The Solution Structure and Internal Motions of a Fragment of the Cytidine-rich Strand of the Human Telomere Anh Tua à n Phan, Maurice Gue  ron* and Jean-Louis Leroy Groupe de Biophysique de l'Ecole Polytechnique, et de l'UMR 7643 du CNRS 91128 Palaiseau, France We present the solution structure of d(CCCTA 2 CCCTA 2 CCCTA 2 CCCT), a fragment of the vertebrate telomere which folds intramolecularly. The four cytidine stretches form an i-motif which includes six intercalated C  C  pairs and terminates with the cytidines at the 5 0 extremity of each stretch. Above, the second TA 2 linker loops across one of the narrow grooves, while at the bottom, the ®rst and third linkers loop across the wide grooves. At 30  C, the spectra of the ®rst and third linkers are quasi-degenerate. Severe broadening at lower temperature indicates that this results from motional averaging between at least two structures of each bottom loop, and makes it impossible to solve the con®guration of the bottom loops directly, in contrast to the rest of the structure. We therefore turned to the modi®ed sequence d(CCCTA 2 5mCCC- TA 2 CCC UA 2 CCCT) in which the two base substitutions (underlined) break the quasi-symmetry between linkers 1 and 3. The three loops fol- low approximately the hairpin ``second pattern'' of Hilbers. In the ®rst loop, T4 is in the syn orientation, whereas its analog in the third loop, U16, oriented anti, is in a central location, where it interacts with bases of both loops, thus contributing to their tight association. The only motion is a syn/anti ¯ip of A18 in the third loop. Returning to the telomere fragment, we show that each of the bottom loops switches between the structures identi®ed in the ®rst and third loops of the modi®ed structure. The motions are concerted, and the resulting con®gurations of the bottom loop cluster present a bulge to either right (T4 syn) or left (T16 syn). # 2000 Academic Press Keywords: DNA solution structure; i-motif; interacting loops; loop motion; NMR, telomere *Corresponding author Introduction The i-motif is an intercalated structure of nucleic acids formed by the association of two parallel- stranded duplexes built of hemi-protonated cyti- dine-cytidine base pairs (Gehring et al., 1993; Chen et al., 1994). Its discovery raised the standard ques- tions: what are its structural properties, what are the requirements for its formation, does it occur in biological conditions, are there biological functions and/or pharmacological implications? The i-motif was ®rst observed by NMR in the tetramer of d(TCCCCC) (Gehring et al., 1993). The original report pointed out that it might also form by intramolecular folding of a single-strand DNA sequence containing four stretches of a few cyti- dines, such as are found in the C-rich strand of tel- omeres. Telomeric sequences of Tetrahymena and of vertebrates were indeed shown to fold into a monomeric i-motif which persisted at pH 7 despite the requirement for cytidine hemi-protonation, pointing to the possibility of intracellular occur- ence. However the spectra were not good enough for a structural study (Ahmed et al., 1994; Leroy et al., 1994). E-mail address of the corresponding author: mg@pmc.polytechnique.fr Abbreviations used: NOESY, nuclear Overhauser enhancement spectroscopy; TOCSY, total correlation spectroscopy; COSY, correlation spectroscopy; HSQC, hetero-nuclear single quantum correlation; NOE, nuclear Overhauser enhancement. doi:10.1006/jmbi.2000.3613 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 299, 123±144 0022-2836/00/010123±22 $35.00/0 # 2000 Academic Press