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.

Covalent Bi-Modular Parallel and Antiparallel G-Quadruplex DNA Nanocostructs Reduce Viability of Patient Glioma Primary Cell Cultures.

G-quadruplex oligonucleotides (GQs) exhibit specific anti-proliferative activity in human cancer cell lines, and they can selectively inhibit the viability/proliferation of cancer cell lines vs. non-cancer ones. This ability could be translated into a cancer treatment, in particular for glioblastoma multiform (GBM), which currently has a poor prognosis and low-efficiency therapeutic treatments. A novel bi-modular GQ, bi-(AID-1-T), a twin of the previously described three-quartet AID-1-T, was designed and studied in terms of both its structure and function. A covalent conjugation of two AID-1-Ts via three thymidine link, TTT, did not interfere with its initial GQ structure. A comparison of bi-(AID-1-T) with its mono-modular AID-1-T, mono-modular two-quartet HD1, and bi-modular bi-HD1, as well as conventional two-quartet AS1411, was made. Among the five GQs studied, bi-(AID-1-T) had the highest anti-proliferative activity for the neural cancer cell line U87, while not affecting the control cell line, human embryonic fibroblasts. GQs, for the first time, were tested on several primary glioma cultures from patient surgical samples. It turned out that the sensitivity of the patient primary glioma cultures toward GQs varied, with an apparent IC50 of less than 1 µM for bi-(AID-1-T) toward the most sensitive G11 cell culture (glioma, Grade III).

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.

SERS-Based Biosensors for Virus Determination with Oligonucleotides as Recognition Elements.

Viral infections are among the main causes of morbidity and mortality of humans; sensitive and specific diagnostic methods for the rapid identification of viral pathogens are required. Surface-enhanced Raman spectroscopy (SERS) is one of the most promising techniques for routine analysis due to its excellent sensitivity, simple and low-cost instrumentation and minimal required sample preparation. The outstanding sensitivity of SERS is achieved due to tiny nanostructures which must be assembled before or during the analysis. As for specificity, it may be provided using recognition elements. Antibodies, complimentary nucleic acids and aptamers are the most usable recognition elements for virus identification. Here, SERS-based biosensors for virus identification with oligonucleotides as recognition elements are reviewed, and the potential of these biosensors is discussed.

Energy Transfer as A Driving Force in Nucleic Acid⁻Protein Interactions.

Many nucleic acid-protein structures have been resolved, though quantitative structure-activity relationship remains unclear in many cases. Thrombin complexes with G-quadruplex aptamers are striking examples of a lack of any correlation between affinity, interface organization, and other common parameters. Here, we tested the hypothesis that affinity of the aptamer-protein complex is determined with the capacity of the interface to dissipate energy of binding. Description and detailed analysis of 63 nucleic acid-protein structures discriminated peculiarities of high-affinity nucleic acid-protein complexes. The size of the amino acid sidechain in the interface was demonstrated to be the most significant parameter that correlates with affinity of aptamers. This observation could be explained in terms of need of efficient energy transfer from interacting residues. Application of energy dissipation theory provided an illustrative tool for estimation of efficiency of aptamer-protein complexes. These results are of great importance for a design of efficient aptamers.

Bimodular Antiparallel G-Quadruplex Nanoconstruct with Antiproliferative Activity.

Oligonucleotides with an antiproliferative activity for human cancer cells have attracted attention over the past decades; many of them have a G-quadruplex structure (GQ), and a cryptic target. In particular, DNA oligonucleotide HD1, a minimal GQ, could inhibit proliferation of some cancer cell lines. The HD1 is a 15-nucleotide DNA oligonucleotide that folds into a minimal chair-like monomolecular antiparallel GQ structure. In this study, for eight human cancer cell lines, we have analyzed the antiproliferative activities of minimal bimodular DNA oligonucleotide, biHD1, which has two HD1 modules covalently linked via single T-nucleotide residue. Oligonucleotide biHD1 exhibits a dose-dependent antiproliferative activity for lung cancer cell line RL-67 and cell line of central nervous system cancer U87 by MTT-test and Ki-67 immunoassay. The study of derivatives of biHD1 for the RL-67 and U87 cell lines revealed a structure-activity correlation of GQ folding and antiproliferative activity. Therefore, a covalent joining of two putative GQ modules within biHD1 molecule provides the antiproliferative activity of initial HD1, opening a possibility to design further GQ multimodular nanoconstructs with antiproliferative activity-either as themselves or as carriers.

Selective ruthenium labeling of the tryptophan residue in the bee venom Peptide melittin.

Melittin is a membrane-active peptide from bee venom with promising antimicrobial and anticancer activity. Herein we report on a simple and selective method for labeling of the tryptophan residue in melittin by the organometallic fragment [(C5 H5 )Ru](+) in aqueous solution and in air. Ruthenium coordination does not disturb the secondary structure of the peptide (as verified by 2D NMR spectroscopy), but changes the pattern of its intermolecular interactions resulting in an 11-fold decrease of hemolytic activity. The high stability of the organometallic conjugate allowed the establishment of the biodistribution of the labeled melittin in mice by inductively coupled plasma MS analysis of ruthenium.

