2554–2563 Nucleic Acids Research, 2007, Vol. 35, No. 8 doi:10.1093/nar/gkm138 Published online 4 April 2007 NMR evaluation of ammonium ion movement within a unimolecular G-quadruplex in solution Peter Podbevsˇ ek1, Nicholas V. Hud2 and Janez Plavec1,* 1Slovenian NMR Center, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia and 2School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, USA Received January 25, 2007; Revised February 21, 2007; Accepted February 22, 2007 ABSTRACT d[G4(T4G4)3] has been folded into a unimolecular G-quadruplex in the presence of 15NH4þ ions. NMR spectroscopy confirmed that its topology is the same as the solution state structure determined earlier by Wang and Patel (J. Mol. Biol., 1995; 251: 76–94) in the presence of Naþ ions. The d[G4(T4G4)3] G-quadruplex exhibits four G-quartets with three 15NH4þ-ion-binding sites (O1, I and O2). Quantitative analysis utilizing 15NH4þ ions as a NMR probe clearly demonstrates that there is no unidirectional 15NHþ 4 ion movement through the central cavity of the G-quadruplex. 15NH4þ ions move back and forth between the binding sites within the G-quadruplex and exchange with ions in bulk solution. 15NH4þ ion movement is controlled by the thermodynamic preferences of individual binding sites, steric restraints of the G-quartets for 15NH4þ ion passage and diagonal versus edge-type arrangement of the T4 loops. The movement of 15NH4þ ions from the interior of the G-quadruplex to bulk solution is faster than exchange within the G-quadruplex. The structural details of the G-quadruplex define stiff- ness of individual G-quartets that intimately affects 15NH4þ ion movement. The stiffness of G-quartets and steric hindrance imposed by thymine residues in the loops contribute to the 5-fold difference in the exchange rate constants through the outer G-quartets. INTRODUCTION It has been known for several decades that GMP and guanine polymers form viscous gels in aqueous solutions (2). G-quartets were postulated to be the building blocks of four stranded assemblies or G-quadruplexes. However, these structures were mostly viewed with curiosity, as there was no evident application, nor was their biological relevance known at that time. More recently, interest for G-quadruplex structures has increased due to the role of G-rich DNA in telomere sequences and gene promoter regions (3–10). G-quadruplex formation has been asso- ciated with human diseases (e.g. cancer, HIV or diabetes) (4,11–15) and the sequences that form these structures are now considered therapeutic targets (16–18). G-quartets are assembled from four coplanary arranged guanine bases that are held together by Hoogsteen hydrogen bonds (2–4,19–22). In the center of each G-quartet are four closely spaced carbonyl groups whose electrostatic repulsions are reduced by the coordi- nation of cations (23). G-quadruplexes are formed by the stacking of two or more G-quartets. The strand stoichio- metry of a G-quadruplex is primarily determined by the number of G-rich repeats in the quadruplex-forming sequence. Short oligonucleotides with a single G-rich segment usually form parallel, tetramolecular G-quadruplexes. In such structures, the glycosidic bonds of all guanines are in the anti conformation. Longer oligonucleotides with two or more G-rich segments can form bi- or unimolecular G-quadruplex structures (1,24–27). For unimolecular quadruplexes, the four G-rich segments of a DNA strand can still align in a parallel fashion, if the intervening bases form loops with a double-chain reversal conformation. Alternatively, the G-rich segments of bi- and unimolecular G-quadruplexes can align themselves in an anti-parallel fashion and form the so-called ‘fold-back’ quadruplexes. In these structures, guanine glycosidic bonds alternate between syn and anti conformations around a G-quartet and along an oligo- nucleotide strand. In fold-back G-quadruplexes, the loops connecting G-rich segments run diagonally across the face or along the edges of the outer G-quartets. G-quadruplexes are typically stabilized by monovalent cations, but some divalent cations are also known to stabilize G-quadruplex structures (28–31). Not surpris- ingly, various cations stabilize G-quadruplexes diﬀerently (23,32). The presence of diﬀerent cations can lead to conformational plurality (33,34). Our recent studies have suggested that the G-quadruplex topologies and 3D structures of some G-rich oligonucleotides with two G-rich repeats are more susceptible to the presence of *To whom correspondence should be addressed. Tel: þ1-386 1-47-60-353; Fax: þ386 1-47-60-300; Email: janez.plavec@ki.si 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.