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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created an urgent need for new technologies to treat COVID-19. Here we report a 2′-fluoro protected RNA aptamer that binds with high affinity to the receptor binding domain (RBD) of SARS-CoV-2 spike protein, thereby preventing its interaction with the host receptor ACE2. A trimerized version of the RNA aptamer matching the three RBDs in each spike complex enhances binding affinity down to the low picomolar range. Binding mode and specificity for the aptamer–spike interaction is supported by biolayer interferometry, single-molecule fluorescence microscopy, and flow-induced dispersion analysis in vitro. Cell culture experiments using virus-like particles and live SARS-CoV-2 show that the aptamer and, to a larger extent, the trimeric aptamer can efficiently block viral infection at low concentration. Finally, the aptamer maintains its high binding affinity to spike from other circulating SARS-CoV-2 strains, suggesting that it could find widespread use for the detection and treatment of SARS-CoV-2 and emerging variants.

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.

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.

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 synthetic single-stranded DNA or RNA sequences selected from combinatorial oligonucleotide libraries through the well-known in vitro selection and iteration process, SELEX. The last three decades have witnessed a sudden boom in aptamer research, owing to their unique characteristics, like high specificity and binding affinity, low immunogenicity and toxicity, and ease in synthesis with negligible batch-to-batch variation. Aptamers can specifically bind to the targets ranging from small molecules to complex structures, making them suitable for a myriad of diagnostic and therapeutic applications. In analytical scenarios, aptamers are used as molecular probes instead of antibodies. They have the potential in the detection of biomarkers, microorganisms, viral agents, environmental pollutants, or pathogens. For therapeutic purposes, aptamers can be further engineered with chemical stabilization and modification techniques, thus expanding their serum half-life and shelf life. A vast number of antagonistic aptamers or aptamer-based conjugates have been discovered so far through the in vitro selection procedure. However, the aptamers face several challenges for its successful clinical translation, and only particular aptamers have reached the marketplace so far. Aptamer research is still in a growing stage, and a deeper understanding of nucleic acid chemistry, target interaction, tissue distribution, and pharmacokinetics is required. In this review, we discussed aptamers in the current diagnostics and theranostics applications, while addressing the challenges associated with them. The report also sheds light on the implementation of aptamer conjugates for diagnostic purposes and, finally, the therapeutic aptamers under clinical investigation, challenges therein, and their future directions.

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.

Aptamers have shown potential for diagnosing clinical markers and targeted treatment of diseases. However, their limited stability and short half-life hinder their broader applications. Here, a real sample assisted capture-SELEX strategy is proposed to enhance the aptamer stability, using the selection of specific aptamer towards PD-L1 as an example. Through this developed selection strategy, the aptamer Apt-S1 with higher binding affinity and specificity towards PD-L1 was obtained as compared to the aptamer Apt-A2 which was screened by the traditional capture-SELEX strategy. Moreover, Apt-S1 exhibited a greater PD-L1 binding associated conformational change than Apt-A2, indicating its suitability as a biorecognition element. These findings highlight the potential of Apt-S1 in clinical applications requiring robust and specific targeting of PD-L1. Significantly, Apt-S1 exhibited a lower degradation rate in 10% diluted serum or pure human serum, under the physiological temperature and pH value, compared to Apt-A2. This observation suggested that Apt-S1 possesses higher stability and is more resistant to damage caused by the serum environmental factors, highlighting the superior stability of Apt-S1 over Apt-A2. Furthermore, defatted and deproteinized serum were used to investigate the potential reasons for the improved stability of Apt-S1. The results hinted that the pre-adaptation to nucleases present in serum during the selection process might have contributed to its higher stability. With its improved stability, higher affinity and specificity, Apt-S1 holds great potential for applications in PD-L1 assisted cancer diagnosis and treatment. Meanwhile, the results obtained in this work provide further evidence of the advantages of the real capture-SELEX strategy in improving aptamer stability compared to the traditional strategy.

