Radiochemical Synthesis of 4-[18F]FluorobenzylAzide and Its Conjugation with EGFR-Specific Aptamers.

Central nervous system tumors related to gliomas are of neuroectodermal origin and cover about 30% of all primary brain tumors. Glioma is not susceptible to any therapy and surgical attack remains one of the main approaches to its treatment. Preoperative tumor imaging methods, such as positron emission tomography (PET), are currently used to distinguish malignant tissue to increase the accuracy of glioma removal. However, PET is lacking a specific visualization of cells possessing certain molecular markers. Here, we report an application of aptamers to enhancing specificity in imaging tumor cells bearing the epidermal growth factor receptor (EGFR). Glioblastoma is characterized by increased EGFR expression, as well as mutations of this receptor associated with active division, migration, and adhesion of tumor cells. Since 2021, EGFR has been included into the WHO classification of gliomas as a molecular genetic marker. To obtain conjugates of aptamers GR20 and GOL1-specific to EGFR, a 4-[18F]fluorobenzylazide radiotracer was used as a synthon. For the production of the synthon, a method of automatic synthesis on an Eckert & Ziegler research module was adapted and modified using spirocyclic iodonium ylide as a precursor. Conjugation of 4-[18F]fluorobenzylazide and alkyne-modified aptamers was carried out using Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) with/without the TBTA ligand. As a result, it was possible to obtain 18F-labelled conjugates with 97% radiochemical purity for [18F]FB-GR20 and 98% for [18F]FB-GOL1. The obtained conjugates can be used for further studies in PET analysis on model animals with grafted glioblastoma.

RNA Aptamers for Theranostics of Glioblastoma of Human Brain.

Conventional approaches for studying and molecular typing of tumors include PCR, blotting, omics, immunocytochemistry, and immunohistochemistry. The last two methods are the most used, as they enable detecting both tumor protein markers and their localizations within the cells. In this study, we have investigated a possibility of using RNA aptamers, in particular, 2'-F-pyrimidyl-RNA aptamer ME07 (48 nucleotides long), specific to the receptor of epidermal growth factor (EGFR, ErbB1, Her1), as an alternative to monoclonal antibodies for aptacytochemistry and aptahistochemistry for human glioblastoma multiforme (GBM). A specificity of binding of FAM-ME07 to the receptor on the tumor cells has been demonstrated by flow cytometry; an apparent dissociation constant for the complex of aptamer - EGFR on the cell has been determined; a number of EGFR molecules has been semi-quantitatively estimated for the tumor cell lines having different amount of EGFR: A431 (106 copies per cell), U87 (104 copies per cell), MCF7 (103 copies per cell), and ROZH, primary GBM cell culture derived from patient (104 copies per cell). According to fluorescence microscopy, FAM-ME07 interacts directly with the receptors on A431 cells, followed by its internalization into the cytoplasm and translocation to the nucleolus; this finding opens a possibility of ME07 application as an escort aptamer for a delivery of therapeutic agents into tumor cells. FAM-ME07 efficiently stains sections of GBM clinical specimens, which enables an identification of EGFR-positive clones within a heterogeneous tumor; and providing a potential for further studying animal models of GBM.

The Functional Role of Loops and Flanking Sequences of G-Quadruplex Aptamer to the Hemagglutinin of Influenza a Virus.

Nucleic acid aptamers are generally accepted as promising elements for the specific and high-affinity binding of various biomolecules. It has been shown for a number of aptamers that the complexes with several related proteins may possess a similar affinity. An outstanding example is the G-quadruplex DNA aptamer RHA0385, which binds to the hemagglutinins of various influenza A virus strains. These hemagglutinins have homologous tertiary structures but moderate-to-low amino acid sequence identities. Here, the experiment was inverted, targeting the same protein using a set of related, parallel G-quadruplexes. The 5'- and 3'-flanking sequences of RHA0385 were truncated to yield parallel G-quadruplex with three propeller loops that were 7, 1, and 1 nucleotides in length. Next, a set of minimal, parallel G-quadruplexes with three single-nucleotide loops was tested. These G-quadruplexes were characterized both structurally and functionally. All parallel G-quadruplexes had affinities for both recombinant hemagglutinin and influenza virions. In summary, the parallel G-quadruplex represents a minimal core structure with functional activity that binds influenza A hemagglutinin. The flanking sequences and loops represent additional features that can be used to modulate the affinity. Thus, the RHA0385-hemagglutinin complex serves as an excellent example of the hypothesis of a core structure that is decorated with additional recognizing elements capable of improving the binding properties of the aptamer.

