NMR study of RNA G-quadruplex regulatory motif involved in inflammation processesŠtrbac, Jelena (Avtor) Plavec, Janez (Mentor) G-quadruplexG4NMRTMPRSS2COVID-19SARS-CoV-2PhenDC3Transmembrane serine protease 2 (TMPRSS2) is a protein of great importance for the development of virus-induced COVID-19 disease. Large number of TMPRSS2 are expressed on the surface of human respiratory tract epithelial cells. It was proven that by catalytic cleavage of the S protein of the SARS-CoV-2 virus this enzyme enables viral activation for the fusion of the virion with cellular membrane, i.e., the entry of the virus into the human cell. Therefore, inhibition of TMPRSS2 could be used as the tool for the management of SARS-CoV-2 infection. It is known that the formation of secondary structures in the 5'-UTR region of mRNA can inhibit protein translation. Particularly important motifs that affect translation are G-quadruplexes (G4). G4s are tetrahelical structures formed by sequences rich in guanine residues, which are arranged in G-tracts separated by short (up to seven residues) stretches of random nucleotides. G-rich region is present in the 5'- UTR region of TMPRSS2. Using various analytical techniques, primarily high-resolution nuclear magnetic resonance, we confirmed that G-rich sequence from the 5'-UTR region of TMPRSS2 mRNA could form a family of G4 structures in the presence of potassium ions at conditions close to physiological (pH 7, T of 40°C). Using mutational analysis, we showed that G4s of parallel topology are formed, which have high thermal stability (Tm is about 70°C). We performed a detailed NMR study of G4 formed by mutated oligonucleotide corresponding to one of folds present in the wild type ensemble. We also investigated the complex of selected G4 with PhenDC3 ligand. We observed changes in NMR spectra which confirmed binding of PhenDC3 to outer G-quartets without changing topology. According to literature data TMPRSS2 could be inhibited without decreasing epithelial cell viability. Therefore, stabilization of G4 in 5'-UTR region of mRNA which could affect protein translation could be considered as potential approach for the development of treatment for COVID-19 disease.20222022-09-23 14:00:06Magistrsko delo/naloga141149sl