Superstructural self-assembly of G-quadruplex structure, formed by a synthetic homopurine oligonucleotide.

Four guanine can associate in a planar structure, named G-quartet, held together via Hoogsteen hydrogen bonds. The G-quartets, in guanine-rich sequences, can stack forming four stranded structures, the so-called “quadruplexes”, stabilized by monovalent cations. The self-interaction of G-quartets can give rise also to super-molecular structures called “G-wires” or “frayed wires”, shown by the synthetic guanine-rich oligonucleotides G4T2G4 and A15G15, respectively (1,2). The understanding of G-quadruplex structures closely concerns two different research fields of increasing interest: the biological significance of G-rich sequences in genome organization and the development of biomaterials for nanotechnology applications because of the higher rigidity of G-quadruplex with respect to canonical duplex DNA (3), which has been already adopted for construction of complex two- and three-dimensional nanostructures (4). During the study of a homopurine oligonucleotide, (5’-GGAAAGGAAGGGGAGGGG-3’), PHT, belonging to the telomerase gene promoter, we found by AFM imaging that this sequence has the ability to self-assembly. This finding is of interest from nanotechnological point of view since it suggests the possibility of a new type of G-wires in the case of homopurine sequences. We have studied the structural organization of PHT sequence by AFM imaging and we have found out that it can organize linear superstructures of different lengths on mica surface, characterized by the recurrence of single elements spaced every 14,2 nm. It is worth noting that the single units have height about 1,6 nm that is almost twice the duplex DNA, used as standard. To highlight the structures involved, we have carried out electrophoretic mobility shift assays (EMSA) in different salt conditions. The obtained results suggest that the superstructures building block can be an intermolecular G-quadruplex structure, differently stabilized by Na+ and K+ cations. Surprisingly, the super-structural organization of G-quadruplex blocks appears regulated by the presence of negative phosphate charges at 5’ sequence termini. Circular dichroism studies support both the presence of intermolecular G-quadruplex and the different influence of Na+ and K+ cations on their thermodynamic stability. DMS interference assay proves the self-assembly in a G-quadruplex structure of the 3’ terminal region of the PHT, characterized by the presence of two stretches of four guanines, which appear to be involved in Hoogsteen hydrogen bonds. Possible molecular models for the global structure and the oligomerization process are under investigation. 1. T. Marsh, J. Vesenka and E. Henderson. NAR (1995) 23, 696-700. 2. E. Protozanova, R.B. Macgregor. Biochemistry (1996) 35, 16638-16645. 3. H.Cohen, T. Sapir, N. Borovok, T. Molotsky, R. Di Felice, A. B. Kotlyar and D. Porath, Nano Letters (2007) 7, 981-986. 4. N. C. Seeman, Trends in Biochemical Sciences (2005) 30, 119-125.