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.

Effects of Endohedral Gd-Containing Fullerenols with a Different Number of Oxygen Substituents on Bacterial Bioluminescence.

Gadolinium (Gd)-containing fullerenols are perspective agents for magnetic resonance imaging and cancer research. They combine the unique paramagnetic properties of Gd with solubility in water, low toxicity and antiradical activity of fullerenols. We compared the bioeffects of two Gd-containing fullerenols with a different number of oxygen groups-20 and 42: Gd@C82O20H14 and Gd@C82O42H32. The bioluminescent bacteria-based assay was applied to monitor the toxicity of fullerenols, bioluminescence was applied as a signal physiological parameter, and bacterial enzyme-based assay was used to evaluate the fullerenol effects on enzymatic intracellular processes. Chemiluminescence luminol assay was applied to monitor the content of reactive oxygen species (ROS) in bacterial and enzymatic media. It was shown that Gd@C82O42H32 and Gd@C82O20H14 inhibited bacterial bioluminescence at >10-1 and >10-2 gL-1, respectively, revealing a lower toxicity of Gd@C82O42H32. Low-concentration (10-3-10-1 gL-1) bacterial bioluminescence activation by Gd@C82O42H32 was observed, while this activation was not found under exposure to Gd@C82O20H14. Additional carboxyl groups in the structure of Gd@C82O42H32 were determined by infrared spectroscopy and confirmed by quantum chemical calculations. The groups were supposed to endow Gd@C82O42H32 with higher penetration ability through the cellular membrane, activation ability, lower toxicity, balancing of the ROS content in the bacterial suspensions, and lower aggregation in aqueous media.

Synthesis, Photo- and Electroluminescence of New Polyfluorene Copolymers Containing Dicyanostilbene and 9,10-Dicyanophenanthrene in the Main Chain.

Using palladium-catalyzed Suzuki polycondensation, we synthesized new light-emitting fluorene copolymers containing the dicyano derivatives of stilbene and phenanthrene and characterized them by gel permeation chromatography, UV-vis absorption spectroscopy, spectrofluorimetry, and cyclic voltammetry. The photoluminescence spectra of the synthesized polymers show significant energy transfer from the fluorene segments to the dicyanostilbene and 9,10-dicyanophenanthrene units, which is in agreement with the data of theoretical calculations. OLEDs based on these polymers were fabricated with an ITO/PEDOT-PSS (35 nm)/p-TPD (30 nm)/PVK (5 nm)/light emitting layer (70-75 nm)/PF-PO (20 nm)/LiF (1 nm)/Al (80 nm) configuration. Examination of their electroluminescence revealed that copolymers of fluorene with dicyanostilbene show yellow-green luminescence, while polymers with 9,10-dicyanophenanthrene have a greenish-blue emission. The 9,10-dicyanophenanthrene units have a more rigid structure compared to dicyanostilbene and, in OLEDs based on them, an increase in maximum brightness is observed with an increase in the content of the additive to the polymer chain. In particular, the device using fluorene copolymer with 9,10-dicyanophenanthrene (2.5 mol%) exhibited a maximum brightness of 9230 cd/m2 and a maximum current efficiency of 3.33 cd/A.

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.

Ca2+-Triggered Coelenterazine-Binding Protein Renilla: Expected and Unexpected Features.

Ca2+-triggered coelenterazine-binding protein (CBP) is a natural form of the luciferase substrate involved in the Renilla bioluminescence reaction. It is a stable complex of coelenterazine and apoprotein that, unlike coelenterazine, is soluble and stable in an aquatic environment and yields a significantly higher bioluminescent signal. This makes CBP a convenient substrate for luciferase-based in vitro assay. In search of a similar substrate form for the luciferase NanoLuc, a furimazine-apoCBP complex was prepared and verified against furimazine, coelenterazine, and CBP. Furimazine-apoCBP is relatively stable in solution and in a frozen or lyophilized state, but as distinct from CBP, its bioluminescence reaction with NanoLuc is independent of Ca2+. NanoLuc turned out to utilize all the four substrates under consideration. The pairs of CBP-NanoLuc and coelenterazine-NanoLuc generate bioluminescence with close efficiency. As for furimazine-apoCBP-NanoLuc pair, the efficiency with which it generates bioluminescence is almost twice lower than that of the furimazine-NanoLuc. The integral signal of the CBP-NanoLuc pair is only 22% lower than that of furimazine-NanoLuc. Thus, along with furimazine as the most effective NanoLuc substrate, CBP can also be recommended as a substrate for in vitro analytical application in view of its water solubility, stability, and Ca2+-triggering "character".

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.

Unusual shift in the visible absorption spectrum of an active ctenophore photoprotein elucidated by time-dependent density functional theory.

