Generation and Selection of Specific Aptamers Targeting Brucella Species through an Enhanced Cell-SELEX Methodology

Brucellae are Gram-negative, aerobic, non-motile coccobacilli causing brucellosis in man and animals. The disease is one of the most significant yet neglected global zoonoses. Especially in developing countries, brucellosis is causing public health problems and economic losses to private animal owners and national revenues. Composed of oligonucleotides, aptamers are chemical analogues of antibodies that are promising components for developing aptamer-based rapid, sensitive, and specific tests to identify the Brucella group of bacteria. For this purpose, aptamers were generated and selected by an enhanced protocol of cell systematic evolution of ligands by exponential enrichment (cell-SELEX). This enhanced cell-SELEX procedure involved the combination of both conventional and toggle cell-SELEX to boost the specificity and binding affinity to whole Brucella cells. This procedure, combined with high-throughput sequencing of the resulting aptamer pools, comprehensive bioinformatics analysis, and wet lab validation assays, led to the selection of a highly sensitive and specific aptamer for those Brucella species known to circulate in Egypt. The isolated candidate aptamer showed dissociation constant (K_D) values of 43.5 ± 11, 61.5 ± 8, and 56 ± 10.8 nM for B. melitensis, B. abortus, and B. suis, respectively. This is the first development of a Brucella-specific aptamer using an enhanced combination of conventional and toggle cell-SELEX to the authors’ best knowledge.     

Aptamers for Anti-Viral Therapeutics and Diagnostics

Viral infections cause a host of fatal diseases and seriously affect every form of life from bacteria to humans. Although most viral infections can receive appropriate treatment thereby limiting damage to life and livelihood with modern medicine and early diagnosis, new types of viral infections are continuously emerging that need to be properly and timely treated. As time is the most important factor in the progress of many deadly viral diseases, early detection becomes of paramount importance for effective treatment. Aptamers are small oligonucleotide molecules made by the systematic evolution of ligands by exponential enrichment (SELEX). Aptamers are characterized by being able to specifically bind to a target, much like antibodies. However, unlike antibodies, aptamers are easily synthesized, modified, and are able to target a wider range of substances, including proteins and carbohydrates. With these advantages in mind, many studies on aptamer-based viral diagnosis and treatments are currently in progress. The use of aptamers for viral diagnosis requires a system that recognizes the binding of viral molecules to aptamers in samples of blood, serum, plasma, or in virus-infected cells. From a therapeutic perspective, aptamers target viral particles or host cell receptors to prevent the interaction between the virus and host cells or target intracellular viral proteins to interrupt the life cycle of the virus within infected cells. In this paper, we review recent attempts to use aptamers for the diagnosis and treatment of various viral infections.     

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.     

Optimization of Short RNA Aptamers for TNBC Cell Targeting

Triple-negative breast cancer (TNBC) is an aggressive cancer with limited targeted therapies. RNA aptamers, suitably chemically modified, work for therapeutic purposes in the same way as antibodies. We recently generated 2′Fluoro-pyrimidines RNA-aptamers that act as effective recognition elements for functional surface signatures of TNBC cells. Here, we optimized three of them by shortening and proved the truncated aptamers as optimal candidates to enable active targeting to TNBC. By using prediction of secondary structure to guide truncation, we identified structural regions that account for the binding motifs of the full-length aptamers. Their chemical synthesis led to short aptamers with superb nuclease resistance, which specifically bind to TNBC target cells and rapidly internalize into acidic compartments. They interfere with the growth of TNBC cells as mammospheres, thus confirming their potential as anti-tumor agents. We propose sTN145, sTN58 and sTN29 aptamers as valuable tools for selective TNBC targeting and promising candidates for effective treatments, including therapeutic agents and targeted delivery nanovectors.     

Molecular Contrast Optical Coherence Tomography and Its Applications in Medicine

The growing need to understand the molecular mechanisms of diseases has prompted the revolution in molecular imaging techniques along with nanomedicine development. Conventional optical coherence tomography (OCT) is a low-cost in vivo imaging modality that provides unique high spatial and temporal resolution anatomic images but little molecular information. However, given the widespread adoption of OCT in research and clinical practice, its robust molecular imaging extensions are strongly desired to combine with anatomical images. A range of relevant approaches has been reported already. In this article, we review the recent advances of molecular contrast OCT imaging techniques, the corresponding contrast agents, especially the nanoparticle-based ones, and their applications. We also summarize the properties, design criteria, merit, and demerit of those contrast agents. In the end, the prospects and challenges for further research and development in this field are outlined.     

