2562 Nucleic Acids Research, 2007, Vol. 35, No. 8 longitudinal relaxation which depends on the structure of G-quadruplex. The combined eﬀect of 15NH4þ ion move- ment and T1 relaxation is manifested through autocorre- lation peaks which persist till mixing times as long as 3 s in d[G4(T4G4)3] quadruplex. The current study adds insight to our understanding of the role of cations for stability as well as ﬂexibility of G-quadruplex structures which are important factors in a control of assembly and dis- assembly of this unique DNA structures. CONCLUSION d[G4(T4G4)3] has been folded into unimolecular G-quadruplex in the presence of 15NHþ ions. NMR 4 study conﬁrmed that its topology is consistent with the 3D structure determined earlier by Wang and Patel (1) in the presence of Naþ ions. d[G4(T4G4)3] G-quadruplex exhibits four G-quartets with three 15NH4þ-ion-binding sites. 15NH4þ ions have been utilized as NMR probes to localize cations within the intramolecular d[G4(T4G4)3] quadru- plex and follow their movement amongst the preferred binding sites within G-quadruplex and with bulk solution. Quantitative analysis has clearly shown that there is no unidirectional 15NH4þ ion movement through the central cavity of the G-quadruplex. 15NH4þ ion movement is controlled by thermodynamic preferences of individual binding sites, steric restraints of G-quartets for 15NH4þ ion passage and structure of T4 loops. The analysis of volumes of two well-resolved cross-peaks (O1I and O2I) corre- sponding to the movement of 15NH4þ ions from the two outer binding sites into the inner binding site aﬀorded the exchange rate constants of 0.05 and 0.07 s 1 at 283 K. Temperature rise results in the increase in rate constants to 0.66 and 0.50 s 1 for O1I and O2I processes at 313 K, respectively. Volumes of cross-peaks are further tuned by T1 relaxation. The O2B exchange rate constant of 0.75 s 1 at 293 K demonstrates that 15NH4þ ion exchange with bulk is faster than exchange within the G-quadruplex. Furthermore, the comparison of O2B and O1B exchange rate constants shows that O2B process is roughly ﬁve times faster at 293 K. The results presented in this article show that structural details deﬁne stiﬀness of G-quadruplex structure that intimately aﬀects cation (15NHþ) move- 4 ment. In this way, experimental NMR data on dynamics of cation movement can serve as a probe into (non)ﬂex- ibility of speciﬁc regions of quadruplex structures. ACKNOWLEDGEMENTS We thank Slovenian Research Agency (ARRS) and the Ministry of Higher Education, Science and Technology of the Republic of Slovenia (Grant Nos. P1-0242-0104 and J1-6140-0104) for their ﬁnancial support. Financial sup- port from NATO Collaborative Programs Section (CLG grant 979520) to the authors is gratefully acknowledged. Funding to pay the Open Access publication charges for this article was provided by ARRS. Conﬂict of interest statement. None declared. REFERENCES 1. Wang,Y. and Patel,D.J. (1995) Solution structure of the Oxytricha telomeric repeat d[G4(T4G4)3] G-tetraplex. J. Mol. Biol., 251, 76–94. 2. Gellert,M., Lipsett,M. and Davies,D.R. (1962) Helix formation by guanylic acid. Proc. Natl Acad. Sci. USA, 48, 2013–2018. 3. Neidle,S. and Balasubramanian,S. (eds) (2006) Quadruplex Nucleic Acids. The Royal Society of Chemistry, Cambridge. (Monography for a recent review on G-guadruplex nucleic acids). 4. Keniry,M.A. (2001) Quadruplex structures in nucleic acids. Biopolymers, 56, 123–146. 