Targeting “undruggable” targets with intrinsically disordered structures is of great significance for the treatment of disease. The transcription factor c‐Myc controls global gene expression and is an attractive therapeutic target for multiple types of cancers. However, due to the lack of defined ligand binding pockets, targeted c‐Myc have thus far been unsuccessful. Herein, to address the dilemma of lacking ligands, an efficient and high throughput aptamer screening strategy is established, named polystyrene microwell plate‐based systematic evolution of ligands by exponential enrichment (microwell‐SELEX), and identify the specific aptamer (MA9C1) against c‐Myc. The multifunctional aptamer‐based Proteolysis Targeting Chimeras (PROTAC) for proteolysis of the c‐Myc (ProMyc) is developed using the aptamer MA9C1 as the ligand. ProMyc not only significantly degrades c‐Myc by the ubiquitin‐proteasome system, but also reduces the Max protein, synergistically inhibiting c‐Myc transcriptional activity. Combination of the artificial cyclization and anti‐PD‐L1 aptamer (PA1)‐based delivery system, circular PA1‐ProMyc chimeras achieve tumor regression in the xenograft tumor model, laying a solid foundation for the development of efficacious c‐Myc degrader for the clinic. Therefore, this aptamer‐based degrader provides an invaluable potential degrader in drug discovery and anti‐tumor therapy, offering a promising degrader to overcome the challenge of targeting intractable targets. The drugs against “Undruggable” targets remain an enormous challenge in drug discovery. Aptamers are used to address the ligand deficiency and isolate the anti‐c‐Myc aptamer. The aptamer‐based PROTAC that efficiently degrades c‐Myc (ProMyc) is developed. Based on the aptamer (PA1) delivery system, circular PA1‐ProMyc displays powerful antitumor activity in the tumor model, offering a potential degrader for intractable targets.

Lyme borreliosis (LB) is the most prevalent tick-borne illness, with an estimated 700 000 cases annually in the United States and Europe. The LB diagnosis based on a two-tiered serology remains controversial due to its indirect nature and low sensitivity during the early stage of the disease. Aptamers are single-stranded DNA or RNA oligonucleotides that exhibit high selectivity and specificity for their target due to their unique three-dimensional structure. By applying cross-over-SELEX process, an enrichment of DNA oligonucleotide sequences against a surface protein of Borrelia , named CspZ, has been performed and monitored using absorbance at 260 nm, melting curves and NGS analyses. Beyond sequence enrichment, oligonucleotides binding to CspZ were observed during the selection rounds by Dot Blot and beads assays. Thirteen unique and highly redundant oligonucleotide sequences were further characterized using multiple approaches such as Dot Blot, BioLayer Interferometry and Surface Plasmon Resonance. The selected aptamers showed K D values from tens of nanomolar to the micromolar range by BLI and SPR. Two aptamers, Apta9 and Apta10, characterized by flow cytometry and epifluorescence microscopy, were able to specifically recognize Borrelia burgdorferi sensu stricto. This strategy holds promise for the development of an improved diagnostic assay. Lyme borreliosis is challenging to diagnose, particularly in its early stages. Aptamers targeting CspZ from Borrelia , selected through cross-over SELEX, bind both the recombinant protein and bacteria, demonstrating potential for enhanced diagnostics.

Heavy metal pollution has become more and more serious with industrial development and resource exploitation. Because heavy metal ions are difficult to be biodegraded, they accumulate in the human body and cause serious threat to human health. However, the conventional methods to detect heavy metal ions are more strictly to the requirements by detection equipment, sample pretreatment, experimental environment, etc. Aptasensor has the advantages of strong specificity, high sensitivity and simple preparation to detect small molecules, which provides a new direction platform in the detection of heavy metal ions. This paper reviews the selection of aptamers as target for heavy metal ions since the 21th century and aptasensors application for detection of heavy metal ions that were reported in the past five years. Firstly, the selection methods for aptamers with high specificity and high affinity are introduced. Construction methods and research progress on sensor based aptamers as recognition element are also introduced systematically. Finally, the challenges and future opportunities of aptasensors in detecting heavy metal ions are discussed.