Overview of the Therapeutic Potential of Aptamers Targeting Coagulation Factors

Aptamers are single-stranded DNA or RNA sequences that bind target molecules with high specificity and affinity. Aptamers exhibit several notable advantages over protein-based therapeutics. Aptamers are non-immunogenic, easier to synthesize and modify, and can bind targets with greater affinity. Due to these benefits, aptamers are considered a promising therapeutic candidate to treat various conditions, including hematological disorders and cancer. An active area of research involves developing aptamers to target blood coagulation factors. These aptamers have the potential to treat cardiovascular diseases, blood disorders, and cancers. Although no aptamers targeting blood coagulation factors have been approved for clinical use, several aptamers have been evaluated in clinical trials and many more have demonstrated encouraging preclinical results. This review summarized our knowledge of the aptamers targeting proteins involved in coagulation, anticoagulation, fibrinolysis, their extensive applications as therapeutics and diagnostics tools, and the challenges they face for advancing to clinical use.     

Complex target SELEX

Aptamers are non-naturally occurring structured oligonucleotides that may bind to small molecules, peptides, and proteins. Typically, aptamers are generated by an in vitro selection process referred to as SELEX (systematic evolution of ligands by exponential enrichment). Aptamers that bind with high affinity and specificity to proteins that reside on the cell surface have potential utility as therapeutic antagonists, agonists, and diagnostic agents. When the target protein requires the presence of the cell membrane (e.g., G-protein-coupled receptors, ion channels) or a co-receptor to fold properly, it is difficult or impossible to program the SELEX experiment with purified, soluble protein target. Recent advances in which the useful range of SELEX has been extended from comparatively simple purified forms of soluble proteins to complex mixtures of proteins in membrane preparations or in situ on the surfaces of living cells offer the potential to discover aptamers against previously intractable targets. Additionally, in cases in which a cell-type specific diagnostic is sought, the most desirable target on the cell surface may not be known. Successful application of aptamer selection techniques to complex protein mixtures can be performed even in the absence of detailed target knowledge and characterization. This Account presents a review of recent work in which membrane preparations or whole cells have been utilized to generate aptamers to cell surface targets. SELEX experiments utilizing a range of target "scaffolds" are described, including cell fragments, parasites and bacteria, viruses, and a variety of human cell types including adult mesenchymal stem cells and tumor lines. Complex target SELEX can enable isolation of potent and selective aptamers directed against a variety of cell-surface proteins, including receptors and markers of cellular differentiation, as well as determinants of disease in pathogenic organisms, and as such should have wide therapeutic and diagnostic utility.     

Generating Aptamers by Cell-SELEX for Applications in Molecular Medicine

Aptamers are single-stranded oligonucleotides of DNA or RNA that bind to target molecules with high affinity and specificity. Typically, aptamers are generated by an iterative selection process, called systematic evolution of ligands by exponential enrichment (SELEX). Recent advancements in SELEX technology have extended aptamer selection from comparatively simple mixtures of purified proteins to whole living cells, and now cell-based SELEX (or cell-SELEX) can isolate aptamers that bind to specific target cells. Combined with nanotechnology, microchips, microfluidic devices, RNAi and other advanced technologies, cell-SELEX represents an integrated platform providing ultrasensitive and highly specific tools for clinical medicine. In this review, we describe the recent progress made in the application of cell-SELEX for diagnosis, therapy and biomarker discovery.     

