Structure- and interaction-based design of anti-SARS-CoV-2 aptamers

Aptamer selection against novel infections is a complicated and time-consuming approach. Synergy can be achieved by using computational methods together with experimental procedures. This study aims to develop a reliable methodology for a rational aptamer in silico et vitro design. The new approach combines multiple steps: (1) Molecular design, based on screening in a DNA aptamer library and directed mutagenesis to fit the protein tertiary structure; (2) 3D molecular modeling of the target; (3) Molecular docking of an aptamer with the protein; (4) Molecular dynamics (MD) simulations of the complexes; (5) Quantum-mechanical (QM) evaluation of the interactions between aptamer and target with further analysis; (6) Experimental verification at each cycle for structure and binding affinity by using small-angle X-ray scattering, cytometry, and fluorescence polarization. By using a new iterative design procedure, structure- and interaction-based drug design (SIBDD), a highly specific aptamer to the receptorbinding domain of the SARS-CoV-2 spike protein, was developed and validated. The SIBDD approach enhances speed of the high-affinity aptamers development from scratch, using a target protein structure. The method could be used to improve existing aptamers for stronger binding. This approach brings to an advanced level the development of novel affinity probes, functional nucleic acids. It offers a blueprint for the straightforward design of targeting molecules for new pathogen agents and emerging variant

Monitoring of breast cancer progression via aptamer-based detection of circulating tumor cells in clinical blood samples

Introduction: Breast cancer (BC) diagnostics lack noninvasive methods and procedures for screening and monitoring disease dynamics. Admitted CellSearch® is used for fluid biopsy and capture of circulating tumor cells of only epithelial origin. Here we describe an RNA aptamer (MDA231) for detecting BC cells in clinical samples, including blood. The MDA231 aptamer was originally selected against triple-negative breast cancer cell line MDA-MB-231 using cell-SELEX.Methods: The aptamer structure in solution was predicted using mFold program and molecular dynamic simulations. The affinity and specificity of the evolved aptamers were evaluated by flow cytometry and laser scanning microscopy on clinical tissues from breast cancer patients. CTCs were isolated form the patients’ blood using the developed method of aptamer-based magnetic separation. Breast cancer origin of CTCs was confirmed by cytological, RT-qPCR and Immunocytochemical analyses.Results: MDA231 can specifically recognize breast cancer cells in surgically resected tissues from patients with different molecular subtypes: triple-negative, Luminal A, and Luminal B, but not in benign tumors, lung cancer, glial tumor and healthy epithelial from lungs and breast. This RNA aptamer can identify cancer cells in complex cellular environments, including tumor biopsies (e.g., tumor tissues vs. margins) and clinical blood samples (e.g., circulating tumor cells). Breast cancer origin of the aptamer-based magnetically separated CTCs has been proved by immunocytochemistry and mammaglobin mRNA expression.Discussion: We suggest a simple, minimally-invasive breast cancer diagnostic method based on non-epithelial MDA231 aptamer-specific magnetic isolation of circulating tumor cells. Isolated cells are intact and can be utilized for molecular diagnostics purposes

Aptamers Increase Biocompatibility and Reduce the Toxicity of Magnetic Nanoparticles Used in Biomedicine

Aptamer-based approaches are very promising tools in nanomedicine. These small single-stranded DNA or RNA molecules are often used for the effective delivery and increasing biocompatibility of various therapeutic agents. Recently, magnetic nanoparticles (MNPs) have begun to be successfully applied in various fields of biomedicine. The use of MNPs is limited by their potential toxicity, which depends on their biocompatibility. The functionalization of MNPs by ligands increases biocompatibility by changing the charge and shape of MNPs, preventing opsonization, increasing the circulation time of MNPs in the blood, thus shielding iron ions and leading to the accumulation of MNPs only in the necessary organs. Among various ligands, aptamers, which are synthetic analogs of antibodies, turned out to be the most promising for the functionalization of MNPs. This review describes the factors that determine MNPs’ biocompatibility and affect their circulation time in the bloodstream, biodistribution in organs and tissues, and biodegradation. The work also covers the role of the aptamers in increasing MNPs’ biocompatibility and reducing toxicity

Aptamer-conjugated superparamagnetic ferroarabinogalactan nanoparticles for targeted magnetodynamic therapy of cancer

Nanotechnologies involving physical methods of tumor destruction using functional oligonucleotides are promising for targeted cancer therapy. Our study presents magnetodynamic therapy for selective elimination of tumor cells in vivo using DNA aptamer-functionalized magnetic nanoparticles exposed to a low frequency alternating magnetic field. We developed an enhanced targeting approach of cancer cells with aptamers and arabinogalactan. Aptamers to fibronectin (AS-14) and heat shock cognate 71 kDa protein (AS-42) facilitated the delivery of the nanoparticles to Ehrlich carcinoma cells, and arabinogalactan (AG) promoted internalization through asialoglycoprotein receptors. Specific delivery of the aptamer-modified FeAG nanoparticles to the tumor site was confirmed by magnetic resonance imaging (MRI). After the following treatment with a low frequency alternating magnetic field, AS-FeAG caused cancer cell death in vitro and tumor reduction in vivo. Histological analyses showed mechanical disruption of tumor tissues, total necrosis, cell lysis, and disruption of the extracellular matrix. The enhanced targeted magnetic theranostics with the aptamer conjugated superparamagnetic ferroarabinogalactans opens up a new venue for making biocompatible contrasting agents for MRI imaging and performing non-invasive anti-cancer therapies with a deep penetrated magnetic field