GQIcombi application to subdue glioma via differentiation therapy.

Current therapy protocols fail to cure high-grade gliomas and prevent recurrence. Therefore, novel approaches need to be developed. A re-programing of glioma cell fate is an alternative attractive way to stop tumor growth. The two-step protocol applies the antiproliferative GQ bi-(AID-1-T) and small molecule inducers with BDNF to trigger neural differentiation into terminally differentiated cells, and it is very effective on GB cell cultures. This original approach is a successful example of the "differentiation therapy". To demonstrate a versatility of this approach, in this publication we have extended a palette of cell cultures to gliomas of II, III and IV Grades, and proved an applicability of that version of differential therapy for a variety of tumor cells. We have justified a sequential mode of adding of GQIcombi components to the glioma cells. We have shown a significant retardation of tumor growth after a direct injection of GQIcombi into the tumor in rat brain, model 101/8. Thus, the proposed strategy of influencing on cancer cell growth is applicable to be further translated for therapy use.

The Bi-(AID-1-T) G-Quadruplex Has a Janus Effect on Primary and Recurrent Gliomas: Anti-Proliferation and Pro-Migration.

High-grade gliomas are considered an incurable disease. Despite all the various therapy options available, patient survival remains low, and the tumor usually returns. Tumor resistance to conventional therapy and stimulation of the migratory activity of surviving cells are the main factors that lead to recurrent tumors. When developing new treatment approaches, the effect is most often evaluated on standard and phenotypically depleted cancer cell lines. Moreover, there is much focus on the anti-proliferative effect of such therapies without considering the possible stimulation of migratory activity. In this paper, we studied how glioma cell migration changes after exposure to bi-(AID-1-T), an anti-proliferative aptamer. We investigated the effect of this aptamer on eight human glioma cell cultures (Grades III and IV) that were derived from patients' tumor tissue; the difference between primary and recurrent tumors was taken into account. Despite its strong anti-proliferative activity, bi-(AID-1-T) was shown to induce migration of recurrent tumor cells. This result shows the importance of studying the effect of therapeutic molecules on the invasive properties of glioma tumor cells in order to reduce the likelihood of inducing tumor recurrence.

Laser-Synthesized Germanium Nanoparticles as Biodegradable Material for Near-Infrared Photoacoustic Imaging and Cancer Phototherapy.

Biodegradable nanomaterials can significantly improve the safety profile of nanomedicine. Germanium nanoparticles (Ge NPs) with a safe biodegradation pathway are developed as efficient photothermal converters for biomedical applications. Ge NPs synthesized by femtosecond-laser ablation in liquids rapidly dissolve in physiological-like environment through the oxidation mechanism. The biodegradation of Ge nanoparticles is preserved in tumor cells in vitro and in normal tissues in mice with a half-life as short as 3.5 days. Biocompatibility of Ge NPs is confirmed in vivo by hematological, biochemical, and histological analyses. Strong optical absorption of Ge in the near-infrared spectral range enables photothermal treatment of engrafted tumors in vivo, following intravenous injection of Ge NPs. The photothermal therapy results in a 3.9-fold reduction of the EMT6/P adenocarcinoma tumor growth with significant prolongation of the mice survival. Excellent mass-extinction of Ge NPs (7.9 L g-1 cm-1 at 808 nm) enables photoacoustic imaging of bones and tumors, following intravenous and intratumoral administrations of the nanomaterial. As such, strongly absorbing near-infrared-light biodegradable Ge nanomaterial holds promise for advanced theranostics.

A Combined Effect of G-Quadruplex and Neuro-Inducers as an Alternative Approach to Human Glioblastoma Therapy.

Cancer cell reprogramming based on treatment with G-quadruplex, having antiproliferative power, along with small molecules able to develop iPSCs into neurons, could create a novel approach to diminish the chance of glioblastoma recurrence and circumvent tumor resistance to conventional therapy. In this research, we have tested several combinations of factors to affect both total cell cultures, derived from tumor tissue of patients after surgical resection and two subfractions of this cell culture after dividing them into CD133-enriched and CD133-depleted populations (assuming CD133 to be a marker of glioblastoma stem-like cells). CD133+ and CD133- cells exhibit different responses to the same combinations of factors; CD133+ cells have stem-like properties and are more resistant. Therefore, the ability to affect CD133+ cells provides a possibility to circumvent resistance to conventional therapy and to build a promising strategy for translation to improve the treatment of patients with glioblastoma.

Bimodular thrombin aptamers with two types of non-covalent locks.

