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Aptamers are structural single-stranded oligonucleotides generated in vitro to bind to a specific target molecule. Aptamers’ versatility can be enhanced with nucleic acid mimics (NAMs) during or after a selection process, also known as systematic evolution of ligands by exponential enrichment (SELEX). We address advantages and limitations of the technologies used to generate NAM aptamers, especially the applicability of existing engineered polymerases to replicate NAMs and methodologies to improve aptamers after SELEX. We also discuss the limitations of existing methods for sequencing NAM sequences and bioinformatic tools to predict NAM aptamer structures. As a conclusion, we suggest that NAM aptamers might successfully compete with molecular tools based on proteins such as antibodies for future application. Nucleic acid mimics (NAMs) add new conformational motifs and chemical groups that improve the binding affinity and stability of aptamers, boosting their applicability in vivo.De novo systematic evolution of ligands by exponential enrichment (SELEX) is limited by access to unnatural nucleotides and enzymes that can use unnatural nucleotides as substrates. Engineered polymerases can perform de novo selection of NAM aptamers, but the protocol still is complex and time consuming.The post-SELEX approach lacks the complexity of de novo SELEX because NAM aptamers are chemically synthetized. However, inserting unnatural nucleotides often disturbs aptamer binding interactions, and the effects of such modifications are difficult to establish.Computational tools to predict tertiary structure and docking elucidate aptamer–target interactions and allow tailored modifications that reduce the experimental time of the conventional trial-and-error approach.

Aptamers are versatile molecular tools with broad applications including biosensing, bioimaging, drug delivery, therapeutics, and diagnostics.Chemical modification of aptamers is an important research area for enhancing their performance. Although recent progress has been made in modified aptamers, there are still obstacles to overcome.Next-generation sequencing enables the identification of numerous aptamer candidates as potential target binders. However, the throughput of the binding affinity and specificity assays, as well as the procedures for the validation and characterization of modified aptamer–target binding, remain time-consuming and labor-intensive.Modified aptamers can recognize different classes of molecular targets. Nevertheless, their application in vivo is currently limited and requires further exploration.Recent significant advances in the aforementioned key directions provide a solid foundation and a new paradigm for aptamer research in both fundamental and applied biosciences. Aptamers are single-stranded oligonucleotides that bind to their targets via specific structural interactions. To improve the properties and performance of aptamers, modified nucleotides are incorporated during or after a selection process such as systematic evolution of ligands by exponential enrichment (SELEX). We summarize the latest modified nucleotides and strategies used in modified (mod)-SELEX and post-SELEX to develop modified aptamers, highlight the methods used to characterize aptamer–target interactions, and present recent progress in modified aptamers that recognize different targets. We discuss the challenges and perspectives in further advancing the methodologies and toolsets to accelerate the discovery of modified aptamers, improve the throughput of aptamer-target characterization, and expand the functional diversity and complexity of modified aptamers.

Aptamers are short single stranded DNA or RNA oligonucleotides that can recognize analytes with extraordinary target selectivity and affinity. Despite their promising properties and diagnostic potential, the number of commercial applications remains scarce. In order to endow them with novel recognition motifs and enhanced properties, chemical modification of aptamers has been pursued. This review focuses on chemical modifications, aimed at increasing the binding affinity for the aptamer’s target either in a non-covalent or covalent fashion, hereby improving their application potential in a diagnostic context. An overview of current methodologies will be given, thereby distinguishing between pre- and post-SELEX (Systematic Evolution of Ligands by Exponential Enrichment) modifications.

IntroductionMany bacteria are responsible for infections in humans and plants, being found in vegetables, water, and medical devices. Most bacterial detection methods are time-consuming and take days to give the result. Aptamers are a promising alternative for a quick and reliable measurement technique to detect bacteria present in food products. Selected aptamers are DNA or RNA oligonucleotides that can bind with bacteria or other molecules with affinity and specificity for the target cells by the SELEX or cell-SELEX technique. This method is based on some rounds to remove the non-ligand oligonucleotides, leaving the aptamers specific to bind to the selected bacteria. Compared with conventional methodologies, the detection approach using aptamers is a rapid, low-cost form of analysis. ObjectiveThis review summarizes obtention methods and applications of aptamers in the food industry and biotechnology. Besides, different techniques with aptamers are presented, which enable more effective target detection.ConclusionApplications of aptamers as biosensors, or the association of aptamers with nanomaterials, may be employed in analyses by colorimetric, fluorescence, or electrical devices. Additionally, more efficient ways of sample preparation are presented, which can support food safety to provide human health, with a low-cost method for contaminant detection.Key points• Aptamers are promising for detecting contaminants outbreaks.• Studies are needed to identify aptamers for different targets.