Methods and Applications of In Silico Aptamer Design and Modeling

Aptamers are nucleic acid analogues of antibodies with high affinity to different targets, such as cells, viruses, proteins, inorganic materials, and coenzymes. Empirical approaches allow the design of in vitro aptamers that bind particularly to a target molecule with high affinity and selectivity. Theoretical methods allow significant expansion of the possibilities of aptamer design. In this study, we review theoretical and joint theoretical-experimental studies dedicated to aptamer design and modeling. We consider aptamers with different targets, such as proteins, antibiotics, organophosphates, nucleobases, amino acids, and drugs. During nucleic acid modeling and in silico design, a full set of in silico methods can be applied, such as docking, molecular dynamics (MD), and statistical analysis. The typical modeling workflow starts with structure prediction. Then, docking of target and aptamer is performed. Next, MD simulations are performed, which allows for an evaluation of the stability of aptamer/ligand complexes and determination of the binding energies with higher accuracy. Then, aptamer/ligand interactions are analyzed, and mutations of studied aptamers made. Subsequently, the whole procedure of molecular modeling can be reiterated. Thus, the interactions between aptamers and their ligands are complex and difficult to understand using only experimental approaches. Docking and MD are irreplaceable when aptamers are studied in silico.     

Artificial Intelligence in Aptamer–Target Binding Prediction

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.     

Structural Biology for the Molecular Insight between Aptamers and Target Proteins

Aptamers are promising therapeutic and diagnostic agents for various diseases due to their high affinity and specificity against target proteins. Structural determination in combination with multiple biochemical and biophysical methods could help to explore the interacting mechanism between aptamers and their targets. Regrettably, structural studies for aptamer–target interactions are still the bottleneck in this field, which are facing various difficulties. In this review, we first reviewed the methods for resolving structures of aptamer–protein complexes and for analyzing the interactions between aptamers and target proteins. We summarized the general features of the interacting nucleotides and residues involved in the interactions between aptamers and proteins. Challenges and perspectives in current methodologies were discussed. Approaches for determining the binding affinity between aptamers and target proteins as well as modification strategies for stabilizing the binding affinity of aptamers to target proteins were also reviewed. The review could help to understand how aptamers interact with their targets and how alterations such as chemical modifications in the structures affect the affinity and function of aptamers, which could facilitate the optimization and translation of aptamers-based theranostics.     

Nucleic acid aptamers-from selection in vitro to applications in vivo

Aptamers are nucleic acid ligands which are isolated from combinatorial oligonucleotide libraries by in vitro selection. They exhibit highly complex and sophisticated molecular recognition properties and are capable of binding tightly and specifically to targets ranging from small molecules to complex multimeric structures. Besides their promising application as molecular sensors, many aptamers targeted against proteins are also able to interfere with the proteins' biological function. Recently developed techniques facilitate the intracellular application of aptamers and their use as in vivo modulators of cellular physiology. Using these approaches, one can quickly obtain highly specific research reagents that act on defined intracellular targets in the context of the living cell.     

High-Throughput Selection and Characterisation of Aptamers on Optical Next-Generation Sequencers

Aptamers feature a number of advantages, compared to antibodies. However, their application has been limited so far, mainly because of the complex selection process. ‘High-throughput sequencing fluorescent ligand interaction profiling’ (HiTS–FLIP) significantly increases the selection efficiency and is consequently a very powerful and versatile technology for the selection of high-performance aptamers. It is the first experiment to allow the direct and quantitative measurement of the affinity and specificity of millions of aptamers simultaneously by harnessing the potential of optical next-generation sequencing platforms to perform fluorescence-based binding assays on the clusters displayed on the flow cells and determining their sequence and position in regular high-throughput sequencing. Many variants of the experiment have been developed that allow automation and in situ conversion of DNA clusters into base-modified DNA, RNA, peptides, and even proteins. In addition, the information from mutational assays, performed with HiTS–FLIP, provides deep insights into the relationship between the sequence, structure, and function of aptamers. This enables a detailed understanding of the sequence-specific rules that determine affinity, and thus, supports the evolution of aptamers. Current variants of the HiTS–FLIP experiment and its application in the field of aptamer selection, characterisation, and optimisation are presented in this review.     

