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.     

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.     

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.     

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.     

Molecular Selection,Modification and Development of Therapeutic Oligonucleotide Aptamers

Monoclonal antibodies are the dominant agents used in inhibition of biological target molecules for disease therapeutics, but there are concerns of immunogenicity, production, cost and stability. Oligonucleotide aptamers have comparable affinity and specificity to targets with monoclonal antibodies whilst they have minimal immunogenicity, high production, low cost and high stability, thus are promising inhibitors to rival antibodies for disease therapy. In this review, we will compare the detailed advantages and disadvantages of antibodies and aptamers in therapeutic applications and summarize recent progress in aptamer selection and modification approaches. We will present therapeutic oligonucleotide aptamers in preclinical studies for skeletal diseases and further discuss oligonucleotide aptamers in different stages of clinical evaluation for various disease therapies including macular degeneration, cancer, inflammation and coagulation to highlight the bright commercial future and potential challenges of therapeutic oligonucleotide aptamers.     

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.     

Effect of Locked-Nucleic Acid on a Biologically Active G-Quadruplex. A Structure-Activity Relationship of the Thrombin Aptamer

Here we tested the ability to augment the biological activity of the thrombin aptamer, d(GGTTGGTGTGGTTGG), by using locked nucleic acid (LNA) to influence its G-quadruplex structure. Compared to un-substituted control aptamer, LNA-containing aptamers displayed varying degrees of thrombin inhibition. Aptamers with LNA substituted in either positions G5, T7, or G8 showed decreased thrombin inhibition, whereas LNA at position G2 displayed activity comparable to un-substituted control aptamer. Interestingly, the thermal stability of the substituted aptamers does not correlate to activity – the more stable aptamers with LNA in position G5, T7, or G8 showed the least thrombin inhibition, while a less stable aptamer with LNA at G2 was as active as the un-substituted aptamer. These results suggest that LNA substitution at sites G5, T7, and G8 directly perturbs aptamer-thrombin affinity. This further implies that for the thrombin aptamer, activity is not dictated solely by the stability of the G-quadruplex structure, but by specific interactions between the central TGT loop and thrombin and that LNA can be tolerated in a biologically active nucleic acid structure albeit in a position dependent fashion.     

Combined 3D-QSAR, Molecular Docking and Molecular Dynamics Study on Derivatives of Peptide Epoxyketone and Tyropeptin-Boronic Acid as Inhibitors Against the β5 Subunit of Human 20S Proteasome

An abnormal ubiquitin-proteasome is found in many human diseases, especially in cancer, and has received extensive attention as a promising therapeutic target in recent years. In this work, several in silico models have been built with two classes of proteasome inhibitors (PIs) by using 3D-QSAR, homology modeling, molecular docking and molecular dynamics (MD) simulations. The study resulted in two types of satisfactory 3D-QSAR models, i.e., the CoMFA model (Q(2) = 0.462, R(2) (pred) = 0.820) for epoxyketone inhibitors (EPK) and the CoMSIA model (Q(2) = 0.622, R(2) (pred) = 0.821) for tyropeptin-boronic acid derivatives (TBA). From the contour maps, some key structural factors responsible for the activity of these two series of PIs are revealed. For EPK inhibitors, the N-cap part should have higher electropositivity; a large substituent such as a benzene ring is favored at the C6-position. In terms of TBA inhibitors, hydrophobic substituents with a larger size anisole group are preferential at the C8-position; higher electropositive substituents like a naphthalene group at the C3-position can enhance the activity of the drug by providing hydrogen bond interaction with the protein target. Molecular docking disclosed that residues Thr60, Thr80, Gly106 and Ser189 play a pivotal role in maintaining the drug-target interactions, which are consistent with the contour maps. MD simulations further indicated that the binding modes of each conformation derived from docking is stable and in accord with the corresponding structure extracted from MD simulation overall. These results can offer useful theoretical references for designing more potent PIs.     

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.     

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.     