Key Points Nucleic acid aptamers, often termed chemical antibodies, are short, single-stranded DNA or RNA molecules (20–100 nucleotides in length) with defined structures that can specifically bind to a molecular target via three-dimensional structures. Similarly to the way antibodies bind to antigens, aptamers specifically recognize and bind to their cognate targets through unique three-dimensional structures. SELEX (systematic evolution of ligands by exponential enrichment) is a gold-standard methodology for generating aptamers, in which an iterative selection procedure — including binding, partitioning, recovery and re-amplification steps — is conducted. Specific sequences (that is, aptamers) can be enriched and dominate the population of library species. Aptamer-based therapeutics typically exploit one of three strategies: an aptamer can serve as an antagonist for blocking the interaction of disease-associated targets (for example, receptor–ligand interactions); an aptamer can serve as an agonist for activating the function of target receptors; or a cell-type-specific aptamer can serve as a carrier for delivering other therapeutic agents to the target cells or tissue. There are three aptamers designated for use in ophthalmology, including one drug approved by the US Food and Drug Administration (FDA) (pegaptanib (Macugen)), and two in late-stage development (ACR-1905 and E-10030). Six RNA and four DNA aptamers have undergone clinical trials for the treatment of various conditions, including macular degeneration, coagulation, oncology and inflammation. All aptamers that have entered clinical trials so far act as antagonists. Nucleic acid aptamers offer several advantages over traditional antibodies, but their clinical translation has been delayed by several factors, including insufficient potency, lack of safety data and high production costs. Here, Zhou and Rossi provide an overview of aptamer generation, focusing on recent technological advances and clinical development, as well as challenges and lessons learned. Nucleic acid aptamers, often termed 'chemical antibodies', are functionally comparable to traditional antibodies, but offer several advantages, including their relatively small physical size, flexible structure, quick chemical production, versatile chemical modification, high stability and lack of immunogenicity. In addition, many aptamers are internalized upon binding to cellular receptors, making them useful targeted delivery agents for small interfering RNAs (siRNAs), microRNAs and conventional drugs. However, several crucial factors have delayed the clinical translation of therapeutic aptamers, such as their inherent physicochemical characteristics and lack of safety data. This Review discusses these challenges, highlighting recent clinical developments and technological advances that have revived the impetus for this promising class of therapeutics.

SELEX (systematic evolution of ligands by exponential enrichment) is a process involving the progressive isolation of high selective ssDNA/RNA from a combinatorial single-stranded oligonucleotide library through repeated rounds of binding, partitioning and amplification. SELEX-derived single-stranded DNA/RNA molecules, called aptamers, are selected against a wide range of targets, including purified proteins, live cells, tissues, microorganisms, small molecules and so on. With the development of SELEX technology over the last two decades, various modified SELEX processes have been arisen. A majority of aptamers are selected against purified proteins through traditional SELEX. Unfortunately, more and more evidence showed aptamers selected against purified membrane proteins failed to recognize their targets in live cells. Cell-SELEX could develop aptamers against a particular target cell line to discriminate this cell line from others. Therefore, cell-SELEX has been widely used to select aptamers for the application of both diagnosis and therapy of various diseases, especially for cancer. In this review, the advantages and limitations of cell-SELEX and SELEX against purified protein will be compared. Various modified cell-SELEX techniques will be summarized, and application of cell-SELEX in cancer diagnosis and therapy will be discussed.

Aptamers are short oligonucleotides with single-stranded regions or peptides that recently started to transform the field of diagnostics. Their unique ability to bind to specific target molecules with high affinity and specificity is at least comparable to many traditional biorecognition elements. Aptamers are synthetically produced, with a compact size that facilitates deeper tissue penetration and improved cellular targeting. Furthermore, they can be easily modified with various labels or functional groups, tailoring them for diverse applications. Even more uniquely, aptamers can be regenerated after use, making aptasensors a cost-effective and sustainable alternative compared to disposable biosensors. This review delves into the inherent properties of aptamers that make them advantageous in established diagnostic methods. Furthermore, we will examine some of the limitations of aptamers, such as the need to engage in bioinformatics procedures in order to understand the relationship between the structure of the aptamer and its binding abilities. The objective is to develop a targeted design for specific targets. We analyse the process of aptamer selection and design by exploring the current landscape of aptamer utilisation across various industries. Here, we illuminate the potential advantages and applications of aptamers in a range of diagnostic techniques, with a specific focus on quartz crystal microbalance (QCM) aptasensors and their integration into the well-established ELISA method. This review serves as a comprehensive resource, summarising the latest knowledge and applications of aptamers, particularly highlighting their potential to revolutionise diagnostic approaches.

