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.     

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.     

Diagnostics and Therapeutics in Targeting HER2 Breast Cancer: A Novel Approach

Breast cancer is one of the most commonly occurring cancers in women globally and is the primary cause of cancer mortality in females. BC is highly heterogeneous with various phenotypic expressions. The overexpression of HER2 is responsible for 15–30% of all invasive BC and is strongly associated with malignant behaviours, poor prognosis and decline in overall survival. Molecular imaging offers advantages over conventional imaging modalities, as it provides more sensitive and specific detection of tumours, as these techniques measure the biological and physiological processes at the cellular level to visualise the disease. Early detection and diagnosis of BC is crucial to improving clinical outcomes and prognosis. While HER2-specific antibodies and nanobodies may improve the sensitivity and specificity of molecular imaging, the radioisotope conjugation process may interfere with and may compromise their binding functionalities. Aptamers are single-stranded oligonucleotides capable of targeting biomarkers with remarkable binding specificity and affinity. Aptamers can be functionalised with radioisotopes without compromising target specificity. The attachment of different radioisotopes can determine the aptamer’s functionality in the treatment of HER2(+) BC. Several HER2 aptamers and investigations of them have been described and evaluated in this paper. We also provide recommendations for future studies with HER2 aptamers to target HER2(+) BC.     

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.     

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.     

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.     

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.     

The Functional Role of Loops and Flanking Sequences of G-Quadruplex Aptamer to the Hemagglutinin of Influenza a Virus

Nucleic acid aptamers are generally accepted as promising elements for the specific and high-affinity binding of various biomolecules. It has been shown for a number of aptamers that the complexes with several related proteins may possess a similar affinity. An outstanding example is the G-quadruplex DNA aptamer RHA0385, which binds to the hemagglutinins of various influenza A virus strains. These hemagglutinins have homologous tertiary structures but moderate-to-low amino acid sequence identities. Here, the experiment was inverted, targeting the same protein using a set of related, parallel G-quadruplexes. The 5′- and 3′-flanking sequences of RHA0385 were truncated to yield parallel G-quadruplex with three propeller loops that were 7, 1, and 1 nucleotides in length. Next, a set of minimal, parallel G-quadruplexes with three single-nucleotide loops was tested. These G-quadruplexes were characterized both structurally and functionally. All parallel G-quadruplexes had affinities for both recombinant hemagglutinin and influenza virions. In summary, the parallel G-quadruplex represents a minimal core structure with functional activity that binds influenza A hemagglutinin. The flanking sequences and loops represent additional features that can be used to modulate the affinity. Thus, the RHA0385–hemagglutinin complex serves as an excellent example of the hypothesis of a core structure that is decorated with additional recognizing elements capable of improving the binding properties of the aptamer.     

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.     

Role of RNA Motifs in RNA Interaction with Membrane Lipid Rafts: Implications for Therapeutic Applications of Exosomal RNAs

RNA motifs may promote interactions with exosomes (EXO-motifs) and lipid rafts (RAFT-motifs) that are enriched in exosomal membranes. These interactions can promote selective RNA loading into exosomes. We quantified the affinity between RNA aptamers containing various EXO- and RAFT-motifs and membrane lipid rafts in a liposome model of exosomes by determining the dissociation constants. Analysis of the secondary structure of RNA molecules provided data about the possible location of EXO- and RAFT-motifs within the RNA structure. The affinity of RNAs containing RAFT-motifs (UUGU, UCCC, CUCC, CCCU) and some EXO-motifs (CCCU, UCCU) to rafted liposomes is higher in comparison to aptamers without these motifs, suggesting direct RNA-exosome interaction. We have confirmed these results through the determination of the dissociation constant values of exosome-RNA aptamer complexes. RNAs containing EXO-motifs GGAG or UGAG have substantially lower affinity to lipid rafts, suggesting indirect RNA-exosome interaction via RNA binding proteins. Bioinformatics analysis revealed RNA aptamers containing both raft- and miRNA-binding motifs and involvement of raft-binding motifs UCCCU and CUCCC. A strategy is proposed for using functional RNA aptamers (fRNAa) containing both RAFT-motif and a therapeutic motif (e.g., miRNA inhibitor) to selectively introduce RNAs into exosomes for fRNAa delivery to target cells for personalized therapy.     