Integrated in Silico and Experimental Approach towards the Design of a Novel Recombinant Protein Containing an Anti-HER2 scFv

Biological therapies, such as recombinant proteins, are nowadays amongst the most promising approaches towards precision medicine. One of the most innovative methodologies currently available aimed at improving the production yield of recombinant proteins with minimization of costs relies on the combination of in silico studies to predict and deepen the understanding of the modified proteins with an experimental approach. The work described herein aims at the design and production of a biomimetic vector containing the single-chain variable domain fragment (scFv) of an anti-HER2 antibody fragment as a targeting motif fused with HIV gp41. Molecular modeling and docking studies were performed to develop the recombinant protein sequence. Subsequently, the DNA plasmid was produced and HEK-293T cells were transfected to evaluate the designed vector. The obtained results demonstrated that the plasmid construction is robust and can be expressed in the selected cell line. The multidisciplinary integrated in silico and experimental strategy adopted for the construction of a recombinant protein which can be used in HER2+-targeted therapy paves the way towards the production of other therapeutic proteins in a more cost-effective way.     

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.     

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.     

Nucleic acid aptamers as tools and drugs: recent developments

Nucleic acid aptamers are molecules that bind to their ligands with high affinity and specificity. Unlike other functional nucleic acids such as antisense oligonucleotides, ribozymes, or siRNAs, aptamers almost never exert their effects on the genetic level. They manipulate their target molecules such as gene products or epitopes directly and site specifically, leaving nontargeted protein functions intact. In a similar way to antibodies, aptamers bind to many different kinds of target molecules with high specificity and can be made to order, but as a result of their different biochemical nature and size they can also be used complementary to antibodies. In some cases, aptamers might be more suitable or more specific than antibody approaches or small molecules, both as scientific and biotechnological tools and as therapeutic agents. Recent examples of characterization of aptamers as tools for scientific research to study regulatory circuits, as tools in diagnostic or biosensor development, and as therapeutic agents are discussed.     

A comparative reverse docking strategy to identify potential antineoplastic targets of tea functional components and binding mode

The main functional components of green tea, such as epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG) and epicatechin (EC), are found to have a broad antineoplastic activity. The discovery of their targets plays an important role in revealing the antineoplastic mechanism. Therefore, to identify potential target proteins for tea polyphenols, we have taken a comparative virtual screening approach using two reverse docking systems, one based on Autodock software and the other on Tarfisdock. Two separate in silico workflows were implemented to derive a set of target proteins related to human diseases and ranked by the binding energy score. Several conventional clinically important proteins with anti-tumor effects are screened out from the PDTD protein database as the potential receptors by both procedures. To further analyze the validity of docking results, we study the binding mode of EGCG and the potential target protein Leukotriene A4 hydrolase in detail. We indicate that interactions mediated by electrostatic and hydrogen bond play a key role in ligand binding. EGCG binds to the enzyme with certain orientation and conformation that is suitable for nucleophilic attacks by several electrical residues inside the enzyme's activity cavity. This study provides useful information for studying the antitumor mechanism of tea's functional components. The comparative reverse docking strategy presented generates a tractable set of antineoplastic proteins for future experimental validation as drug targets against tumors.     

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.     

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.     

In Silico Evaluation of the Binding Site of Fucosyltransferase 8 and First Attempts to Synthesize an Inhibitor with Drug-Like Properties

Core fucosylation of N-glycans is catalyzed by fucosyltransferase 8 and is associated with various types of cancer. Most reported fucosyltransferase inhibitors contain non-drug-like features, such as charged groups. New starting points for the development of inhibitors of fucosyltransferase 8 using a fragment-based strategy are presented. Firstly, we discuss the potential of a new putative binding site of fucosyltransferase 8 that, according to a molecular dynamics (MD) simulation, is made accessible by a significant motion of the SH3 domain. This might enable the design of completely new inhibitor types for fucosyltransferase 8. Secondly, we have performed a docking study targeting the donor binding site of fucosyltransferase 8, and this yielded two fragments that were linked and trimmed in silico. The resulting ligand was synthesized. Saturation transfer difference (STD) NMR confirmed binding of the ligand featuring a pyrazole core that mimics the guanine moiety. This ligand represents the first low-molecular-weight compound for the development of inhibitors of fucosyltransferase 8 with drug-like properties.