Aptamer-Based Viability Impedimetric Sensor for Bacteria

The development of an aptamer-based viability impedimetric sensor for bacteria (AptaVISens-B) is presented. Highly specific DNA aptamers to live <i>Salmonella typhimurium</i> were selected via the cell-systematic evolution of ligands by exponential enrichment (SELEX) technique. Twelve rounds of selection were performed; each comprises a positive selection step against viable <i>S. typhimurium</i> and a negative selection step against heat killed <i>S. typhimurium</i> and a mixture of related pathogens, including <i>Salmonella enteritidis</i>, <i>Escherichia coli</i>, <i>Staphylococcus aureus</i>, <i>Pseudomonas aeruginosa</i>, and <i>Citrobacter freundii</i> to ensure the species specificity of the selected aptamers. The DNA sequence showing the highest binding affinity to the bacteria was further integrated into an impedimetric sensor via self-assembly onto a gold nanoparticle-modified screen-printed carbon electrode (GNP-SPCE). Remarkably, this aptasensor is highly selective and can successfully detect <i>S. typhimurium</i> down to 600 CFU mL^–1 (equivalent to 18 live cells in 30 μL of assay volume) and distinguish it from other <i>Salmonella</i> species, including <i>S. enteritidis</i> and <i>S. choleraesuis</i>. This report is envisaged to open a new venue for the aptamer-based viability sensing of a variety of microorganisms, particularly viable but nonculturable (VBNC) bacteria, using a rapid, economic, and label-free electrochemical platform

Four steps for revealing and adjusting the 3D structure of aptamers in solution by small-angle X-ray scattering and computer simulation

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Aptamer-Based Impedimetric Sensor for Bacterial Typing

The development of an aptamer-based impedimetric sensor for typing of bacteria (AIST-B) is presented. Highly specific DNA aptamers to <i>Salmonella enteritidis</i> were selected via Cell-SELEX technique. Twelve rounds of selection were performed; each comprises a positive selection step against <i>S. enteritidis</i> and a negative selection step against a mixture of related pathogens, including <i>Salmonella typhimurium</i>, <i>Escherichia coli</i>, <i>Staphylococcus aureus</i>, <i>Pseudomonas aeruginosa</i>, and <i>Citrobacter freundii</i>, to ensure the species-specificity of the selected aptamers. After sequencing of the pool showing the highest binding affinity to <i>S. enteritidis</i>, a DNA sequence of high affinity to the bacteria was integrated into an impedimetric sensor via self-assembly onto a gold nanoparticles-modified screen-printed carbon electrode (GNPs-SPCE). Remarkably, this aptasensor is highly selective and can successfully detect <i>S. enteritidis</i> down to 600 CFU mL^–1 (equivalent to 18 CFU in 30 μL assay volume) in 10 min and distinguish it from other Salmonella species, including <i>S. typhimurium</i> and <i>S. choleraesuis</i>. This report is envisaged to open a new venue for the aptamer-based typing of a variety of microorganisms using a rapid, economic, and label-free electrochemical platform

Laser Cleaning Improves Stem Cell Adhesion on the Dental Implant Surface during Peri-Implantitis Treatment

Dental implant therapy is a well-accepted treatment modality. Despite good predictability and success in the early stages, the risk of postplacement inflammation in the long-term periods remains an urgent problem. Surgical access and decontamination with chemical and mechanical methods are more effective than antibiotic therapy. The search for the optimal and predictable way for peri-implantitis treatment remains relevant. Here, we evaluated four cleaning methods for their ability to preserve the implant’s surface for adequate mesenchymal stem cell adhesion and differentiation. Implants isolated after peri-implantitis were subjected to cleaning with diamond bur; Ti-Ni alloy brush, air-flow, or Er,Cr:YSGG laser and cocultured with mice MSC for five weeks. Dental bur and titanium brushes destroyed the implants’ surfaces and prevented MSC attachment. Air-flow and laser minimally affected the dental implant surface microroughness, which was initially designed for good cell adhesion and bone remodeling and to provide full microbial decontamination. Anodized with titanium dioxide and sandblasted with aluminum oxide, acid-etched implants appeared to be better for laser treatment. In implants sandblasted with aluminum oxide, an acid-etched surface better preserves its topology when treated with the air-flow. These cleaning methods minimally affect the implant’s surface, so it maintains the capability to absorb osteogenic cells for further division and differentiation