Molecular Docking and Molecular Dynamics Simulation Studies of Triterpenes from Vernonia patula with the Cannabinoid Type 1 Receptor

A molecular docking approach was employed to evaluate the binding affinity of six triterpenes, namely epifriedelanol, friedelin, α-amyrin, α-amyrin acetate, β-amyrin acetate, and bauerenyl acetate, towards the cannabinoid type 1 receptor (CB1). Molecular docking studies showed that friedelin, α-amyrin, and epifriedelanol had the strongest binding affinity towards CB1. Molecular dynamics simulation studies revealed that friedelin and α-amyrin engaged in stable non-bonding interactions by binding to a pocket close to the active site on the surface of the CB1 target protein. The studied triterpenes showed a good capacity to penetrate the blood–brain barrier. These results help to provide some evidence to justify, at least in part, the previously reported antinociceptive and sedative properties of Vernonia patula.     

Binding of RNA Aptamers to Membrane Lipid Rafts: Implications for Exosomal miRNAs Transfer from Cancer to Immune Cells

Intraluminal vesicles (ILVs) are released into the extracellular space as exosomes after the fusion of multivesicular bodies (MVBs) with the plasma membrane. miRNAs are delivered to the raft-like region of MVB by RNA-binding proteins (RBPs). RNA loading into exosomes can be either through direct interaction between RNA and the raft-like region of the MVB membrane, or through interaction between an RBP–RNA complex with this raft-like region. Selection of RNA aptamers that bind to lipid raft region of liposomal membranes was performed using the selection-amplification (SELEX) method. The pool of RNA aptamers was isolated, and the binding of this pool to lipid-raft regions was demonstrated. Sequencing of clones from rafted liposome-eluted RNAs showed sequences apparently of independent origin. Bioinformatics analysis revealed the most frequent raft-motifs present within these sequences. Four raft RNA motifs, one of them an EXO motif, have been identified. These motifs appear to be most frequent both in the case of raft RNA aptamers and in the case of exosomal pro-tumoral miRNAs transferred from cancer cells to macrophages, natural killer cells and dendritic cells, thus suggesting that the selection for incorporation of these miRNAs into ILVs is based on their affinity to the raft-like region of the MVB membrane.     

Charge-Transfer Interactions Stabilize G-Quadruplex-Forming Thrombin Binding Aptamers and Can Improve Their Anticoagulant Activity

In the search for optimized thrombin binding aptamers (TBAs), we herein describe the synthesis of a library of TBA analogues obtained by end-functionalization with the electron-rich 1,5-dialkoxy naphthalene (DAN) and the electron-deficient 1,8,4,5-naphthalenetetra-carboxylic diimide (NDI) moieties. Indeed, when these G-rich oligonucleotides were folded into the peculiar TBA G-quadruplex (G4) structure, effective donor–acceptor charge transfer interactions between the DAN and NDI residues attached to the extremities of the sequence were induced, providing pseudo-cyclic structures. Alternatively, insertion of NDI groups at both extremities produced TBA analogues stabilized by π–π stacking interactions. All the doubly-modified TBAs were characterized by different biophysical techniques and compared with the analogues carrying only the DAN or NDI residue and unmodified TBA. These modified TBAs exhibited higher nuclease resistance, and their G4 structures were markedly stabilized, as evidenced by increased T_m values compared to TBA. These favorable properties were also associated with improved anticoagulant activity for one DAN/NDI-modified TBA, and for one NDI/NDI-modified TBA. Our results indicated that TBA pseudo-cyclic structuring by ad hoc designed end-functionalization represents an efficient approach to improve the aptamer features, while pre-organizing and stabilizing the G4 structure but allowing sufficient flexibility to the aptamer folding, which is necessary for optimal thrombin recognition.     

