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.     

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.     

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。     

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.     

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.     

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.     

Progress and Challenges in Developing Aptamer-Functionalized Targeted Drug Delivery Systems

Aptamers, which can be screened via systematic evolution of ligands by exponential enrichment (SELEX), are superior ligands for molecular recognition due to their high selectivity and affinity. The interest in the use of aptamers as ligands for targeted drug delivery has been increasing due to their unique advantages. Based on their different compositions and preparation methods, aptamer-functionalized targeted drug delivery systems can be divided into two main categories: aptamer-small molecule conjugated systems and aptamer-nanomaterial conjugated systems. In this review, we not only summarize recent progress in aptamer selection and the application of aptamers in these targeted drug delivery systems but also discuss the advantages, challenges and new perspectives associated with these delivery systems.     

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.     

Aptamers can discriminate alkaline proteins with high specificity

Aptamers are single-stranded nucleic acids that fold into stable three-dimensional structures with ligand binding sites that are complementary in shape and charge to a desired target. Aptamers are generated by an iterative process known as in vitro selection, which permits their isolation from pools of random sequences. While aptamers have been selected to bind a wide range of targets, it is generally thought that these molecules are incapable of discriminating strongly alkaline proteins due to the attractive forces that govern oppositely charged polymers (e.g., polyelectrolyte effect). Histones, eukaryotic proteins that make up the core structure of nucleosomes are attractive targets for exploring the binding properties of aptamers because these proteins have positively charged surfaces that bind DNA through noncovalent sequence-independent interactions. Previous selections by our lab and others have yielded DNA aptamers with high affinity but low specificity to individual histone proteins. Whether this is a general limitation of aptamers is an interesting question with important practical implications in the future development of protein affinity reagents. Here we report the in vitro selection of a DNA aptamer that binds to histone H4 with a K(d) of 13 nM and distinguishes other core histone proteins with 100 to 480-fold selectivity, which corresponds to a ΔΔG of up to 3.4 kcal mol(-1) . This result extends our fundamental understanding of aptamers and their ability to fold into shapes that selectively bind alkaline proteins.     

A Luminescent Cyclometalated Iridium(III) Complex Accumulates in Mitochondria and Induces Mitochondrial Shortening by Conjugation to Specific Protein Targets

We report the cellular properties of a luminescent cyclometalated iridium(III) complex, [Ir(pq)₂(phen‐ITC)](PF₆) (Ir‐ITC; Hpq=2‐phenylquinoline, phen‐ITC=5‐isothiocyanate‐1,10‐phenanthroline), that efficiently and specifically labels mitochondria in living mammalian cells. Ir‐ITC can be covalently conjugated to its protein targets, and its luminescence survived cell lysis, protein extraction, and gel electrophoresis under denaturing conditions. The conjugation of Ir‐ITC with live‐cell proteins is rapid and highly selective; the process requires active cellular metabolism, as the conjugation is abolished at nonphysiological temperature or in the presence of sodium azide. Based on measurements of the luminescence intensity, we have devised a biochemical fractionation procedure that allows the enrichment of the conjugated proteins, and their subsequent separation by two‐dimensional gel electrophoresis (2DGE). Luminescent protein spots were picked from the gel and analyzed by mass spectrometry; this resulted in the identification of 46 proteins. Many of the strongly luminescently labeled proteins are mitochondrial proteins. One of the targets is VDAC1 (voltage‐dependent anion channel 1). Consistent with known phenotypes of VDAC1 deregulation, prolonged exposure of cells to Ir‐ITC led to significant mitochondrial shortening and fragmentation. As far as we know, this is the first report on the molecular characterization of the interactions of a luminescent dye with its biological targets. As many biological dyes exhibit specific intracellular staining patterns, the identification of their molecular targets can help elucidate the mechanisms behind their staining specificities and cytotoxicity. We believe our biochemical approach can be applied to identify the targets of a wide range of fluorescent and luminescent probes.     

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.     

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.     

Synthetic DNA aptamers to detect protein molecular variants in a high-throughput fluorescence quenching assay

Real-time protein detection in homogeneous solutions is necessary in many biotechnology and biomedical studies. The recent development of molecular aptamers, combined with fluorescence techniques, may provide an easy and efficient approach to protein elucidation. This report describes the development of a fluorescence-based assay with synthetic DNA aptamers that can detect and distinguish molecular variants of proteins in biological samples in a high-throughput process. We used an aptamer with high affinity for the B chain of platelet-derived growth factor (PDGF), labeled it with a fluorophore and a quencher at the two termini, and measured fluorescence quenching by PDGF. The specific quenching can be used to detect PDGF at picomolar concentrations even in the presence of serum and other cell-derived proteins in cell culture media. This is the first successful application of a synthetic aptamer for the detection of tumor-related proteins directly from the tumor cells. We also show that three highly related molecular variants of PDGF (AA, AB, and BB dimers) can be distinguished from one another in this single-step assay, which can be readily adapted to a microtiter plate assay for high-throughput analysis. The use of fluorescence quenching as a measure of binding between the DNA probe and the target protein eliminates potential false signals that may arise in traditional fluorescence enhancement assays as a result of degradation of the DNA aptamer by contaminating nucleases in biological specimens. This assay is applicable to proteins that are not naturally DNA binding. The excellent specificity, ultrahigh sensitivity, and simplicity of this one-step assay addresses a growing need for high-throughput methods that detect changes in the expression of gene products and their variants in cell cultures and biological specimens.     

