiled. Affinity (measured as the dissociation constant, Kd) of the most abundant aptamers were quantified using Enzyme-Linked OligoNucleotide Assay (ELONA)-based methods. The obtained Kd values of the best aptamers were in the nanomolar range, as low as 0.4 nM, thus evidencing a very high affinity for HCV core protein. Additionally, the two most prevalent and high affinity aptamers were assayed in Huh-7.5 reporter cell lines infected with HCV, where they decreased both the viral progeny titer and the extracellular viral RNA level, while increasing the amount of intracellular viral RNA [Torres-Vázquez et al. (2022), J. Mol. Biol. 434: 167501]. Our results suggest that these aptamers inhibit HCV capsid assembly and virion formation, making them good candidate molecules for the design of novel therapeutic approaches for hepatitis C.
3D-printed adaptor for aptamer-based lateral flow assays visualization
#JToscano #AptOx22
Nancy J Ruiz-Pérez 1 and Julia D Toscano-Garibay 2
1 Independent Researcher. Mexico city, Mexico
2 Dirección de Planeación, Enseñanza e Investigación. Hospital Regional de Alta Especialidad de Ixtapaluca, Mexico State, Mexico
Additive manufacturing is an industrial technique developed for rapid prototyping and nowadays it has the advantage of creating almost unlimited shapes from any computational designs. Recently it has also been used for the tooling and small-scale production of biomedical devices. In here, we designed and 3D-printed a small cellphone adaptor to visualize aptamer-target complexes captured on lateral flow assay strips. We used a biotinylated version of an aptamer (IGA3) that recognized insulin fixated on nitrocellulose membranes. After migration, retained aptamer was incubated with streptavidin-conjugated HRP, revealed using ECL reagents and signal was detected with a cellphone CMOS-camera. The use of AM for the design of diagnostic devices is an accessible alternative to reduce research costs, ultimately leading to the fabrication of customized equipment for local health applications.
Aptamer-based biosensing characterisation and design complemented by molecular dynamics simulations
#STungsirisurp #AptOx22
Sireethorn Tungsirisurp1, Robert M. Ziolek2, Christian D. Lorenz2, Nunzianda Frascione1
1 Department of Analytical, Environmental and Forensic Sciences, King’s College London, London, SE1 9NH, UK
2 Department of Physics, King’s College London, London, WC2R 2LS, 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. Here, we demonstrate that molecular dynamics (MD) simulations can complement existing experimental data to characterise and visualise an aptamer structure, its target binding interaction, and assist in aptamer-based biosensing design. A previously reported DNA aptamer against SARS-CoV-2 virus is used in this study. Experimentally, various biophysical techniques were used to characterise the aptamer binding properties against the receptor binding domain (RBD) protein. These include enzymatically linked oligonucleotide assay (ELONA) and surface plasmon resonance (SPR) for binding affinity and kinetics evaluation, and circular dichroism (CD) for structural study of the aptamer and aptamer-target complex. Web-based servers such as MFold and QGRS Mapper were also used for the aptamer secondary structure and G-quadruplex formation predictions, respectively. An automated in silico modelling workflow has been developed to supplement these experimental and simulated data to allow visualisation of the aptamer’s tertiary structure, including G-quadruplex, and its interaction with the target RBD protein. It is believed that this, combined with the experimental work, will allow a more in-depth assessment of aptamer-target interactions and appropriate aptamer-based biosensing design.