核酸适配体在生化分离及检测领域中的研究进展

核酸适配体是一种能够特异性地识别目标物的寡聚核苷酸,可以是RNA也可以是DNA。较其他识别分子而言,适配体具有性质稳定、易合成、易标记、分子量较小和目标分子广泛等优势。目前核酸适配体主要被应用在检测、分离纯化和医疗三大领域,本文主要论述了适配体在生化分离及检测领域中的研究进展。     

In Vitro Selection of New DNA Aptamers for Human Vascular Endothelial Growth Factor 165

Two DNA aptamers that bind the heparin-binding domain (HBD) of the human vascular endothelial growth factor 165 (VEGF-165) have been previously reported. Although VEGF-165 is a homodimeric protein and the two aptamers have different sequences and secondary structures, the aptamers appear to occupy the same binding site and cannot form a 2 : 1 aptamer/protein complex, thus making them unsuitable for creating a higher-affinity dimeric DNA aptamer. This has motivated us to conduct a new in vitro selection experiment to search for new VEGF-165-binding DNA aptamers with different properties. We undertook a multistream selection strategy in which the concentration of VEGF-165 was varied significantly. We carried out 11 rounds of selection, and next-generation sequencing was conducted for every round in each stream. From comprehensive sequence analysis, we identified four classes of DNA aptamers, of which two were reported before, but two are new DNA aptamers. One of the new aptamers exhibits a unique property that has never been observed before: it is capable of forming the 2 : 1 aptamer/protein complex with VEGF-165. This work has expanded the repertoire of VEGF-165-binding DNA aptamers and creates a possibility to engineer a higher affinity homodimeric aptamer for VEGF-165.     

Cross-Binding of Adenosine by Aptamers Selected Using Theophylline

We recently reported that some adenosine binding aptamers can also bind caffeine and theophylline with around 20-fold lower affinities. This discovery led to the current work to examine the cross-binding of adenosine to theophylline aptamers. For the DNA aptamer for theophylline, cross-binding to adenosine was observed, and the affinity was 18 to 38-fold lower for adenosine based on assays using isothermal titration calorimetry and ThT fluorescence spectroscopy. The binding complexes were characterized using NMR spectroscopy, and both adenosine and theophylline showed an overall similar binding structure to the DNA theophylline aptamer, although small differences were also observed. In contrast, the RNA aptamer did not show binding to adenosine, although both aptamers have very similar relative selectivity for various methylxanthines including caffeine. After a negative selection, a few new aptamers with completely different primary sequences for theophylline were obtained and they did not show binding to adenosine. Thus, there are many ways for aptamers to bind theophylline and some can have cross-binding to adenosine. In biology, theophylline, caffeine, and adenosine can bind to the same protein receptors to regulate sleep, and their binding to the same DNA motifs may suggest an early role of nucleic acids in similar regulatory functions.     

Characterization of Aptamer-Protein Complexes by X-ray Crystallography and Alternative Approaches

Aptamers are oligonucleotide ligands, either RNA or ssDNA, selected for high-affinity binding to molecular targets, such as small organic molecules, proteins or whole microorganisms. While reports of new aptamers are numerous, characterization of their specific interaction is often restricted to the affinity of binding (K(D)). Over the years, crystal structures of aptamer-protein complexes have only scarcely become available. Here we describe some relevant technical issues about the process of crystallizing aptamer-protein complexes and highlight some biochemical details on the molecular basis of selected aptamer-protein interactions. In addition, alternative experimental and computational approaches are discussed to study aptamer-protein interactions.     

A Novel Family of Winged-Helix Single-Stranded DNA-Binding Proteins from Archaea

The winged helix superfamily comprises a large number of structurally related nucleic acid-binding proteins. While these proteins are often shown to bind dsDNA, few are known to bind ssDNA. Here, we report the identification and characterization of Sul7s, a novel winged-helix single-stranded DNA binding protein family highly conserved in Sulfolobaceae. Sul7s from Sulfolobus islandicus binds ssDNA with an affinity approximately 15-fold higher than that for dsDNA in vitro. It prefers binding oligo(dT)₃₀ over oligo(dC)₃₀ or a dG-rich 30-nt oligonucleotide, and barely binds oligo(dA)₃₀. Further, binding by Sul7s inhibits DNA strand annealing, but shows little effect on the melting temperature of DNA duplexes. The solution structure of Sul7s determined by NMR shows a winged helix-turn-helix fold, consisting of three α-helices, three β-strands, and two short wings. It interacts with ssDNA via a large positively charged binding surface, presumably resulting in ssDNA deformation. Our results shed significant light on not only non-OB fold single-stranded DNA binding proteins in Archaea, but also the divergence of the winged-helix proteins in both function and structure during evolution.     

