Engineering of materials with various bio-nanoapplications can rely on robust self-assembly of DNA e.g., short, guanine-rich oligonucleotides can self-assemble to long nanostructures, G-wires. Their structural details and self-assembly mechanism are crucial for optimization of G-wire’s properties, however they are poorly understood. Using NMR, we obtained a detailed insight into G-wire formation. We utilized this knowledge for modifications of parent oligonucleotide and thus preparation of G-wires of different properties. For visualization and further characterization we employed complementary methods.