Overlapping but distinct: a new model for G-quadruplex biochemical specificity
#MVolek #AptOx22
Martin Volek 1,2, Sofia Kolesniková 1,3, Kateřina Švehlová 1,2, Pavel Srb 1, Ráchel Sgallová 1,4, Tereza Streckerová 1,3, Juan A Redondo 1, Václav Veverka 1,5 and Edward A Curtis 1
1 Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague 166 10, Czech Republic
2 Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Prague 128 44, Czech Republic
3 Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague 166 28, Czech Republic
4 Department of Low-Temperature Physics, Faculty of Mathematics and Physics, Charles University in Prague, Prague 180 00, Czech Republic
5 Department of Cell Biology, Faculty of Science, Charles University in Prague, Prague 128 44, Czech Republic
G-quadruplexes are noncanonical nucleic acid structures formed by stacked guanine tetrads. They are capable of a range of functions and thought to play widespread biological roles. This diversity raises an important question: what determines the biochemical specificity of G-quadruplex structures? The answer is particularly important from the perspective of biological regulation because genomes can contain hundreds of thousands of G-quadruplexes with a range of functions. Here we analyze the specificity of each sequence in a 496-member library of variants of a reference G-quadruplex with respect to five functions. Our analysis shows that the sequence requirements of G-quadruplexes with these functions are different from one another, with some mutations altering biochemical specificity by orders of magnitude. Mutations in tetrads have larger effects than mutations in loops, and changes in specificity are correlated with changes in multimeric state. To complement our biochemical data we determined the solution structure of a monomeric G-quadruplex from the library. The stacked and accessible tetrads rationalize why monomers tend to promote a model peroxidase reaction and generate fluorescence. Our experiments support a model in which the sequence requirements of G-quadruplexes with different functions are overlapping but distinct. This has implications for biological regulation, bioinformatics, and drug design. Acknowledgment: This work was supported by the Chemical biology for drugging undruggable targets (ChemBioDrug) [CZ.02.1.01/0.0/0.0/16 019/0000729] from the European Regional Development Fund (OP RDE).
Toward cell-targeted therapeutics in osteosarcoma: A smart paclitaxel-aptamer conjugate
#DXie #AptOx22
Duoli Xie 1, Zhuqian Wang 1, Chao Liang 1,2, De-an Guo 3, Aiping Lu 1
1 Law Sau Fai Institute for Advancing Translational Medicine in Bone and Joint Diseases, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong SAR, 999077, China
2 Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen 518055, Guangdong, China
3 National Engineering Laboratory for Standardization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica of the Chinese Academy of Sciences, Shanghai 201203, China
Osteosarcoma (OS) is the eighth most common primary tumor among childhood malignancies and accounts for 8.9% of cancer-related deaths in children. Even though clinical outcome of OS has significantly improved with the introduction of chemotherapeutics, 35–40% OS patients poorly respond to chemotherapeutics. Recently, we screened natural products (triptolide (TP) or paclitaxel) as highly sensitive drugs to OS in vitro. However, the poor cell targeting, severe toxicity and weak water solubility are bottlenecks for clinical translation of natural products. Aptamers selected by systematic evolution of ligands by exponential enrichment (SELEX) are single-stranded oligonucleotides that can bind to targets with high affinity and selectivity. Aptamers have been widely used as targeting moieties in selective delivery of therapeutic agents. We screened an aptamer LC6 which could target OS cells but not hepatocytes and peripheral blood cells (PBCs). We attached LC6 aptamer to TP via a contrived acid-sensitive linkage (vinyl ether bond), which could help maintain the LC6-TP conjugate at physiological pH but rupture in the acidic environment around OS cells for releasing TP to work. In vivo data showed that LC6 aptamer facilitated selectively targeting OS and subsequently promoting the antitumor activity for TP with less toxicity. We also synthesized a highly water-soluble LC6-paclitaxel conjugate that could selectively deliver paclitaxel to OS. By connecting LC6 aptamer to the active hydroxyl group at 2′ position of paclitaxel via a cathepsin B sensitive dipeptide bond, LC6-paclitexel remained stable and inactive in the circulation. LC6 aptamer facilitated t`he uptake of the conjugated paclitaxel specifically into OS cells. Once inside cells, the dipeptide bond linker of LC6-paclitaxel is cleaved by cathepsin B and then the conjugated paclitaxel is released for action. In vivo data showed that LC6-paclitaxel conjugate had decreased toxicity and improved antitumor activity. This study will facilitate the clinical translation of conjugates of aptamers and natural products for OS treatment. This work was supported by the Croucher Foundation (CAS14BU/CAS14201 to A.L.), the National Natural Science Foundation Council of China (82172386 to C.L. and 81922081 to C.L.), Department of Education of Guangdong Province (2021KTSCX104 to C.L.) and the Science, Technology and Innovation Commission of Shenzhen (JCYJ20210324104201005 to C.L.).
