Accelerated Discovery of Potent Bioactive anti‐TNFα Aptamers by Microbead‐Assisted Capillary Electrophoresis (MACE)‐SELEX
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Dysregulation of tumor necrosis factor-α (TNFα), a pro-inflammatory cytokine, causes several diseases, making it an important therapeutic target. Here, we identified a novel DNA aptamer against human TNFα using in vitro selection, which included a high exclusion pressure process against non-binding and weak binders through microbead-assisted capillary electrophoresis (MACE) in only three rounds. Among the 15 most enriched aptamers, Apt14 exhibited the highest inhibitory activity for the interaction between TNFα and its cognate receptor in mouse L929 cells. For further improving the bioactivity of the aptamer, dimerization programed by hybridization was evaluated, resulting in the Apt14 dimer exhibited a twofold higher binding affinity and stronger inhibition compared to the monomer counterpart. Rapid identification of bioactive aptamers using MACE in combination with facile dimerization by hybridization accelerates the discovery of novel bioactive aptamers, paving the way toward replacing current monoclonal antibody therapy with the less expensive and non-immunogenic aptamer therapy.Keywords:
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Aptamers are oligonucleotide receptors with great potential for sensing and therapeutic applications. They are isolated from random libraries through an in vitro method termed systematic evolution of ligands by exponential enrichment (SELEX). Although SELEX-based methods have been widely employed over several decades, many aspects of the experimental process remain poorly understood in terms of how to adjust the selection conditions to obtain aptamers with the desired set of binding characteristics. As a result, SELEX is often performed with arbitrary parameters that tend to produce aptamers with insufficient affinity and/or specificity. Having a better understanding of these basic principles could increase the likelihood of obtaining high-quality aptamers. Here, we have systematically investigated how altering the selection stringency in terms of target concentration─which is essentially the root source of selection pressure for aptamer isolation─affects the outcome of SELEX. By performing four separate trials of SELEX for the same small-molecule target, we experimentally prove that the use of excessively high target concentrations promotes enrichment of low-affinity binders while also suppressing the enrichment of high-affinity aptamers. These findings should be broadly applicable across SELEX methods, given that they share the same core operating principle, and will be crucial for guiding selections to obtain high-quality aptamers in the future.
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Aptamers are usually created by in vitro selection using a strategy termed systematic evolution of ligands by exponential enrichment (SELEX). Although numerous SELEX alternatives with improved selection efficiency have been developed, the overall success rate of SELEX at present is still not very ideal, which remains a great obstacle to aptamer-based research and application. In this study, an efficient and facile SELEX method was developed for in vitro screening of protein-binding aptamers, applying epitope-imprinted magnetic nanoparticles (MNPs) that exhibit highly favorable binding properties as a general affinity platform. As a proof of the principle, myoglobin (Mb) and β2-microglobulin were employed as two target proteins. Two satisfied aptamers toward each target protein, with the dissociation constant at the 10-8 M level and cross-reactivity less than 16.5%, were selected within three rounds, taking only 1 day. A dual aptamer-based fluorescence sandwich assay was constructed using a pair of the selected aptamers. The resulting assay allowed for quantitatively detecting Mb in human serum and distinguishing acute myocardial infarction patients from healthy individuals. The epitope-imprinted MNP-based SELEX is straightforward and generally applicable for a wide range of target proteins, providing a promising aptamer selection tool for aptamer-based research and real-world applications.
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Aptamers are a group of synthetic single-stranded nucleic acids. They are generated from a random library of single-stranded DNA or RNA by a technology named systematic evolution of ligands by exponential enrichment (SELEX). SELEX is a repetitive process to select and identify suitable aptamers that show high affinity and specificity towards target cells. Great strides have been achieved in the design, construction, and use of aptamers up to this point. However, only a small number of aptamer-based applications have achieved widespread commercial and clinical acceptance. Additionally, finding more effective ways to acquire aptamers with high affinity remains a challenge. Therefore, it is crucial to thoroughly examine the existing dearth and advancement in aptamer-related technologies. This review focuses on aptamers that are generated by SELEX to detect pathogenic microorganisms and mammalian cells, as well as in cell-internalizing SELEX for diagnostic and therapeutic purposes. The development of novel aptamer-based biosensors using optical and electrical methods for microbial detection is reported. The applications and limitations of aptamers are also discussed.
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Aptamers are nucleic acid oligomers with distinct conformational shapes that allow binding targets with high affinity and specificity. Selective Evolution of Ligands by Exponential Enrichment (SELEX); an in vitro selection process to develop aptamers, has been invented in 1990. Despite more than 20 years have passed after its discovery, products of SELEX technology are in use in medicine. In this review we discuss why we need aptamers not only in therapeutic but also in diagnostic applications; and also critical points in SELEX technology. Finally; we present the aptamers in use and some patented aptamers awaiting approval.
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