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.     

Aptamer-Functionalized Hybrid Nanostructures for Sensing,Drug Delivery,Catalysis and Mechanical Applications

Sequence-specific nucleic acids exhibiting selective recognition properties towards low-molecular-weight substrates and macromolecules (aptamers) find growing interest as functional biopolymers for analysis, medical applications such as imaging, drug delivery and even therapeutic agents, nanotechnology, material science and more. The present perspective article introduces a glossary of examples for diverse applications of aptamers mainly originated from our laboratory. These include the introduction of aptamer-functionalized nanomaterials such as graphene oxide, Ag nanoclusters and semiconductor quantum dots as functional hybrid nanomaterials for optical sensing of target analytes. The use of aptamer-functionalized DNA tetrahedra nanostructures for multiplex analysis and aptamer-loaded metal-organic framework nanoparticles acting as sense-and-treat are introduced. Aptamer-functionalized nano and microcarriers are presented as stimuli-responsive hybrid drug carriers for controlled and targeted drug release, including aptamer-functionalized SiO₂ nanoparticles, carbon dots, metal-organic frameworks and microcapsules. A further application of aptamers involves the conjugation of aptamers to catalytic units as a means to mimic enzyme functions “nucleoapzymes”. In addition, the formation and dissociation of aptamer-ligand complexes are applied to develop mechanical molecular devices and to switch nanostructures such as origami scaffolds. Finally, the article discusses future challenges in applying aptamers in material science, nanotechnology and catalysis.     

Functional Nucleic Acids Under Unusual Conditions

Functional nucleic acids (FNAs), including naturally occurring ribozymes and riboswitches as well as artificially created DNAzymes and aptamers, have been popular molecular toolboxes for diverse applications. Given the high chemical stability of nucleic acids and their ability to fold into diverse sequence-dependent structures, FNAs are suggested to be highly functional under unusual reaction conditions. This review will examine the progress of research on FNAs under conditions of low pH, high temperature, freezing conditions, and the inclusion of organic solvents and denaturants that are known to disrupt nucleic acid structures. The FNA species to be discussed include ribozymes, riboswitches, G-quadruplex-based peroxidase mimicking DNAzymes, RNA-cleaving DNAzymes, and aptamers. Research within this space has not only revealed the hidden talents of FNAs but has also laid important groundwork for pursuing these intriguing functional macromolecules for unique applications.     

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.     

The Three S's for Aptamer‐Mediated Control of DNA Nanostructure Dynamics: Shape,Self‐Complementarity,and Spatial Flexibility

DNA aptamers are ideal tools to enable modular control of the dynamics of DNA nanostructures. For molecular recognition, they have a particular advantage over antibodies in that they can be integrated into DNA nanostructures in a bespoke manner by base pairing or nucleotide extension without any complex bioconjugation strategy. Such simplicity will be critical upon considering advanced therapeutic and diagnostic applications of DNA nanostructures. However, optimizing DNA aptamers for functional control of the dynamics of DNA nanostructure can be challenging. Herein, we present three considerations—shape, self‐complementarity, and spatial flexibility—that should be paramount upon optimizing aptamer functionality. These lessons, learnt from the growing number of aptamer–nanostructure reports thus far, will be helpful for future studies in which aptamers are used to control the dynamics of nucleic acid nanostructures.     

Bacterially Derived Antibody Binders as Small Adapters for DNA-PAINT Microscopy

Current optical super-resolution implementations are capable of resolving features spaced just a few nanometers apart. However, translating this spatial resolution to cellular targets is limited by the large size of traditionally employed primary and secondary antibody reagents. Recent advancements in small and efficient protein binders for super-resolution microscopy, such as nanobodies or aptamers, provide an exciting avenue for the future; however, their widespread availability is still limited. To address this issue, here we report the combination of bacterial-derived binders commonly used in antibody purification with DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT) microscopy. The small sizes of these protein binders, relative to secondary antibodies, make them an attractive labeling alternative for emerging superresolution techniques. We present here a labeling protocol for DNA conjugation of bacterially derived proteins A and G for DNA-PAINT, having assayed their intracellular performance by targeting primary antibodies against tubulin, TOM20, and the epidermal growth factor receptor (EGFR) and quantified the increases in obtainable resolution.     

Bioaffinity detection of pathogens on surfaces

The demand for improved technologies capable of rapidly detecting pathogens with high sensitivity and selectivity in complex environments continues to be a significant challenge that helps drive the development of new analytical techniques. Surface-based detection platforms are particularly attractive as multiple bioaffinity interactions between different targets and corresponding probe molecules can be monitored simultaneously in a single measurement. Furthermore, the possibilities for developing new signal transduction mechanisms alongside novel signal amplification strategies are much more varied. In this article, we describe some of the latest advances in the use of surface bioaffinity detection of pathogens. Three major sections will be discussed: (i) a brief overview on the choice of probe molecules such as antibodies, proteins and aptamers specific to pathogens and surface attachment chemistries to immobilize those probes onto various substrates, (ii) highlighting examples among the current generation of surface biosensors, and (iii) exploring emerging technologies that are highly promising and likely to form the basis of the next generation of pathogenic sensors.