A DNA Aptameric Ligand of Human Transferrin Receptor Generated by Cell-SELEX

General cancer-targeted ligands that can deliver drugs to cells have been given considerable attention. In this paper, a high-affinity DNA aptamer (HG1) generally binding to human tumor cells was evolved by cell-SELEX, and was further optimized to have 35 deoxynucleotides (HG1-9). Aptamer HG1-9 could be taken up by live cells, and its target protein on a cell was identified to be human transferrin receptor (TfR). As a man-made ligand of TfR, aptamer HG1-9 was demonstrated to bind at the same site of human TfR as transferrin with comparable binding affinity, and was proved to cross the epithelium barrier through transferrin receptor-mediated transcytosis. These results suggest that aptamer HG1-9 holds potential as a promising ligand to develop general cancer-targeted diagnostics and therapeutics.     

Identification and Engineering of Aptamers for Theranostic Application in Human Health and Disorders

An aptamer is a short sequence of synthetic oligonucleotides which bind to their cognate target, specifically while maintaining similar or higher sensitivity compared to an antibody. The in-vitro selection of an aptamer, applying a conjoining approach of chemistry and molecular biology, is referred as Systematic Evolution of Ligands by Exponential enrichment (SELEX). These initial products of SELEX are further modified chemically in an attempt to make them stable in biofluid, avoiding nuclease digestion and renal clearance. While the modification is incorporated, enough care should be taken to maintain its sensitivity and specificity. These modifications and several improvisations have widened the window frame of aptamer applications that are currently not only restricted to in-vitro systems, but have also been used in molecular imaging for disease pathology and treatment. In the food industry, it has been used as sensor for detection of different diseases and fungal infections. In this review, we have discussed a brief history of its journey, along with applications where its role as a therapeutic plus diagnostic (theranostic) tool has been demonstrated. We have also highlighted the potential aptamer-mediated strategies for molecular targeting of COVID-19. Finally, the review focused on its future prospective in immunotherapy, as well as in identification of novel biomarkers in stem cells and also in single cell proteomics (scProteomics) to study intra or inter-tumor heterogeneity at the protein level. Small size, chemical synthesis, low batch variation, cost effectiveness, long shelf life and low immunogenicity provide advantages to the aptamer over the antibody. These physical and chemical properties of aptamers render them as a strong biomedical tool for theranostic purposes over the existing ones. The significance of aptamers in human health was the key finding of this review.     

Chemical Modifications for a Next Generation of Nucleic Acid Aptamers

In the past three decades, in vitro systematic evolution of ligands by exponential enrichment (SELEX) has yielded many aptamers for translational applications in both research and clinical settings. Despite their promise as an alternative to antibodies, the low success rate of SELEX (∼30 %) has been a major bottleneck that hampers the further development of aptamers. One hurdle is the lack of chemical diversity in nucleic acids. To address this, the aptamer chemical repertoire has been extended by introducing exotic chemical groups, which provide novel binding functionalities. This review will focus on how modified aptamers can be selected and evolved, with illustration of some successful examples. In particular, unique chemistries are exemplified. Various strategies of incorporating modified building blocks into the standard SELEX protocol are highlighted, with a comparison of the differences between pre-SELEX and post-SELEX modifications. Nucleic acid aptamers with extended functionality evolved from non-natural chemistries will open up new vistas for function and application of nucleic acids.     

In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element against Atrazine

Widespread use of the chlorotriazine herbicide, atrazine, has led to serious environmental and human health consequences. Current methods of detecting atrazine contamination are neither rapid nor cost-effective. In this work, atrazine-specific single-stranded DNA (ssDNA) molecular recognition elements (MRE) were isolated. We utilized a stringent Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology that placed the greatest emphasis on what the MRE should not bind to. After twelve rounds of SELEX, an atrazine-specific MRE with high affinity was obtained. The equilibrium dissociation constant (K_d) of the ssDNA sequence is 0.62 ± 0.21 nM. It also has significant selectivity for atrazine over atrazine metabolites and other pesticides found in environmentally similar locations and concentrations. Furthermore, we have detected environmentally relevant atrazine concentrations in river water using this MRE. The strong affinity and selectivity of the selected atrazine-specific ssDNA validated the stringent SELEX methodology and identified a MRE that will be useful for rapid atrazine detection in environmental samples.     

De novo ssRNA Aptamers against the SARS-CoV-2 Main Protease: In Silico Design and Molecular Dynamics Simulation

Herein, we have generated ssRNA aptamers to inhibit SARS-CoV-2 M^pro, a protease necessary for the SARS-CoV-2 coronavirus replication. Because there is no aptamer 3D structure currently available in the databanks for this protein, first, we modeled an ssRNA aptamer using an entropic fragment-based strategy. We refined the initial sequence and 3D structure by using two sequential approaches, consisting of an elitist genetic algorithm and an RNA inverse process. We identified three specific aptamers against SARS-CoV-2 M^pro, called MApta^pro, MApta^pro-IR1, and MApta^pro-IR2, with similar 3D conformations and that fall in the dimerization region of the SARS-CoV-2 M^pro necessary for the enzymatic activity. Through the molecular dynamic simulation and binding free energy calculation, the interaction between the MApta^pro-IR1 aptamer and the SARS-CoV-2 M^pro enzyme resulted in the strongest and the highest stable complex; therefore, the ssRNA MApta^pro-IR1 aptamer was selected as the best potential candidate for the inhibition of SARS-CoV-2 M^pro and a perspective therapeutic drug for the COVID-19 disease.     

