Guanine-rich sequences with a typical form of GnXaGnXbGnXcGn, where Gn are guanine tracts; n ≥ 2; 7 ≥ a/b/c ≥ 0 and X is any nucleotide, are capable of forming G-quadruplex structures. They are common in the regulatory regions of viral genomes, but rare in the SARS-CoV-2 genome. Ligands for viral G-quadruplexes have great therapeutic potential. Atypical G-quadruplex structures can contain bulges and hairpin loops which may represent additional binding sites for ligands. Atypical G-quadruplex forming sequences were searched for in the SARS-CoV-2 genome, for which we wrote an updated algorithm based on the G4Hunter algorithm named G4HunterHairpin. This is the first known algorithm to include the search for hairpins in interrupted G-tracts. Using G4HunterHairpin, we found oligonucleotide sequences in the SARS-CoV-2 genome with predicted folding into atypical G-quadruplex structures. We synthesised 13 oligonuclotides and analysed them using 1D 1H NMR spectroscopy. The formation of hairpins or G-quadruplexes was observed, with simultaneous formation of both structures observed for oligonucleotide V8-30. The latter was further characterised by 2D 1H-1H NOESY spectroscopy, from which the parallel topology of the G-quadruplex was determined and certain base pairs in the hairpin were assigned.
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