We systematically investigated the influence of locked nucleic acid (LNA), unlock nucleic acid (UNA), and 2'-O-methyl-RNA (2'-O-Me-RNA) residues on the thermal stability, structure folding topology, biological activity and enzymatic resistance of three sequence-related DNA G-quadruplexes. In order to better understand the mechanism of action of the studied modifications, a single-position substitution in the loops or G-tetrads was performed and their influence was analyzed for a total of twenty-seven modified G-quadruplex variants. The studies show that the influence of each modification on the physicochemical properties of G-quadruplexes is position-dependent, due to mutual interactions between G-tetrads, loops, and additional guanosine at 5' or 3' end. Nevertheless, the anticancer activity of the modified G-quadruplexes is determined by their structure, thus also by the local changes of chemical character of sugar moieties, what might influence the specific interactions with therapeutic targets. In general, UNA modifications are efficient modulators of the G-quadruplex thermodynamic stability, however they are poor tools to improve the anticancer properties. In contrast, LNA and 2'-O-Me-RNA modified G-quadruplexes demonstrated certain antiproliferative potential and might be used as molecular tools for designing novel G-quadruplex-based therapeutics.