Macromolecular Interactions: Aptamers

Due to their small size, stability, and cost-effectiveness compared to antibodies, aptamers developed by systematic evolution of ligands by exponential enrichment (SELEX) are promising candidates for the detection of small molecules. In SELEX, a small target molecule is usually covalently immobilized on a surface to separate bound from unbound nucleic acid sequences. However, this immobilization alters the molecule, that is, additional chemical entities are added and the electron distribution is altered, compromising the enrichment properties. To overcome this problem, a capture SELEX method has been successfully developed in which the RNA or DNA libraries are bound to a surface via a complementary oligodeoxynucleotide, and the soluble ligand is used to capture nucleic acids that bind to it from that surface. Herein, the development of an automated version of the capture SELEX method for the identification of RNA aptamers that bind small molecules is described. This method is fully automated and performs up to 12 iterative selection cycles without manual interference in 72 h. The approach is therefore suitable as rapid route to aptamers and enables resource-efficient test selections to assess "aptamerogenicity" of a target.

Theranostics cover emerging technologies for cell biomarking for disease diagnosis and targeted introduction of drug ingredients to specific malignant sites. Theranostics development has become a significant biomedical research endeavor for effective diagnosis and treatment of diseases, especially cancer. An efficient biomarking and targeted delivery strategy for theranostic applications requires effective molecular coupling of binding ligands with high affinities to specific receptors on the cancer cell surface. Bioaffinity offers a unique mechanism to bind specific target and receptor molecules from a range of non-targets. The binding efficacy depends on the specificity of the affinity ligand toward the target molecule even at low concentrations. Aptamers are fragments of genetic materials, peptides, or oligonucleotides which possess enhanced specificity in targeting desired cell surface receptor molecules. Aptamer-target binding results from several inter-molecular interactions including hydrogen bond formation, aromatic stacking of flat moieties, hydrophobic interaction, electrostatic, and van der Waals interactions. Advancements in Systematic Evolution of Ligands by Exponential Enrichment (SELEX) assay has created the opportunity to artificially generate aptamers that specifically bind to desired cancer and tumor surface receptors with high affinities. This article discusses the potential application of molecular dynamics (MD) simulation to advance aptamer-mediated receptor targeting in targeted cancer therapy. MD simulation offers real-time analysis of the molecular drivers of the aptamer-receptor binding and generate optimal receptor binding conditions for theranostic applications. The article also provides an overview of different cancer types with focus on receptor biomarking and targeted treatment approaches, conventional molecular probes, and aptamers that have been explored for cancer cells targeting.

The influence of food matrices on aptamer selection by SELEX (systematic evolution of ligands by exponential enrichment) targeting the norovirus P-Domain

Computational predictive approaches for interaction and structure of aptamers

Screening and Identification of Aptamers Against Pulmonary Surfactant Protein A

Aptamers are single-stranded DNA (ssDNA) affinity reagents capable of substituting conventional antibodies in processes of molecular recognition. Their higher affinities, lower costs of production and longer shelf lives of aptamers are making them increasingly popular and replacing conventional antibodies in the fields of diagnostics and therapeutics. Systematic evolution of ligands by exponential enrichment (SELEX) is a well-established and efficient technology for generation of aptamers with high affinity against various targets including whole cells, isolated proteins, and small molecules. This study is the first to report efforts in exploring SELEX for isolating protein-binding aptamers using a quantitative PCR and NGS-based approach in a local context. A low-cost selection platform was developed by coating microwell plates with human serum albumin, the target protein of interest, for quantitative and qualitative solid phase immunoassays. Changes in affinity and diversity were monitored through analysis of amplification plots, melt curves, remelt curves and high-resolution melt curve (HRM). Following eight selection cycles, the enriched DNA was subjected to high-throughput sequencing, and the two most abundant sequences were identified. The sequences were evaluated through in-silico binding assays, which resulted in comparable binding affinities expected for aptamers. Among the monitoring techniques, the amplification curve analysis was a valuable tool in understanding changes in pool affinity. Although the melt curve initially lacked sufficient resolution in the early stages of SELEX, the re-melt curve and HRM analysis accurately reflected pool diversity during this time. Thus, we demonstrate that it is feasible to use locally available technology for the successful development of aptamers. This highlights the potential to produce affinity reagents locally on a commercial scale in the future.