Kinetic and stoichiometric characterisation of streptavidin-binding aptamers

Aptamers are oligonucleotide ligands that are selected for high-affinity binding to molecular targets. Only limited knowledge relating to relations between structural and kinetic properties that define aptamer-target interactions is available. To this end, streptavidin-binding aptamers were isolated and characterised by distinct analytical techniques. Binding kinetics of five broadly similar aptamers were determined by surface plasmon resonance (SPR); affinities ranged from 35-375 nM with large differences in association and dissociation rates. Native mass spectrometry showed that streptavidin can accommodate up to two aptamer units. In a 3D model of one aptamer, conserved regions are exposed, strongly suggesting that they directly interact with the biotin-binding pockets of streptavidin. Mutational studies confirmed both conserved regions to be crucial for binding. An important result is the observation that the most abundant aptamer in our selections is not the tightest binder, emphasising the importance of having insight into the kinetics of complex formation. To find the tightest binder it might be better to perform fewer selection rounds and to focus on post-selection characterisation, through the use of complementary approaches as described in this study.     

Light-up Hoechst-DNA aptamer pair: generation of an aptamer-selective fluorophore from a conventional DNA-staining dye

We have designed a strategy to generate a light-up fluorophore-aptamer pair based on a down-modification of a conventional DNA-staining dye to suppress its affinity to the original dsDNA targets, followed by reselection of aptamers that would bind to the modified dye. Following this line, we prepared a micropolarity-sensitive Hoechst derivative possessing two tBu groups with low affinity to the usual AT-rich dsDNA targets. DNA aptamers selected in vitro from a random pool worked as triggers to enhance the fluorescence of an otherwise nonfluorescent Hoechst derivative, and the shortened 25-mer sequence showed remarkable enhancement (light-up). The 25-mer sequence was split into binary aptamer probes, thus enabling us to detect a target nucleic acid sequence with a single-nucleotide resolution by use of unmodified DNA as a probe.     

One Solution for All: Searching for Universal Aptamers for Constantly Mutating Spike Proteins of SARS-CoV-2

Aptamers that can recognize the spike (S) protein of SARS-CoV-2 with high affinity and specificity are useful molecules towards the development of diagnostics and therapeutics to fight COVID-19. However, this S protein is constantly mutating, producing variants of concern (VoCs) that can significantly weaken the binding by aptamers initially engineered to recognize the S protein of the wildtype virus or a specific VoC. One strategy to overcome this problem is to develop universal aptamers that are insensitive to all or most of the naturally emerging mutations in the protein. We have recently demonstrated this concept by subjecting a pool of S protein-binding DNA aptamers for one-round parallel-SELEX experiments targeting 5 different S protein variants for binding-based sequence enrichment, followed by bioinformatic analysis of the enriched pools. This effort has led to the identification of a universal aptamer that recognizes 8 different variants of the spike protein with equally excellent affinity.     

Functional self-assembled DNA nanostructures for molecular recognition

Nucleic acids present a wonderful toolkit of structural motifs for nanoconstruction. Functional DNA nanostructures can enable protein recognition by the use of aptamers attached to a basic core shape formed by DNA self-assembly. Here, we present a facile, programmable strategy for the assembly of discrete aptamer-tagged DNA shapes and nanostructures that can function for molecular recognition and binding in an aqueous environment. These nanostructures, presented here to bind two different protein targets, are easily synthesized in large numbers, and are portable and stable over long periods of time. This construction modality can facilitate on-demand production of libraries of diverse shapes to recognize and bind proteins or catalyze reactions via functional nucleic acid tags.     