Target Affinity and Structural Analysis for a Selection of Norovirus Aptamers

Aptamers, single-stranded oligonucleotides that specifically bind a molecule with high affinity, are used as ligands in analytical and therapeutic applications. For the foodborne pathogen norovirus, multiple aptamers exist but have not been thoroughly characterized. Consequently, there is little research on aptamer-mediated assay development. This study characterized seven previously described norovirus aptamers for target affinity, structure, and potential use in extraction and detection assays. Norovirus-aptamer affinities were determined by filter retention assays using norovirus genotype (G) I.1, GI.7, GII.3, GII.4 New Orleans and GII.4 Sydney virus-like particles. Of the seven aptamers characterized, equilibrium dissociation constants for GI.7, GII.3, GII.4 New Orleans and GII.4 Sydney ranged from 71 ± 38 to 1777 ± 1021 nM. Four aptamers exhibited affinity to norovirus GII.4 strains; three aptamers additionally exhibited affinity toward GII.3 and GI.7. Aptamer affinity towards GI.1 was not observed. Aptamer structure analysis by circular dichroism (CD) spectroscopy showed that six aptamers exhibit B-DNA structure, and one aptamer displays parallel/antiparallel G-quadruplex hybrid structure. CD studies also showed that biotinylated aptamer structures were unchanged from non-biotinylated aptamers. Finally, norovirus aptamer assay feasibility was demonstrated in dot-blot and pull-down assays. This characterization of existing aptamers provides a knowledge base for future aptamer-based norovirus detection and extraction assay development and aptamer modification.     

A Web Server for Designing Molecular Switches Composed of Two Interacting RNAs

The programmability of RNA–RNA interactions through intermolecular base-pairing has been successfully exploited to design a variety of RNA devices that artificially regulate gene expression. An in silico design for interacting structured RNA sequences that satisfies multiple design criteria becomes a complex multi-objective problem. Although multi-objective optimization is a powerful technique that explores a vast solution space without empirical weights between design objectives, to date, no web service for multi-objective design of RNA switches that utilizes RNA–RNA interaction has been proposed. We developed a web server, which is based on a multi-objective design algorithm called MODENA, to design two interacting RNAs that form a complex in silico. By predicting the secondary structures with RactIP during the design process, we can design RNAs that form a joint secondary structure with an external pseudoknot. The energy barrier upon the complex formation is modeled by an interaction seed that is optimized in the design algorithm. We benchmarked the RNA switch design approaches (MODENA+RactIP and MODENA+RNAcofold) for the target structures based on natural RNA-RNA interactions. As a result, MODENA+RactIP showed high design performance for the benchmark datasets.     

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.     

Structural Aspects and Prediction of Calmodulin-Binding Proteins

Calmodulin (CaM) is an important intracellular protein that binds Ca²⁺ and functions as a critical second messenger involved in numerous biological activities through extensive interactions with proteins and peptides. CaM’s ability to adapt to binding targets with different structures is related to the flexible central helix separating the N- and C-terminal lobes, which allows for conformational changes between extended and collapsed forms of the protein. CaM-binding targets are most often identified using prediction algorithms that utilize sequence and structural data to predict regions of peptides and proteins that can interact with CaM. In this review, we provide an overview of different CaM-binding proteins, the motifs through which they interact with CaM, and shared properties that make them good binding partners for CaM. Additionally, we discuss the historical and current methods for predicting CaM binding, and the similarities and differences between these methods and their relative success at prediction. As new CaM-binding proteins are identified and classified, we will gain a broader understanding of the biological processes regulated through changes in Ca²⁺ concentration through interactions with CaM.