Nuclear magnetic resonance study of G-quadruplex structures formed by d(TG(3)T) and its modified analogs containing a 5'-5' or 3'-3' inversion of polarity sites, namely d(3'TG5'-5'G(2)T3'), d(3'T5'-5'G(3)T3') and d(5'TG3'-3'G(2)T5') demonstrates formation of G-quadruplex structures with tetrameric topology and distinct cation-binding preferences. All oligonucleotides are able to form quadruplex structures with two binding sites, although the modified oligonucleotides also form, in variable amounts, quadruplex structures with only one bound cation. The inter-quartet cavities at the inversion of polarity sites bind ammonium ions less tightly than a naturally occurring 5'-3' backbone. Exchange of (15) ions between G-quadruplex and bulk solution is faster at the 3'-end in comparison to the 5'-end. In addition to strand directionality, cation movement is influenced by formation of an all-syn G-quartet. Formation of such quartet has been observed also for the parent d(TG(3)T) that besides the canonical quadruplex with only all-anti G-quartets, forms a tetramolecular parallel quadruplex containing one all-syn G-quartet, never observed before in unmodified quadruplex structures.