The Potential of Aptamer-Mediated Liquid Biopsy for Early Detection of Cancer

The early detection of cancer favors a greater chance of curative treatment and long-term survival. Exciting new technologies have been developed that can help to catch the disease early. Liquid biopsy is a promising non-invasive tool to detect cancer, even at an early stage, as well as to continuously monitor disease progression and treatment efficacy. Various methods have been implemented to isolate and purify bio-analytes in liquid biopsy specimens. Aptamers are short oligonucleotides consisting of either DNA or RNA that are capable of binding to target molecules with high specificity. Due to their unique properties, they are considered promising recognition ligands for the early detection of cancer by liquid biopsy. A variety of circulating targets have been isolated with high affinity and specificity by facile modification and affinity regulation of the aptamers. In this review, we discuss recent progress in aptamer-mediated liquid biopsy for cancer detection, its associated challenges, and its future potential for clinical applications.     

Aptamer modules as sensors and detectors

Aptamers comprise a range of molecular recognition scaffolds that can be engineered to bind to a legion of different proteins and other targets with excellent specificity and affinity. Because these non-natural oligonucleotides are accessible entirely synthetically, aptamers can be equipped with all sorts of reporter groups and can be coupled to many different carriers, surfaces, nanoparticles, or other biomolecules. They can be used in a highly modular fashion and often recognize their targets by a mechanism in which the aptamer undergoes considerable structural rearrangement, which can be exploited for transducing a binding event into a signal. As a consequence, aptamers have been adapted to a huge variety of "read-out configurations" and are increasingly used as capture agents in many different bioanalytical methods. But despite considerable success with these applications, many remaining challenges must still be overcome for the more widespread incorporation of aptasensors in clinical and environmental biosensing and diagnostics to take place. Some particularly noteworthy progress on this front is currently being made with aptasensor configurations that can be used for the multiplexed sensing of many analytes in parallel. In this Account, we describe some of the concepts involved in transducing the binding of a ligand into a signal through various physico-chemical interactions. Research in this area usually involves the combination of the molecular biology of proteins and nucleic acids with biotechnology, synthetic chemistry, physical chemistry, and surface physics. We begin with a brief introduction of the properties and characteristics that qualify aptamers as capture agents for many different analytes and their suitability as highly versatile biosensor components. We then address approaches that apply to surface acoustic wave configurations, drawing largely from our own contributions to aptasensor development, before moving on to describe previous and recent progress in multiplexed aptasensors. Obtaining proteome-wide profiles in cells, organs, organisms, or full populations requires the ability to accurately measure many different analytes in small sample volumes over a broad dynamic range. Multiplexed sensing is an invaluable tool in this endeavor. We discuss what we consider the biggest obstacles to the broader clinical use of aptasensor-based diagnostics and our perspective on how they can be surmounted. Finally,we explore the tremendous potential of aptamer-based sensors that can specifically discriminate between diseased and healthy cells. Progress in these areas will greatly expand the range of aptasensor applications, leading to enhanced diagnosis of diseases in clinical practice and, ultimately, improved patient care.     

DNA-Nanoscaffold-Assisted Selection of Femtomolar Bivalent Human α-Thrombin Aptamers with Potent Anticoagulant Activity

Multivalent aptamers that interact with their target proteins through multiple sites exhibit much stronger binding strengths than their monovalent counterparts. In this work, we have designed a single-stranded DNA (ssDNA) library (10¹⁵ molecules, each 145 nt) based on a predefined DNA nanostructure designed to present two random-loop sites for bivalent aptamer evolution. From this library, a group of ultra-strong bivalent aptamers against human α-thrombin (with apparent K_D values of ≈340 fm ) were easily identified through a simple seven-round conventional systematic evolution of ligands by exponential enrichment (SELEX) procedure. The dominant bivalent aptamers consist of two components, one binding to exosite I and the other to exosite II. The best of these bivalent aptamers show strong allosteric attenuation of the thrombin cleavage activity and also display an extremely potent anticoagulation effect in human plasma, demonstrating their great potential in therapeutic applications. The method developed here can easily be adapted to conventional SELEX techniques, opening a new route for fast selection of multivalent aptamers with superior binding affinity for other targets.