Characterizing Aptamer Interaction with the Oncolytic Virus VV-GMCSF-Lact.

Aptamers are currently being investigated for their potential to improve virotherapy. They offer several advantages, including the ability to prevent the aggregation of viral particles, enhance target specificity, and protect against the neutralizing effects of antibodies. The purpose of this study was to comprehensively investigate an aptamer capable of enhancing virotherapy. This involved characterizing the previously selected aptamer for vaccinia virus (VACV), evaluating the aggregation and molecular interaction of the optimized aptamers with the recombinant oncolytic virus VV-GMCSF-Lact, and estimating their immunoshielding properties in the presence of human blood serum. We chose one optimized aptamer, NV14t_56, with the highest affinity to the virus from the pool of several truncated aptamers and built its 3D model. The NV14t_56 remained stable in human blood serum for 1 h and bound to VV-GMCSF-Lact in the micromolar range (Kd ≈ 0.35 µM). Based on dynamic light scattering data, it has been demonstrated that aptamers surround viral particles and inhibit aggregate formation. In the presence of serum, the hydrodynamic diameter (by intensity) of the aptamer-virus complex did not change. Microscale thermophoresis (MST) experiments showed that NV14t_56 binds with virus (EC50 = 1.487 × 109 PFU/mL). The analysis of the amplitudes of MST curves reveals that the components of the serum bind to the aptamer-virus complex without disrupting it. In vitro experiments demonstrated the efficacy of VV-GMCSF-Lact in conjunction with the aptamer when exposed to human blood serum in the absence of neutralizing antibodies (Nabs). Thus, NV14t_56 has the ability to inhibit virus aggregation, allowing VV-GMCSF-Lact to maintain its effectiveness throughout the storage period and subsequent use. When employing aptamers as protective agents for oncolytic viruses, the presence of neutralizing antibodies should be taken into account.

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.

Discovery of DNA aptamers targeting SARS-CoV-2 nucleocapsid protein and protein-binding epitopes for label-free COVID-19 diagnostics.

The spread of COVID-19 has affected billions of people across the globe, and the diagnosis of viral infection still needs improvement. Because of high immunogenicity and abundant expression during viral infection, SARS-CoV-2 nucleocapsid (N) protein could be an important diagnostic marker. This study aimed to develop a label-free optical aptasensor fabricated with a novel single-stranded DNA aptamer to detect the N protein. The N-binding aptamers selected using asymmetric-emulsion PCR-SELEX and their binding affinity and cross-reactivity were characterized by biolayer interferometry. The tNSP3 aptamer (44 nt) was identified to bind the N protein of wild type and Delta and Omicron variants with high affinity (KD in the range of 0.6-3.5 nM). Utilizing tNSP3 to detect the N protein spiked in human saliva evinced the potential of this aptamer with a limit of detection of 4.5 nM. Mass spectrometry analysis was performed along with molecular dynamics simulation to obtain an insight into how tNSP3 binds to the N protein. The identified epitope peptides are localized within the RNA-binding domain and C terminus of the N protein. Hence, we confirmed the performance of this aptamer as an analytical tool for COVID-19 diagnosis.

Structure and Vibrational Spectroscopy of C82 Fullerenol Valent Isomers: An Experimental and Theoretical Joint Study.

Gd@C82OxHy endohedral complexes for advanced biomedical applications (computer tomography, cancer treatment, etc.) were synthesized using high-frequency arc plasma discharge through a mixture of graphite and Gd2O3 oxide. The Gd@C82 endohedral complex was isolated by high-efficiency liquid chromatography and consequently oxidized with the formation of a family of Gd endohedral fullerenols with gross formula Gd@C82O8(OH)20. Fourier-transformed infrared (FTIR) spectroscopy was used to study the structure and spectroscopic properties of the complexes in combination with the DFTB3 electronic structure calculations and infrared spectra simulations. It was shown that the main IR spectral features are formed by a fullerenole C82 cage that allows one to consider the force constants at the DFTB3 level of theory without consideration of gadolinium endohedral ions inside the carbon cage. Based on the comparison of experimental FTIR and theoretical DFTB3 IR spectra, it was found that oxidation of the C82 cage causes the formation of Gd@C82O28H20, with a breakdown of the integrity of the parent C82 cage with the formation of pores between neighboring carbonyl and carboxyl groups. The Gd@C82O6(OOH)2(OH)18 endohedral complex with epoxy, carbonyl and carboxyl groups was considered the most reliable fullerenole structural model.

Aptamers Regulating the Hemostasis System.

The hemostasis system is a complex structure that includes the fibrinolysis system, and Yes this is correct coagulation and anticoagulation parts. Due to the multicomponent nature, it becomes relevant to study the key changes in the functioning of signaling pathways, and develop new diagnostic methods and modern drugs with high selectivity. One of the ways to solve this problem is the development of molecular recognition elements capable of blocking one of the hemostasis systems and/or activating another. Aptamers can serve as ligands for targeting specific clinical needs, promising anticoagulants with minor side effects and significant biological activity. Aptamers with several clotting factors and platelet proteins are used for the treatment of thrombosis. This review is focused on the aptamers used for the correction of the hemostasis system, and their structural and functional features. G-rich nucleic acid aptamers, mostly versatile G-quadruplexes, recognize different components of the hemostasis system and are capable of correcting the functioning.

Nucleic Acid Aptamers Increase the Anticancer Efficiency and Reduce the Toxicity of Cisplatin-Arabinogalactan Conjugates In Vivo.

Cisplatin is an effective drug for treating various cancer types. However, it is highly toxic for both healthy and tumor cells. Therefore, there is a need to reduce its therapeutic dose and increase targeted bioavailability. One of the ways to achieve this could be the coating of cisplatin with polysaccharides and specific carriers for targeted delivery. Nucleic acid aptamers could be used as carriers for the specific delivery of medicine to cancer cells. Cisplatin-arabinogalactan-aptamer (Cis-AG-Ap) conjugate was synthesized based on Cis-dichlorodiammineplatinum, Siberian larch arabinogalactan, and aptamer AS-42 specific to heat-shock proteins (HSP) 71 kDa (Hspa8) and HSP 90-beta (Hsp90ab1). The antitumor effect was estimated using ascites and metastatic Ehrlich tumor models. Cis-AG-Ap toxicity was assessed by blood biochemistry on healthy mice. Here, we demonstrated enhanced anticancer activity of Cis-AG-Ap and its specific accumulation in tumor foci. It was shown that targeted delivery allowed a 15-fold reduction in the therapeutic dose of cisplatin and its toxicity. Cis-AG-Ap sufficiently suppressed the growth of Ehrlich's ascites carcinoma, the mass and extent of tumor metastasis in vivo. Arabinogalactan and the aptamers promoted cisplatin efficiency by enhancing its bioavailability. The described strategy could be very promising for targeted anticancer therapy.

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 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.

11C-radiolabeled aptamer for imaging of tumors and metastases using positron emission tomography- computed tomography.

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.

The role of SAXS and molecular simulations in 3D structure elucidation of a DNA aptamer against lung cancer.

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.