Active hydromedusan and ctenophore Ca2+-regulated photoproteins form complexes consisting of apoprotein and strongly non-covalently bound 2-hydroperoxycoelenterazine (an oxygenated intermediate of coelenterazine). Whereas the absorption maximum of hydromedusan photoproteins is at 460-470 nm, ctenophore photoproteins absorb at 437 nm. Finding out a physical reason for this blue shift is the main objective of this work, and, to achieve it, the whole structure of the protein-substrate complex was optimized using a linear scaling quantum-mechanical method. Electronic excitations pertinent to the spectra of the 2-hydroperoxy adduct of coelenterazine were simulated with time-dependent density functional theory. The dihedral angle of 60° of the 6-(p-hydroxy)-phenyl group relative to the imidazopyrazinone core of 2-hydroperoxycoelenterazine molecule was found to be the key factor determining the absorption of ctenophore photoproteins at 437 nm. The residues relevant to binding of the substrate and its adopting the particular rotation were also identified.

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.

Molecular Epitope Determination of Aptamer Complexes of the Multidomain Protein C-Met by Proteolytic Affinity-Mass Spectrometry.

C-Met protein is a glycosylated receptor tyrosine kinase of the hepatocyte growth factor (HGF), composed of an α and a ß chain. Upon ligand binding, C-Met transmits intracellular signals by a unique multi-substrate docking site. C-Met can be aberrantly activated leading to tumorigenesis and other diseases, and has been recognized as a biomarker in cancer diagnosis. C-Met aptamers have been recently considered a useful tool for detection of cancer biomarkers. Herein we report a molecular interaction study of human C-Met expressed in kidney cells with two DNA aptamers of 60 and 64 bases (CLN0003 and CLN0004), obtained using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure. Epitope peptides of aptamer-C-Met complexes were identified by proteolytic affinity-mass spectrometry in combination with SPR biosensor analysis (PROTEX-SPR-MS), using high-pressure proteolysis for efficient digestion. High affinities (KD , 80-510 nM) were determined for aptamer-C-Met complexes, with two-step binding suggested by kinetic analysis. A linear epitope, C-Met (381-393) was identified for CLN0004, while the CLN0003 aptamer revealed an assembled epitope comprised of two peptide sequences, C-Met (524-543) and C-Met (557-568). Structure modeling of C-Met-aptamers were consistent with the identified epitopes. Specificities and affinities were ascertained by SPR analysis of the synthetic epitope peptides. The high affinities of aptamers to C-Met, and the specific epitopes revealed render them of high interest for cellular diagnostic studies.

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

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.

Two-dimensional hexagonal CrN with promising magnetic and optical properties: A theoretical prediction.

Half-metallic ferromagnetic materials with planar forms are promising for spintronics applications. A wide range of 2D lattices like graphene, h-BN, transition metal dichalcogenides, etc. are non-magnetic or weakly magnetic. Using first principles calculations, the existence of graphene-like hexagonal chromium nitride (h-CrN) with an almost flat atomically thin structure is predicted. We find that freestanding h-CrN has a 100% spin-polarized half-metallic nature with possible ferromagnetic ordering and a high rate of optical transparency. As a possible method for stabilization and synthesis, deposition of h-CrN on 2D MoSe2 or on MoS2 is proposed. The formation of composites retains the half-metallic properties and leads to the reduction of spin-down band gaps to 1.43 and 1.71 eV for energetically favorable h-CrN/MoSe2 and h-CrN/MoS2 configurations, respectively. Calculation of the dielectric functions of h-CrN, h-CrN/MoSe2 and h-CrN/MoS2 exhibit the high transparency of all three low-dimensional nanomaterials. The honeycomb CrN may be considered as a promising fundamental 2D material for a variety of potential applications of critical importance.

Ultraviolet fluorescence of coelenteramide and coelenteramide-containing fluorescent proteins. Experimental and theoretical study.

Coelenteramide-containing fluorescent proteins are products of bioluminescent reactions of marine coelenterates. They are called 'discharged photoproteins'. Their light-induced fluorescence spectra are variable, depending considerably on external conditions. Current work studies a dependence of light-induced fluorescence spectra of discharged photoproteins obelin, aequorin, and clytin on excitation energy. It was demonstrated that photoexcitation to the upper electron-excited states (260-300nm) of the discharged photoproteins initiates a fluorescence peak in the near UV region, in addition to the blue-green emission. To characterize the UV fluorescence, the light-induced fluorescence spectra of coelenteramide (CLM), fluorophore of the discharged photoproteins, were studied in methanol solution. Similar to photoproteins, the CLM spectra depended on photoexcitation energy; the additional peak (330nm) in the near UV region was observed in CLM fluorescence at higher excitation energy (260-300nm). Quantum chemical calculations by time depending method with B3LYP/cc-pVDZ showed that the conjugated pyrazine-phenolic fragment and benzene moiety of CLM molecule are responsible for the additional UV fluorescence peak. Quantum yields of CLM fluorescence in methanol were 0.028±0.005 at 270-340nm photoexcitation. A conclusion was made that the UV emission of CLM might contribute to the UV fluorescence of the discharged photoproteins. The study develops knowledge on internal energy transfer in biological structures - complexes of proteins with low-weight aromatic molecules.