|Reference : Exploring the Formation Pathways of DNA G-Quadruplex Architectures|
|Scientific congresses and symposiums : Unpublished conference|
|Life sciences : Biochemistry, biophysics & molecular biology|
Physical, chemical, mathematical & earth Sciences : Chemistry
|Exploring the Formation Pathways of DNA G-Quadruplex Architectures|
|Rosu, Frédéric [Université de Liège - ULg > Département de chimie (sciences) > GIGA-R : Laboratoire de spectrométrie de masse (L.S.M.) >]|
|Poncelet, Harmonie [Université de Liège - ULg > Département de Chimie (Sciences) > GIGA-R : Laboratoire de Spectrométrie de Masse > >]|
|Teulade-Fichou, Marie-Paule [Collège de France, Paris > > > >]|
|De Pauw, Edwin [Université de Liège - ULg > Département de chimie (sciences) > GIGA-R : Laboratoire de spectrométrie de masse (L.S.M.) >]|
|Gabelica, Valérie [Université de Liège - ULg > Département de chimie (sciences) > GIGA-R : Laboratoire de spectrométrie de masse (L.S.M.) >]|
|2nd European Conference on Chemistry for Life Sciences|
|September 4-7, 2007|
|[en] mass spectrometry ; G-quadruplex ; electrospray ; noncovalent ; affinity ; stoichiometry ; binding|
|[en] Guanine-rich DNA strands can form the so-called G-quadruplex architectures due to the formation of quartets of guanines linked by 8 hydrogen bonds. G-quadruplexes are further stabilized by the inclusion of cations between the G-quartets. The abundance of G-rich regions throughout the genome and their very presence in telomeric regions made G-quadruplexes interesting targets. NMR and crystallographic studies of G-quadruplex structures revealed amazing variety in the G-quadruplex topologies. The next challenge will be to understand the rules governing the formation of the various topologies, in order to predict relevant G-quadruplexes in the genome, and in order to act rationally on their formation or disruption. To date, only few experimental  or theoretical  studies have been devoted to investigating the mechanisms of G-quadruplex formation.
We report here a detailed investigation of DNA G-quadruplex formation pathways using electrospray mass spectrometry (ESI-MS). The sequences TGnT (n = 3-6) were purchased from Eurogentec (Seraing, Beliugm). ESI-MS experiments were performed in the negative ion mode on a Q-TOF Ultima Global (Waters, Manchester, UK). The cation used was ammonium (up to 150 mM). Experiments were performed in the presence and absence of methanol (up to 20%) as co-solvent.
ESI-MS allows counting both the number of strands and the number of cations in each intermediate. We could confirm the presence of transient dimer and trimer intermediates in low abundance. More unexpectedly, ESI-MS also reveals unambiguously the formation of pentamers which contain ammonium cations. The pentamers slowly convert into tetramers. Counting the number of included cations also revealed that, in the case of (TG6T)4, inclusion of four ammonium cations is fast, while the inclusion of the last ammonium ion is very slow. We also found that the addition of methanol (initially added to obtain higher ion intensities) significantly increases the rate of G-quadruplex formation. Finally, we also investigated the role of G-quadruplex ligands in the rate of formation of G-quadruplexes. We could classify the ligands according to their increase of G-quadruplex formation kinetics, and distinguish the intermediates. Interestingly, one ligand showed formation of a higher-order structure by bridging two G-quadruplexes.
Acknowledgement: The authors thank the FRS-FNRS for their support.
 J. Gros et al., Nucleic Acids Res., 2007, doi:10.1093/nar/gkm111.
 R. Stefl et al., Biophys. J., 2003, 85(3), 1787-1804.
|Giga-Systems Biology and Chemical Biology|
|Fonds de la Recherche Scientifique (Communauté française de Belgique) - F.R.S.-FNRS|
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