Two G-quadruplex forming sequences, 5'-TGGGAG and the 17-mer sequence T30177, which exhibit anti-HIV-1 activity on cell lines, were modified using either locked nucleic acids (LNA) or via insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (intercalating nucleic acid, INA) or (R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol (twisted intercalating nucleic acid, TINA). Incorporation of LNA or INA/TINA monomers provide as much as 8-fold improvement of anti-HIV-1 activity. We demonstrate for the first time a detailed analysis of the effect the incorporation of INA/TINA monomers in quadruplex forming oligonucleotides (QFOs) and the effect of LNA monomers in the context of biologically active QFOs. In addition, recent literature reports and our own studies on the gel retardation of the phosphodiester analogue of T30177 led to the conclusion that this sequence forms a parallel, dimeric G-quadruplex. Introduction of the 5'-phosphate inhibits dimerisation of this G-quadruplex as a result of negative charge-charge repulsion. Contrary to that, we found that attachment of the 5'-O-DMT-group produced a more active 17-mer sequence that showed signs of aggregation-forming multimeric G-quadruplex species in solution. Many of the antiviral QFOs in the present study formed more thermally stable G-quadruplexes and also high-order G-quadruplex structures which might be responsible for the increased antiviral activity observed.