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Here, we present DNA aptamers capable of specific binding to glial tumor cells in vitro, ex vivo, and in vivo for visualization diagnostics of central nervous system tumors. We selected the aptamers binding specifically to the postoperative human glial primary tumors and not to the healthy brain cells and meningioma, using a modified process of systematic evolution of ligands by exponential enrichment to cells; sequenced and analyzed ssDNA pools using bioinformatic tools and identified the best aptamers by their binding abilities; determined three-dimensional structures of lead aptamers (Gli-55 and Gli-233) with small-angle X-ray scattering and molecular modeling; isolated and identified molecular target proteins of the aptamers by mass spectrometry; the potential binding sites of Gli-233 to the target protein and the role of post-translational modifications were verified by molecular dynamics simulations. The anti-glioma aptamers Gli-233 and Gli-55 were used to detect circulating tumor cells in liquid biopsies. These aptamers were used for in situ, ex vivo tissue staining, histopathological analyses, and fluorescence-guided tumor and PET/CT tumor visualization in mice with xenotransplanted human astrocytoma. The aptamers did not show in vivo toxicity in the preclinical animal study. This study demonstrates the potential applications of aptamers for precise diagnostics and fluorescence-guided surgery of brain tumors.
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One of the promising novel methods for radical tumor resection at a single-cell level is magneto-mechanical microsurgery (MMM) with magnetic nano- or microdisks modified with cancer-recognizing molecules. A low-frequency alternating magnetic field (AMF) remotely drives and controls the procedure. Here, we present characterization and application of magnetic nanodisks (MNDs) as a surgical instrument ("smart nanoscalpel") at a single-cell level. MNDs with a quasi-dipole three-layer structure (Au/Ni/Au) and DNA aptamer AS42 (AS42-MNDs) on the surface converted magnetic moment into mechanical and destroyed tumor cells. The effectiveness of MMM was analyzed on Ehrlich ascites carcinoma (EAC) cells in vitro and in vivo using sine and square-shaped AMF with frequencies from 1 to 50 Hz with 0.1 to 1 duty-cycle parameters. MMM with the "Nanoscalpel" in a sine-shaped 20 Hz AMF, a rectangular-shaped 10 Hz AMF, and a 0.5 duty cycle was the most effective. A sine-shaped field caused apoptosis, whereas a rectangular-shaped field caused necrosis. Four sessions of MMM with AS42-MNDs significantly reduced the number of cells in the tumor. In contrast, ascites tumors continued to grow in groups of mice and mice treated with MNDs with nonspecific oligonucleotide NO-MND. Thus, applying a "smart nanoscalpel" is practical for the microsurgery of malignant neoplasms.
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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 receptor-binding 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 variants.
Assuntos
Aptâmeros de Nucleotídeos , COVID-19 , Aptâmeros de Nucleotídeos/química , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , SARS-CoV-2 , Técnica de Seleção de Aptâmeros , Glicoproteína da Espícula de CoronavírusRESUMO
Identification of primary tumors and metastasis sites is an essential step in cancer diagnostics and the following treatment. Positron emission tomography-computed tomography (PET/CT) is one of the most reliable methods for scanning the whole organism for malignancies. In this work, we synthesized an 11C-labeled oligonucleotide primer and hybridized it to an anti-cancer DNA aptamer. The 11C-aptamer was applied for in vivo imaging of Ehrlich ascites carcinoma and its metastases in mice using PET/CT. The imaging experiments with the 11C-aptamer determined very small primary and secondary tumors of 3 mm2 and less. We also compared 11C imaging with the standard radiotracer, 2-deoxy-2-[fluorine-18]fluoro-D-glucose (18F-FDG), and found better selectivity of the 11C-aptamer to metastatic lesions in the metabolically active organs than 18F-FDG. 11C radionuclide with an ultra-short (20.38 min) half-life is considered safest for PET/CT imaging and does not cause false-positive results in heart imaging. Its combination with aptamers gives us high-specificity and high-contrast imaging of cancer cells and can be applied for PET/CT-guided drug delivery in cancer therapies.
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Aptamers are short, single-stranded DNA or RNA oligonucleotide molecules that function as synthetic analogs of antibodies and bind to a target molecule with high specificity. Aptamer affinity entirely depends on its tertiary structure and charge distribution. Therefore, length and structure optimization are essential for increasing aptamer specificity and affinity. Here, we present a general optimization procedure for finding the most populated atomistic structures of DNA aptamers. Based on the existed aptamer LC-18 for lung adenocarcinoma, a new truncated LC-18 (LC-18t) aptamer LC-18t was developed. A three-dimensional (3D) shape of LC-18t was reported based on small-angle X-ray scattering (SAXS) experiments and molecular modeling by fragment molecular orbital or molecular dynamic methods. Molecular simulations revealed an ensemble of possible aptamer conformations in solution that were in close agreement with measured SAXS data. The aptamer LC-18t had stronger binding to cancerous cells in lung tumor tissues and shared the binding site with the original larger aptamer. The suggested approach reveals 3D shapes of aptamers and helps in designing better affinity probes.
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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.
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Nucleic acid (NA) aptamers bind to their targets with high affinity and selectivity. The three-dimensional (3D) structures of aptamers play a major role in these non-covalent interactions. Here, we use a four-step approach to determine a true 3D structure of aptamers in solution using small-angle X-ray scattering (SAXS) and molecular structure restoration (MSR). The approach consists of (i) acquiring SAXS experimental data of an aptamer in solution, (ii) building a spatial distribution of the molecule's electron density using SAXS results, (iii) constructing a 3D model of the aptamer from its nucleotide primary sequence and secondary structure, and (iv) comparing and refining the modeled 3D structures with the experimental SAXS model. In the proof-of-principle we analyzed the 3D structure of RE31 aptamer to thrombin in a native free state at different temperatures and validated it by circular dichroism (CD). The resulting 3D structure of RE31 has the most energetically favorable conformation and the same elements such as a B-form duplex, non-complementary region, and two G-quartets which were previously reported by X-ray diffraction (XRD) from a single crystal. More broadly, this study demonstrates the complementary approach for constructing and adjusting the 3D structures of aptamers, DNAzymes, and ribozymes in solution, and could supply new opportunities for developing functional nucleic acids. Graphical abstract.
Assuntos
Aptâmeros de Nucleotídeos/química , Algoritmos , Simulação por Computador , Quadruplex G , Modelos Moleculares , Conformação de Ácido Nucleico , Espalhamento a Baixo Ângulo , Difração de Raios X/métodosRESUMO
Novel nanoscale bioconjugates combining unique plasmonic photothermal properties of gold nanoparticles (AuNPs) with targeted delivery using cell-specific DNA aptamers have a tremendous potential for medical diagnostics and therapy of many cell-based diseases. In this study, we demonstrate the high anti-cancer activity of aptamer-conjugated, 37-nm spherical gold nanoparticles toward Ehrlich carcinoma in tumor-bearing mice after photothermal treatment. The synthetic anti-tumor aptamers bring the nanoparticles precisely to the desired cells and selectively eliminate cancer cells after the subsequent laser treatment. To prove tumor eradication, we used positron emission tomography (PET) utilizing radioactive glucose and computer tomography, followed by histological analysis of cancer tissue. Three injections of aptamer-conjugated AuNPs and 5 min of laser irradiations are enough to make the tumor undetectable by PET. Histological analysis proves PET results and shows lower damage of healthy tissue in addition to a higher treatment efficiency and selectivity of the gold nanoparticles functionalized with aptamers in comparison to control experiments using free unconjugated nanoparticles.