Saxitoxin (STX) is one of the potent marine biotoxins that has high rate of lethality. However, there are no effective treatments at present, and the existing detection methods need to be further explored because of ethical problems or technical limitations. In this work, oligonucleotide aptamers toward STX were screened based on immobilizing libraries on Immobilized Metal-Chelate (IMC), such as Ni-NTA Sepharose, and the IMC-SELEX was conducted by the G-quadruplex library and the random library, respectively. Aptamer 45e (from the G-quadruplex library) and aptamer 75a were obtained after optimization, and aptamer 45e turned out to have a higher affinity toward STX. Furthermore, it was found that the hydrogen bonding and the van der Waals forces (VDW) played major roles in the high efficiency and specificity between STX and 45e by means of molecular docking and dynamics simulation. Based on this, aptamer 45e-1 with the Kd value of 19 nM was obtained by further optimization, which was then used to construct a simple, label-free and real-time optical BLI aptasensor for the detection of STX. This aptasensor showed good reproducibility and stability. In summary, with the advantages of screening aptamers of high efficiency and specificity toward the targets, the proposed IMC-SELEX provides a promising screening strategy for discovering aptamers, which could be used as the potential molecular recognition elements in the fields of biomedicine, food safety and environmental monitoring.

Aptamers are functional single-stranded oligonucleotide fragments isolated from randomized libraries by Systematic Evolution of Ligands by Exponential Enrichment (SELEX), exhibiting excellent affinity and specificity toward targets. Compared with traditional antibody reagents, aptamers display many desirable properties, such as low variation and high flexibility, and they are suitable for artificial and large-scale synthesis. These advantages make aptamers have a broad application potential ranging from biosensors, bioimaging to therapeutics and other areas of application. However, the overall performance of aptamer pre-selected by SELEX screening is far from being satisfactory. To improve aptamer performance and applicability, various post-SELEX optimization methods have been developed in the last decade. In this review, we first discuss the key factors that influence the performance or properties of aptamers, and then we summarize the key strategies of post-SELEX optimization which have been successfully used to improve aptamer performance, such as truncation, extension, mutagenesis and modification, splitting, and multivalent integration. This review shall provide a comprehensive summary and discussion of post-SELEX optimization methods developed in recent years. Moreover, by discussing the mechanism of each approach, we highlight the importance of choosing the proper method to perform post-SELEX optimization. Graphical Abstract

Aptamers are short single-stranded DNA, RNA, or synthetic Xeno nucleic acids (XNA) molecules that can interact with corresponding targets with high affinity. Owing to their unique features, including low cost of production, easy chemical modification, high thermal stability, reproducibility, as well as low levels of immunogenicity and toxicity, aptamers can be used as an alternative to antibodies in diagnostics and therapeutics. Systematic evolution of ligands by exponential enrichment (SELEX), an experimental approach for aptamer screening, allows the selection and identification of in vitro aptamers with high affinity and specificity. However, the SELEX process is time consuming and characterization of the representative aptamer candidates from SELEX is rather laborious. Artificial intelligence (AI) could help to rapidly identify the potential aptamer candidates from a vast number of sequences. This review discusses the advancements of AI pipelines/methods, including structure-based and machine/deep learning-based methods, for predicting the binding ability of aptamers to targets. Structure-based methods are the most used in computer-aided drug design. For this part, we review the secondary and tertiary structure prediction methods for aptamers, molecular docking, as well as molecular dynamic simulation methods for aptamer–target binding. We also performed analysis to compare the accuracy of different secondary and tertiary structure prediction methods for aptamers. On the other hand, advanced machine-/deep-learning models have witnessed successes in predicting the binding abilities between targets and ligands in drug discovery and thus potentially offer a robust and accurate approach to predict the binding between aptamers and targets. The research utilizing machine-/deep-learning techniques for prediction of aptamer–target binding is limited currently. Therefore, perspectives for models, algorithms, and implementation strategies of machine/deep learning-based methods are discussed. This review could facilitate the development and application of high-throughput and less laborious in silico methods in aptamer selection and characterization.