5. Schaﬃtzel,C., Berger,I., Postberg,J., Hanes,J., Lipps,H.J. and Pluckthun,A. (2001) In vitro generated antibodies speciﬁc for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei. Proc. Natl Acad. Sci. USA, 98, 8572–8577. 6. Dai,J.X., Dexheimer,T.S., Chen,D., Carver,M., Ambrus,A., Jones,R.A. and Yang,D.Z. (2006) An intramolecular G-quadruplex structure with mixed parallel/antiparallel G-strands formed in the human BCL-2 promoter region in solution. J. Am. Chem. Soc., 128, 1096–1098. 7. Ambrus,A., Chen,D., Dai,J.X., Jones,R.A. and Yang,D.Z. (2005) Solution structure of the biologically relevant G-quadruplex element in the human c-MYC promoter. Implications for G-quadruplex stabilization. Biochemistry, 44, 2048–2058. 8. Phan,A.T., Kuryavyi,V., Gaw,H.Y. and Patel,D.J. (2005) Small- molecule interaction with a ﬁve-guanine-tract G-quadruplex struc- ture from the human MYC promoter. Nat. Chem. Biol., 1, 167–173. 9. Siddiqui-Jain,A., Grand,C.L., Bearss,D.J. and Hurley,L.H. (2002) Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription. Proc. Natl Acad. Sci. USA, 99, 11593–11598. 10. Phan,A.T., Kuryavyi,V. and Patel,D.J. (2006) DNA architecture: from G to Z. Curr. Opin. Struct. Biol., 16, 288–298. 11. Wyatt,J.R., Vickers,T.A., Roberson,J.L., Buckheit,R.W. and Ecker,D.J. (1994) Combinatorially selected guanosine-quartet structure is a potent inhibitor of human immunodeﬁciency virus envelope-mediated cell fusion. Proc. Natl Acad. Sci. USA, 91, 1356–1360. 12. Jing,N.J., Marchand,C., Liu,J., Mitra,R., Hogan,M.E. and Pommier,Y. (2000) Mechanism of inhibition of HIV-1 integrase by G-tetrad forming oligonucleotides in vitro. J. Biol. Chem., 275, 21460–21467. 13. Lew,A., Rutter,W.J. and Kennedy,G.C. (2000) Unusual DNA structure of the diabetes susceptibility locus IDDM2 and its eﬀect on transcription by the insulin promoter factor Pur-1/MAZ. Proc. Natl Acad. Sci. USA, 97, 12508–12512. 14. Simonsson,T. (2001) G-quadruplex DNA structures – variations on a theme. Biol. Chem., 382, 621–628. 15. Xu,Y. and Sugiyama,H. (2006) Formation of the G-quadruplex and i-motif structures in retinoblastoma susceptibility genes (Rb). Nucleic Acids Res., 34, 949–954. 16. Neidle,S. and Read,M.A. (2001) G-quadruplexes as therapeutic targets. Biopolymers, 56, 195–208. 17. Rezler,E.M., Bearss,D.J. and Hurley,L.H. (2002) Telomeres and telomerases as drug targets. Curr. Opin. Pharmacol., 2, 415–423. 18. Seenisamy,J., Bashyam,S., Gokhale,V., Vankayalapati,H., Sun,D., Siddiqui-Jain,A., Streiner,N., Shin-ya,K., White,E. et al. (2005) Design and synthesis of an expanded porphyrin that has selectivity for the c-MYC G-quadruplex structure. J. Am. Chem. Soc., 127, 2944–2959. 19. Smith,F.W. and Feigon,J. (1992) Quadruplex structure of Oxytricha telomeric DNA oligonucleotides. Nature, 356, 164–168. 20. Laughlan,G., Murchie,A.I.H., Norman,D.G., Moore,M.H., Moody,P.C.E., Lilley,D.M.J. and Luisi,B. (1994) The high- resolution crystal-structure of a parallel-stranded guanine tetraplex. Science, 265, 520–524. 21. Neidle,S. and Parkinson,G.N. (2003) The structure of telomeric DNA. Curr. Opin. Struct. Biol., 13, 275–283. 22. Davis,J.T. (2004) G-quartets 40 years later: from 50-GMP to molecular biology and supramolecular chemistry. Angew. Chem. Int. Ed., 43, 668–698. 23. Hud,N.V. and Plavec,J. (2006). The role of cations in determining quadruplex structure and stability. In Neidle,S. and