Exploring the efficiency of thrombin inhibitors with a quantitative model of the coagulation cascade.

A detailed mathematical description of the coagulation cascade is a challenging task due to a huge set of protein-protein interactions. Simplified models do not permit quantitative description of anticoagulants. The detailed mathematical model presented here was constructed with 98 reactions between 70 species. The model was verified using experimental data on thrombin generation. Four thrombin inhibitors, which have different inhibitory mechanisms, were incorporated into the model. All four thrombin inhibitors delayed prothrombin conversion into thrombin, but did not preclude it. At high inhibitor concentration, thrombin-mediated positive feedback loops were strongly inhibited and the proportion of prothrombin, converted with factor Xa only, was considerably increased. The most potent inhibitor of prothrombin conversion was aptamer NU172, which also binds prothrombin and inhibits its conversion.

Structural and Functional Aspects of G-Quadruplex Aptamers Which Bind a Broad Range of Influenza A Viruses.

An aptamer is a synthetic oligonucleotide with a unique spatial structure that provides specific binding to a target. To date, several aptamers to hemagglutinin of the influenza A virus have been described, which vary in affinity and strain specificity. Among them, the DNA aptamer RHA0385 is able to recognize influenza hemagglutinins with highly variable sequences. In this paper, the structure of RHA0385 was studied by circular dichroism spectroscopy, nuclear magnetic resonance, and size-exclusion chromatography, demonstrating the formation of a parallel G-quadruplex structure. Three derivatives of RHA0385 were designed in order to determine the contribution of the major loop to affinity. Shortening of the major loop from seven to three nucleotides led to stabilization of the scaffold. The affinities of the derivatives were studied by surface plasmon resonance and an enzyme-linked aptamer assay on recombinant hemagglutinins and viral particles, respectively. The alterations in the loop affected the binding to influenza hemagglutinin, but did not abolish it. Contrary to aptamer RHA0385, two of the designed aptamers were shown to be conformationally homogeneous, retaining high affinities and broad binding abilities for both recombinant hemagglutinins and whole influenza A viruses.

Highly sensitive detection of influenza virus with SERS aptasensor.

Highly sensitive and rapid technology of surface enhanced Raman scattering (SERS) was applied to create aptasensors for influenza virus detection. SERS achieves 106-109 times signal amplification, yielding excellent sensitivity, whereas aptamers to hemagglutinin provide a specific recognition of the influenza virus. Aptamer RHA0385 was demonstrated to have essentially broad strain-specificity toward both recombinant hemagglutinins and the whole viruses. To achieve high sensitivity, a sandwich of primary aptamers, influenza virus and secondary aptamers was assembled. Primary aptamers were attached to metal particles of a SERS substrate, and influenza viruses were captured and bound with secondary aptamers labelled with Raman-active molecules. The signal was affected by the concentration of both primary and secondary aptamers. The limit of detection was as low as 1 · 10-4 hemagglutination units per probe as tested for the H3N2 virus (A/England/42/72). Aptamer-based sensors provided recognition of various influenza viral strains, including H1, H3, and H5 hemagglutinin subtypes. Therefore, the aptasensors could be applied for fast and low-cost strain-independent determination of influenza viruses.

Putative Mechanisms Underlying High Inhibitory Activities of Bimodular DNA Aptamers to Thrombin.

Nucleic acid aptamers are prospective molecular recognizing elements. Similar to antibodies, aptamers are capable of providing specific recognition due to their spatial structure. However, the apparent simplicity of oligonucleotide folding is often elusive, as there is a balance between several conformations and, in some cases, oligomeric structures. This research is focused on establishing a thermodynamic background and the conformational heterogeneity of aptamers taking a series of thrombin DNA aptamers having G-quadruplex and duplex modules as an example. A series of aptamers with similar modular structures was characterized with spectroscopic and chromatographic techniques, providing examples of the conformational homogeneity of aptamers with high inhibitory activity, as well as a mixture of monomeric and oligomeric species for aptamers with low inhibitory activity. Thermodynamic parameters for aptamer unfolding were calculated, and their correlation with aptamer functional activity was found. Detailed analysis of thrombin complexes with G-quadruplex aptamers bound to exosite I revealed the similarity of the interfaces of aptamers with drastically different affinities to thrombin. It could be suggested that there are some events during complex formation that have a larger impact on the affinity than the states of initial and final macromolecules. Possible mechanisms of the complex formation and a role of the duplex module in the association process are discussed.