G-rich segments are highly populated in biologicaly crucial DNA regions. A large, still growing repertoire of possible G-quadruplex topologies differing in the orientation of their DNA strands and the types of connecting loops that a G-rich oligonucleotide can adopt has made the prediction of G-quadruplex topology very challenging. We have initiated a study to explore sequence directionality as a factor that is inherently connected with the folding of G-rich oligonucleotides. Intuitively we could expect that reversing the sequence direction from a 5’-3’ to 3’-5’ would cause dramatic changes in topology and/or the number of formed structures. The melting temperatures of G-quadruplexes adopted by oligonucleotides with sequences in the 5’-3’ direction are higher than those of their 3’-5’ counterparts. NMR study has demonstrated that a mere sequence reversal of the G-rich DNA segment exerts a substantial impact on the polymorphic nature of the resulting G-quadruplexes and thus their potential physiological roles.