Introduction: Nanoscaled aptamers (Aps), as short single-stranded DNA or RNA oligonucleotides, are able to bind to their specific targets with high affinity, upon which they are considered as powerful diagnostic and analytical sensing tools (the so-called "aptasensors"). Aptamers are selected from a random pool of oligonucleotides through a procedure known as "systematic evolution of ligands by exponential enrichment".Methods: In this work, the most recent studies in the field of aptasensors are reviewed and discussed with a main focus on the potential of aptasensors for the multianalyte detection(s).Results: Due to the specific folding capability of aptamers in the presence of analyte, aptasensors have substantially successfully been exploited for the detection of a wide range of small and large molecules (e.g., drugs and their metabolites, toxins, and associated biomarkers in various diseases) at very low concentrations in the biological fluids/samples even in presence of interfering species.Conclusion: Biological samples are generally considered as complexes in the real biological media. Hence, the development of aptasensors with capability to determine various targets simultaneously within a biological matrix seems to be our main challenge. To this end, integration of various key scientific dominions such as bioengineering and systems biology with biomedical researches are inevitable.

An RNA Aptamer That Binds Carcinoembryonic Antigen Inhibits Hepatic Metastasis of Colon Cancer Cells in Mice

Nucleic acid aptamers are small single-stranded DNA or RNA oligonucleotide segments, which bind to their targets with high affinity and specificity via unique three-dimensional structures. Aptamers are generated by an iterative in vitro selection process, termed as systematic evolution of ligands by exponential enrichment. Owing to their specificity, non-immunogenicity, non-toxicity, easily modified chemical structure and wide range of targets, aptamers appear to be ideal candidates for various clinical applications (diagnosis or treatment), such as cell detection, target diagnosis, molecular imaging and drug delivery. Several aptamers have entered the clinical pipeline for applications in diseases such as macular degeneration, coronary artery bypass graft surgery and various types of cancer. The aim of this review was to summarize and highlight the clinical applications of aptamers in cancer diagnosis and treatment.

While all anticoagulants have, to a certain extent, novel properties, the development of agents that inhibit specific coagulation proteases through structural affinity and can be inhibited themselves by the concomitant production of antidotes (drug–antidote pair construct) has the potential to revolutionize the field. With the evolution of our thinking toward hemostasis and thrombosis has come new pharmacologic constructs for safe and effective treatment. Aptamers are single-stranded nucleic acids that inhibit a protein’s function by folding into a specific three-dimensional structure that defines high-affinity binding to the target protein (White et al., 2000). The term aptamer (from the Latin aptus, “to fit”) was coined by Ellington and Szostak (1990) following their pioneering work published originally in Nature. Based on iterative selection techniques, aptamers that bind essentially any protein or small molecule can be generated. A high-affinity, specific inhibitor that interacts with functional groups (on both the nucleic acid and the protein) can be constructed if a small amount of pure target is available. The initiation point for aptamer development is a combinatorial library composed of single-stranded nucleic acids (RNA, DNA, or modified RNA), typically containing 20 to 40 randomized positions (1024 different sequences). Isolation of high-affinity nucleic acid ligands involves a process known as SELEX (systemic evolution of ligands by exponential enrichment). The starting library is incubated with the protein of interest. Nucleic acid molecules that adopt conformations that allow target protein binding are subsequently partitioned from other sequences (that do not bind the protein). The bound sequences are removed and amplified by reverse transcription and polymerase chain reaction (PCR) (for RNA-based libraries) or PCR alone (for DNA-based libraries). After repeating the process several times, the selected ligands are secured and evaluated for binding affinity and ability to inhibit activity (of the target protein). Postselection optimization steps typically include (1) reduction in aptamer length (from a starting molecule of 80–100 nucleotides to 40 nucleotides); (2) enhanced stability in biologic systems (achieved by substitution of ribonucleotides with 2-amino, 2´-fluoro, or 2´-0-alkyl nucleotides and protection from exonuclease digestion by 3´ end capping); and (3) reduced renal clearance (achieved by increasing the molecules’ mo lecular weight through site-specific addition of polyethylene glycol moieties or other hydrophobic groups.

