Oligonucleotides carrying cationic peptides have been used to improve hybridization and cellular uptake of oligonucleotides. In this study, a series of four cationic amino acid derivatives were covalently linked at the 3′-termini with the objective of modifying the Tetrahymena telomeric repeat sequence d(TGGGGT). This led to the synthesis of a small cationic G-quadruplex-forming oligonucleotide series containing lysine, ornithine, homo-arginine and arginine moieties. The preparation of the appropriate solid-supports afforded the synthesis of 3′-lysine and 3′-ornithine oligonucleotides which were converted to homo-arginine and arginine conjugates through post-synthetic modifications. Circular dichroism and thermal denaturation experiments confirmed that the presence of the four cationic residues did not affect the formation of stable parallel G-quadruplex structures. Afterwards, a phosphorothioate oligonucleotide targeting Renilla luciferase mRNA was prepared at the 5′-termini of this telomeric sequence which afforded a series of phosphorothioate oligonucleotide/[d(TG4T)]4 hybrid constructs modified with lysine, ornithine, homo-arginine and arginine units after incubation. In addition, the phosphodiester/phosphorothioate mixed backbone contributed to the degradation of the G-quadruplex moiety by exonucleases liberating the antisense sequence. Cell culture analysis of gene expression showed that the formation of self-assembled G-quadruplex nanostructures did not disrupt the antisense mechanism and therefore were able to induce luciferase gene inhibition in mammalian cells without using cationic lipids. Flow cytometry analyses confirmed that fluorescently labelled antisense G4-quadruplex nanostructures were efficiently taken up by HeLa cells. These results suggest that G-quadruplex nanostructures may be used to improve cellular uptake of therapeutic oligonucleotides. © 2016 The Royal Society of Chemistry.