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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.

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 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.

BACKGROUND: Theranostics, also known as personalized medicine, precision medicine, stratified medicine or P4 medicine, is a modern trend in treating diseases. The large part of that is the immunotheranostics, which applies monoclonal antibodies as molecular recognizing elements (MoRE) to interact specifically and block functioning of therapeutically important target. In terms of therapeutic application, being the proteins, antibodies has both advantages and disadvantages: the critical ones are specificity and immunogenisity, respectively. MoRE made of nucleic acids instead of polypeptides is coined as Aptamers. Aptamers are more robust to produce, handle and applied, not mentioning the lack to provoke an immune response. Therefore, some attempts are made to create aptamers to block glioma proliferation. Among them are the following aptamers: for EphB2/3 receptors, for PDGFRβ, to U87-EGFRvIII cells, GBI-10 to tenascin-C, AS1411 to nucleolin, DNA aptamers from whole-cell SELEX. Some applications of aptamers have been developed: surface-immobilized aptamers for cancer cell isolation and cytology, quantum dot-labeled aptamer nanoprobes, aptamer guided gadolinium-loaded liposomes, aptamer functionalization of nanosystems to get through the blood-brain barrier, microRNA-based targeted to eradicate glioblastoma stem-like cells, aptamer conjugated magnetic nanoparticles as nanosurgeons. In this research EGFR has been selected as specific target to explore interaction with aptamers in details for the further possibility to halt proliferation of EGFR positive clones of glioblastoma (GBM). MATERIAL AND METHODS: All published aptamer sequences, both DNA and RNA, have been collected and analyzed in terms of possible structures, gaining to find putative scaffold and recognizing parts of aptamers. All promising candidates were checked for affinity to the immobilized extracellular domain of human EGFR protein by surface plasmon resonance, as well as compare with commercial monoclonal antibodies. Derivatives of active aptamers were made to reveal putative scaffold and recognizing domain, and again check the affinity. Studying of interactions of several aptamers with EGFR positive cell lines and clones of surgical samples of GBM were made. RESULTS: A particular aptamer, NA511, has a very promising properties, both structural and functional. It shows distinctive putative structure which is easy to modify; it exhibits affinity similar to commercial antibodies. Therefore, it could be a promising candidate to translate this MoRE for theranostics of GMB. CONCLUSION: In spite of many aptamers for EGFR had been published till now, most of them do not have an obvious potential for further development. Therefore, a methodology has to be developed yet for translation of the aptamers into the theranostic drugs. The work was supported by Grant RFBR KOMFI 17-00-00162 (K), 17-00-00160.