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In this paper, a method to discriminate between two target RNA sequences that differ by one nucleotide only is presented. The method relies on the formation of alternative structures, i.e., quadruplex-duplex hybrid (QDH) and duplex with dangling ends (Dss), after hybridization of DNA or RNA G-rich oligonucleotides with target sequences containing 5'-GGGCUGG-3' or 5'-GGGCGGG-3' fragments. Using biophysical methods, we studied the effect of oligonucleotide types (DNA, RNA), non-nucleotide modifications (aliphatic linkers or abasic), and covalently attached G4 ligand on the ability of G-rich oligonucleotides to assemble a G-quadruplex motif. We demonstrated that all examined non-nucleotide modifications could mimic the external loops in the G-quadruplex domain of QDH structures without affecting their stability. Additionally, some modifications, in particular the presence of two abasic residues in the G-rich oligonucleotide, can induce the formation of non-canonical QDH instead of the Dss structure upon hybridization to a target sequence containing the GGGCUGG motif. Our results offer new insight into the sequential requirements for the formation of G-quadruplexes and provide important data on the effects of non-nucleotide modifications on G-quadruplex formation.

Impact of a Single Nucleotide Change or Non-Nucleoside Modifications in G-Rich Region on the Quadruplex–Duplex Hybrid Formation