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

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

Molecular Landscape and Therapeutic Strategies in Cholangiocarcinoma: An Integrated Translational Approach towards Precision Medicine

Cholangiocarcinomas (CCAs) are heterogeneous biliary tract malignancies with dismal prognosis, mainly due to tumor aggressiveness, late diagnosis, and poor response to current therapeutic options. High-throughput technologies have been used as a fundamental tool in unveiling CCA molecular landscape, and several molecular classifications have been proposed, leading to various targeted therapy trials. In this review, we aim to analyze the critical issues concerning the status of precision medicine in CCA, discussing molecular signatures and clusters, related to both anatomical classification and different etiopathogenesis, and the latest therapeutic strategies. Furthermore, we propose an integrated approach comprising the CCA molecular mechanism, pathobiology, clinical and histological findings, and treatment perspectives for the ultimate purpose of improving the methods of patient allocations in clinical trials and the response to personalized therapies.     

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

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

膜技术在医药生产中的应用

通过膜过程分类及其分离机理的介绍,论述了膜技术在医药生产中的应用及膜分离系统选用应注意的问题。阐述了膜分离技术在医药生产中的重要性及未来的发展前景。     

Inhibition of Human Urokinase-Type Plasminogen Activator (uPA) Enzyme Activity and Receptor Binding by DNA Aptamers as Potential Therapeutics through Binding to the Different Forms of uPA

Urokinase-type plasminogen activator is widely discussed as a marker for cancer prognosis and diagnosis and as a target for cancer therapies. Together with its receptor, uPA plays an important role in tumorigenesis, tumor progression and metastasis. In the present study, systematic evolution of ligands by exponential enrichment (SELEX) was used to select single-stranded DNA aptamers targeting different forms of human uPA. Selected aptamers allowed the distinction between HMW-uPA and LMW-uPA, and therefore, presumably, have different binding regions. Here, uPAapt-02-FR showed highly affine binding with a K_D of 0.7 nM for HMW-uPA and 21 nM for LMW-uPA and was also able to bind to pro-uPA with a K_D of 14 nM. Furthermore, no cross-reactivity to mouse uPA or tissue-type plasminogen activator (tPA) was measured, demonstrating high specificity. Suppression of the catalytic activity of uPA and inhibition of uPAR-binding could be demonstrated through binding with different aptamers and several of their truncated variants. Since RNA aptamers are already known to inhibit uPA-uPAR binding and other pathological functions of the uPA system, these aptamers represent a novel, promising tool not only for detection of uPA but also for interfering with the pathological functions of the uPA system by additionally inhibiting uPA activity.     

Molecular recognition of small-cell lung cancer cells using aptamers

Early diagnosis is the way to improve the rate of lung cancer survival, but is almost impossible today due to the lack of molecular probes that recognize lung cancer cells sensitively and selectively. We developed a new aptamer approach for the recognition of specific small-cell lung cancer (SCLC) cell-surface molecular markers. Our approach relies on cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX) to evolve aptamers for whole live cells that express a variety of surface markers representing molecular differences among cancer cells. When applied to different lung cancer cells including those from patient samples, these aptamers bind to SCLC cells with high affinity and specificity in various assay formats. When conjugated with magnetic and fluorescent nanoparticles, the aptamer nanoconjugates could effectively extract SCLC cells from mixed cell media for isolation, enrichment, and sensitive detection. These studies demonstrate the potential of the aptamer approach for early lung cancer detection.     

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.     

磁性纳米粒子在生物学及医学领域的应用

磁性纳米粒子由于其生物相客性和低毒性而广泛应用于生物医学领域.阐述了近年来磁性纳米粒子在生物分离、生物检测、靶向药物输送、磁共振成像、肿瘤磁感应热疗等生物学和医学领域中的应用进展,并指出其主要发展方向和亟待解决的问题.     

Melt Extruded Bioresorbable Polymer Composites for Potential Regenerative Medicine Applications

Biodegradable polymers—polyethylene oxide and poly (?-caprolactone)—were melt extruded with β-tricalcium phosphate. Breakdown analysis revealed that the percentage increase in bioceramic caused a prolonged degradation rate, with samples containing 20 wt% β-tricalcium phosphate losing significantly less weight over time in comparison to the control sample. Compression testing of samples following submission in aqueous environments revealed the composites exhibited enhanced strength with increasing bioceramic loading. The mechanical properties were significantly reduced over a period of 5 weeks. It was found that hot-melt extrusion of β-tricalcium phosphate is a viable and effective method of producing novel composite scaffolds with potential for regenerative medicine applications.