Tertiary core rearrangements in a tight binding transfer RNA aptamer
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Keywords:
Riboswitch
Aptamer
Nucleic acid structure
Protein tertiary structure
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SELEX Aptamer Technique
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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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The different functions of RNA have led to great and increasing interest in the field of synthetic biology. One such example of versatile RNA molecules used in synthetic biology is aptamers. Aptamers are able to bind to a wide range of target molecules with a high affinity and specificity, which is why they are often compared to antibodies. Using the SELEX method, they can be generated in vitro against target molecules for a broad range of possible applications. In nature, they work as the sensing domain of riboswitches and regulate gene expression by binding their cognate target molecule. Riboswitches can also be generated synthetically from in vitro generated aptamers.
In the course of this work, a novel RNA aptamer which selectively binds to one of two light-induced isoforms of a specific small molecule ligand (azoCm) was developed. The potential function of such an aptamer as a riboswitch was evaluated.
A previously carried out SELEX experiment against azoCm yielded a specifically binding aptamer (aptamer 42). At the beginning of this work, its secondary structure was analyzed using in-line probing. As it was previously shown aptamer 42 could not work as a riboswitch regulating GFP expression in the model organisms Saccharomyces cerevisiae, other aptamers from the same SELEX were tested under the same conditions using in vivo screening. As no functional riboswitch could be identified, a new SELEX experiment using a new aptamer library was started.
The new affinity SELEX yielded aptamers specifically binding to azoCm. However, in vivo screening of aptamers from this SELEX did not result in an azoCm dependent riboswitch. Therefore, a novel light SELEX method was developed and established. This light SELEX protocol enriched isoform selective aptamers by a light-induced conformational change of azoCm during the SELEX process. As in vivo screening of light SELEX aptamers did not yield a riboswitch again, in vitro binding studies of aptamers from the light SELEX were performed. Comparing three aptamers which showed the highest discrimination between the two isoforms of azoCm to each other led to the discovery of a 13 nt sequence motif that only these aptamers shared. A next-generation sequencing experiment performed with the SELEX and light SELEX rounds revealed that this sequence motif (“light motif”) was specifically enriched during light SELEX rounds, but was gradually depleted during the later, more stringent affinity SELEX. Based on this discovery, a new library for SELEX was designed, containing the partially randomized light motif, as well as entirely randomized flanking regions. SELEX performed with this light motif doped library led to a fast enrichment within five rounds, and aptamers from this SELEX were tested regarding their in vivo functionality. From the aptamers tested in vivo, four showed gene regulatory function, however with a low regulatory factor of 1.25- to 1.35-fold. Based on the best functional aptamer B2, partially randomized aptamer libraries were generated for further in vivo screenings. While the regulatory factor of B2 could not be improved using this approach, a modified version of it called B2-1 could be shown to regulate gene expression in yeast cells in an azoCm dose-dependent manner. To learn more about the aptamer B2-1, its binding affinity was analyzed using isothermal calorimetry. The kD of B2-1 was determined to be 23 nM, depending on the calculation model used. A truncated version of B2-1 showed a low micromolar binding to azoCm, indicating that structurally relevant parts of B2-1 had been deleted during the truncation. However, both B2-1 and its truncated version did selectively bind to only one isoform of azoCm, while binding to the other isoform could not be detected.
The aptamer developed in this work shows a much stronger discrimination between the two isoforms of its light-switchable ligand than previously reported isoform-selective aptamers. It also shows the highest binding affinity to its ligand compared to the isoform-selective aptamers in literature to date. While a riboswitch based on this aptamer shows only slight regulatory function, dose dependent regulation of gene expression could be shown. This work therefore constitutes the first steps towards the generation of a light dependent riboswitch.
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