Oligodeoxynucleotides which include stretches of guanines form a well-known tetrameric structure. We show that the recording of reversible absorbance changes at 295 nm allows to precisely monitor intramolecular guanine (G)-quartet formation and dissociation. Accurate T m and thermodynamic values could be easily extracted from the data, whereas classical recordings at 260 nm led to a much larger uncertainty and in extreme cases, to completely inaccurate measurements. This inverted denaturation profile was observed for all G-quartetforming oligonucleotides studied so far. This technique is very useful in all cases where intramolecular or intermolecular quadruplex formation is suspected.z 1998 Federation of European Biochemical Societies. [11,12], calorimetry [13] and ultraviolet absorption. In the latter case, absorbance of a guanine-rich oligonucleotide is recorded vs. temperature. In most cases, the pro¢les do not provide a precise determination of the melting temperature [14]. This situation led several laboratories to record absorbance at di¡erent wavelengths (272 or 273 nm [15,16], rather than 260 nm). Only in these cases were thermodynamic parameters succesfully determined from UV-melting measurements. Most of the vH³ and vS³ values compiled in the literature were rather the result of CD-melting experiments.We wanted to optimize the quality of the melting pro¢les. Unmodi¢ed oligodeoxynucleotides were obtained and puri¢ed as previously described [17]. All concentrations were estimated by UV absorption [18] and expressed in strand molarity. The absorbance spectrum of the guanine-rich doligonucleotide was recorded at 1 and 90³C (Fig. 1A). As previously noted by Scaria and colleagues [15], the di¡erence spectrum exhibits an isosbestic point around 280 nm, whereas a net hyperchromism is observed upon G-quartet formation at 285 nm or higher wavelength. This di¡erence is maximal at 295 nm. For these reasons, we investigated whether the formation of the G-quartet structure could be followed at 295 nm. In parallel, we recorded the absorbance pro¢le at 260 nm and 295 nm (Fig. 1B). A nice sigmoidal curve was obtained at 295 nm with a melting temperature of 56³C in a 0.1-M NaCl, 10-mM sodium cacodylate bu¡er. A smaller variation (4%) of absorbance was recorded at 260 nm upon G-quartet dissociation. The melting temperature at 295 nm was independent of pH (in the pH 5^7.5 range) and also independent of oligonucleotide concentration (in the 0.5^50-WM strand concentration range, not shown). The later results demonstrated that the formation of this structure was an intramolecular process. The pro¢le was perfectly reversible (cooling and heating curves were superimposed) and reproducible (two cooling pro¢les were also superimposed). As expected for the dissociation of a G-quartet, the T m was dependent on the nature of the monovalent ion. A T m of 63³C was obtained in a 10-mM sodium cacodylate bu¡er with 0.1 M KCl, as compared to 37³C in a 10-mM sodium cacodylate bu¡er with 0.1 M LiCl. Thus, as expected, the e¡ect o...

Telomeres play an important role in cellular aging and cancer. Human telomeric DNA and RNA G‐rich sequences are capable of forming a four‐stranded structure, known as the G‐quadruplex. Such a structure might be important for telomere biology and a good target for drug design. This minireview describes the structural diversity or conservation of DNA and RNA human telomeric G‐quadruplexes, discusses structural views on targeting these G‐quadruplexes and presents some future challenges for structural studies.

G-quadruplex structures formed by DNA at the human telomeres are attractive anticancer targets. Human telomeric sequences can adopt a diverse range of intramolecular G-quadruplex conformations: a parallel-stranded conformation was observed in the crystalline state, while at least four other forms were seen in K(+) solution, raising the question of which conformation is favored in crowded cellular environment. Here, we report the first NMR structure of a human telomeric G-quadruplex in crowded solution. We show that four different G-quadruplex conformations are converted to a propeller-type parallel-stranded G-quadruplex in K(+)-containing crowded solution due to water depletion. This study also reveals the formation of a new higher-order G-quadruplex structure under molecular crowding conditions. Our molecular dynamics simulations of solvent distribution provide insights at molecular level on the formation of parallel-stranded G-quadruplex in environment depleted of water. These results regarding human telomeric DNA can be extended to oncogenic promoters and other genomic G-rich sequences.