An aptamer is a synthetic oligonucleotide, referring to a single-stranded deoxyribonucleic acid or ribonucleic acid ligand produced by synthesis from outside the body using systematic evolution of ligands by exponential enrichment (SELEX) technology. Owing to their special screening process and adjustable tertiary structures, aptamers can bind to multiple targets (small molecules, proteins, and even whole cells) with high specificity and affinity. Moreover, due to their simple preparation and stable modification, they have been widely used to construct biosensors for target detection. The paper-based sensor is a product with a low price, short detection time, simple operation, and other superior characteristics, and is widely used as a rapid detection method. This review mainly focuses on the screening methods of aptamers, paper-based devices, and applicable sensing strategies. Furthermore, the design of the aptamer-based lateral flow assay (LFA), which underlies the most promising devices for commercialization, is emphasized. In addition, the development prospects and potential applications of paper-based biosensors using aptamers as recognition molecules are also discussed.

Aptamers are short single-strand oligonucleotides that can form secondary and tertiary structures, fitting targets with high affinity and specificity. They are so-called "chemical antibodies" and can target specific biomarkers in both diagnostic and therapeutic applications. Systematic evolution of ligands by exponential enrichment (SELEX) is usually used for the enrichment and selection of aptamers, and the targets could be metal ions, small molecules, nucleotides, proteins, cells, or even tissues or organs. Due to the high specificity and distinctive binding affinity of aptamers, aptamer-drug conjugates (ApDCs) have demonstrated their potential role in drug delivery for cancer-targeting therapies. Compared with antibodies which are produced by a cell-based bioreactor, aptamers are chemically synthesized molecules that can be easily conjugated to drugs and modified; however, the conventional ApDCs conjugate the aptamer with an active drug using a linker which may add more concerns to the stability of the ApDC, the drug-releasing efficiency, and the drug-loading capacity. The function of aptamer in conventional ApDC is just as a targeting moiety which could not fully perform the advantages of aptamers. To address these drawbacks, scientists have started using active nucleotide analogs as the cargoes of ApDCs, such as clofarabine, ara-guanosine, gemcitabine, and floxuridine, to replace all or part of the natural nucleotides in aptamer sequences. In turn, these new types of ApDCs, aptamer nucleotide analog drug conjugates, show the strength for targeting efficacy but avoid the complex drug linker designation and improve the synthetic efficiency. More importantly, these classic nucleotide analog drugs have been used for many years, and aptamer nucleotide analog drug conjugates would not increase any unknown druggability risk but improve the target tumor accumulation. In this review, we mainly summarized aptamer-conjugated nucleotide analog drugs in cancer-targeting therapies.

AFBI assay – Aptamer Fluorescence Binding and Internalization assay for cultured adherent cells

Aptamers are biomaterials that bind to a target molecule through a unique structure, and have high applicability in the diagnostic and medical fields. To effectively utilize aptamers, it is important to analyze the structure of the aptamer binding to the target molecule; however, there are difficulties in experimentally identifying this structure. In the modern pharmaceutical industry, computer-driven docking simulations that predict intermolecular binding models are used to select candidates that effectively bind target molecules. Botulinum toxin (BoNT) is the most poisonous neurotoxin produced from the Clostridium botulinum bacteria, and BoNT/C, one of the eight serotypes, causes paralysis in livestock. In this study, the aptamers that bound to BoNT/C were screened via the systematic evolution of ligands by exponential enrichment, and the binding affinity analysis and binding model were evaluated to select optimal aptamers. Based on surface plasmon resonance analysis and molecular operating environment docking simulation, a pair of aptamers that had high binding affinity to BoNT/C and were bound to different BoNT/C sites were selected. A sandwich assay based on this aptamer pair detected the BoNT/C protein to a concentration as low as ~0.2 ng Ml−1. These results show that docking simulations are a useful strategy for screening aptamers that bind to specific targets.

Aptamers from Cell-based Selection for Bioanalysis and Bioimaging