Aptamers are structured oligonucleotides that specifically bind their targets. Oligonucleotides can be assembled in large nanostructures via intermolecular duplexes or G-quadruplexes. Addition of aptamers can be used to create nanostructures that bind specifically certain targets. Here two types of self-assembling locks were used to create bimodular aptamer constructions. Well-known aptamer to thrombin was chosen as a model object. The assembly of duplex locks was more efficient at low concentrations. The functional activity of aptamer modules was nearly the same as in HD1. However, the affinity of bimodular aptamers with G-quadruplex locks to immobilized thrombin was 5-10 times higher.

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.

Pyrene-Modified DNA Aptamers with High Affinity to Wild-Type EGFR and EGFRvIII.

Nucleic acid aptamers have been proven to be a useful tool in many applications. Particularly, aptamers to epidermal growth factor receptor (EGFR) have been successfully used for the recognition of EGFR-expressing cells, the inhibition of EGFR-dependent pathways, and targeted drug delivery into EGFR-positive cells. Several aptamers are able to discriminate wild-type EGFR from its mutant form, EGFRvIII. Aptamers to EGFR have hairpin-like secondary structures with several possible folding variations. Here, an aptamer, previously selected to EGFRvIII, was chosen as a lead compound for extensive post-SELEX maturation. The aptamer was 1.5-fold truncated, the ends of the hairpin stem were appended with GC-pairs to increase thermal stability, and single pyrene modification was introduced into the aptamer to increase affinity to the target protein. Pyrene modification was selected from extensive computer docking studies of a library of thousands of chemicals to EGFR near the EGF-binding interface. The resulting aptamers bound extracellular domains of both variants of EGFR: EGFRwt and EGFRvIII with subnanomolar apparent dissociation constants. Compared with the initial aptamer, affinity to EGFRwt was increased up to 7.5-fold, whereas affinity to EGFRvIII was increased up to 4-fold.

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.

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.

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.

Direct visualization of the oligomeric state of hemagglutinins of influenza virus by high-resolution atomic force microscopy.

High-resolution atomic force microscopy (AFM) is a powerful technique for the direct visualization of single molecules. Here, AFM is applied to characterize the oligomeric state of hemagglutinins of the influenza virus. Hemagglutinins are known to be present in a trimeric form inside the viral envelope. However, recombinant hemagglutinins are also present as large oligomers, which impair the functional activity of the protein. Five commercial recombinant hemagglutinins from the viral strains H1, H3, H5, H7, and H9 were studied with high-resolution AFM. Functionally inactive hemagglutinins were shown to have a higher percentage of large oligomers compared with the proteins with functional activity. Large oligomers were revealed to be unstable; the oligomeric state of hemagglutinin was affected by pH or the presence of ligands. Antibody binding shifts the balance to small oligomers, whereas DNA aptamer induced the formation of large associates.

The Evaluation of Pharmacodynamics and Pharmacokinetics of Anti-thrombin DNA Aptamer RA-36.

Anticoagulants are a vital class of drugs, which are applied for short-term surgical procedures, and for long-term treatments for thrombosis prevention in high risk groups. Several anticoagulant drugs are commercially available, but all have intrinsic disadvantages, e.g., bleeding risks, as well as specific ones, e.g., immune response to peptide/protein drugs. Therefore, the search for novel, efficient and safe anticoagulants is essential. Nucleic acid aptamers are an emerging class of contemporary pharmaceuticals which are fully biocompatible and biodegradable; they have low toxicity, and are as efficient as many protein-based drugs. The anti-thrombin DNA aptamer RA-36 has been created using a combination of rational design and molecular dynamics, showing several extra-features over existing aptamers. Aptamer RA-36 has a bimodular structure; the first G-quadruplex binds and inhibits thrombin, whereas the second G-quadruplex varies the properties of the first. This bimodular structure provides a favorable dose-effect dependence allowing the risk of bleeding to be potentially decreased. Here, the results of efficiency trials of the aptamer are presented. The aptamer RA-36 has a distinctive species specificity; therefore, the careful selection of experimental animals was required. The anticoagulant activity was characterized in rats and monkeys in vivo. Antithrombotic activity was evaluated in the live murine model of the induced thrombosis. Pharmacokinetics was estimated by tracking radionuclide labeled aptamer in rats. The aptamer was thoroughly characterized using bivalirudin as a reference drug. Despite the different profiles of anticoagulant activity, these two compounds could refer to each other, and the corresponding doses could be estimated. Bivalirudin turned out to have 10-fold higher anticoagulant and antithrombotic activity. The difference in activity is easy to explain due to the pharmacokinetic profiles of the substances: the aptamer RA-36 has 20-fold faster elimination from blood with a half-life of 1 min. The entire dataset revealed that the non-modified DNA aptamer could be an alternative to the currently used bivalent peptide inhibitor; the dosage profile could be improved by manipulating aptamer pharmacokinetics. The study has revealed aptamer RA-36 to be one of the most promising candidates for further development as a new generation of anticoagulants.