Structure, internal motions and association–dissociation kinetics of the i-motif dimer of d(5mCCTCACTCC)

At slightly acidic pH, the association of two d(5mCCTCACTCC) strands results in the formation of an i-motif dimer. Using NMR methods, we investigated the structure of [d(5mCCTCACTCC)](2), the internal motion of the base pairs stacked in the i-motif core, the dimer formation and dissociation kinetics versus pH. The excellent resolution of the (1)H and (31)P spectra provided the determination of dihedral angles, which together with a large set of distance restraints, improve substantially the definition of the sugar-phosphate backbone by comparison with previous NMR studies of i-motif structures. [d(5mCCTCACTCC)](2) is built by intercalation of two symmetrical hairpins held together by six symmetrical C•C(+) pairs and by pair T7•T7. The hairpin loops that are formed by a single residue, A5, cross the narrow grooves on the same side of the i-motif core. The base pair intercalation order is C9•C9(+)/5mC1•5mC1(+)/C8•C8(+)/C2•C2(+)/T7.T7/C6•C6(+)/C4•C4(+). The T3 bases are flipped out in the wide grooves. The core of the structure includes four long-lived pairs whose lifetimes at 15°C range from 100 s (C8•C8(+)) to 0.18 s (T7•T7). The formation rate and the lifetime of [d(5mCCTCACTCC)](2) were measured between pH 6.8 and 4.8. The dimer formation rate is three to four magnitude orders slower than that of a B-DNA duplex. It depends on pH, as it must occur for a bimolecular process involving non cooperative association of neutral and protonated residues. In the range of pH investigated, the dimer lifetime, 500 s at 0°C, pH 6.8, varies approximately as 10(−pH)

Solution structures of the i-motif tetramers of d(TCC), d(5methylCCT) and d(T5methylCC): novel NOE connections between amino protons and sugar protons

AbstractBackground: At slightly acid or even neutral pH, oligodeoxynucleotides that include a stretch of cytidines form a tetramer structure in which two parallel-stranded duplexes have their hemi-protonated C·C+ base pairs face-to-face and fully intercalated, in a so-called i-motif, first observed serendipitously in [d(TC5 )]4.Results A high-definition structure of [d(TCC)]4 was computed on the basis of inter-residue distances corresponding to 21 NOESY cross-peaks measured at short mixing times. A similarly defined structure of [d(5mCCT)]4 was also obtained. A small number of very characteristic (amino proton)-(sugar proton) cross-peaks entails the intercalation topology. The structure is generally similar to that of [d(TC5 )]4 . The sequence d(T5mCC) forms two tetramers in comparable proportions. The intercalation topologies are read off the two patterns of (amino proton)-(sugar proton) cross-peaks: one is the same as in the d(TCC) tetramer, the other has the intercalated strands shifted by one base, which avoids the steric hindrance between the methyl groups of the 5mC pairs of the two duplexes. .Conclusion The structures obtained in this work and the procedures introduced to characterize them and to solve the problems linked to the symmetry of the structure provide tools for further exploring the conditions required for formation of the i-motif

i-motif solution structure and dynamics of the d(AACCCC) and d(CCCCAA) tetrahymena telomeric repeats

Using NMR methods, we have resolved the i-motif structures formed by d(AACCCC) and by d(CCCCAA), two versions of the DNA sequence repeated in the telomeric regions of the C-rich strand of tetrahymena chromosomes. Both oligonucleotides form fully symmetrical i-motif tetramers built by intercalation of two hemiprotonated duplexes containing four C•C(+) pairs. The structures are extremely stable. In the tetramer of d(AACCCC), the outermost C•C(+) pairs are formed by the cytidines of the 5′ ends of the cytidine tracts. A2 forms an A2•A2 (H6trans–N7) pair stacked to C3•C3(+) and cross-strand stacked to A1. At 0°C, the lifetimes of the hemiprotonated pairs range from 1 ms for the outermost pair to ∼1 h for the innermost pairs. The tetramer of d(CCCCAA) adopts two distinct intercalation topologies in slow conformational exchange. One, whose outermost C•C(+) pairs are built by the cytidines of the 5′ end and the other by those of the 3′ end. In both topologies, the adenosine bases are fairly well stacked to the adjacent C•C(+) pairs. They are not paired but form symmetrical pseudo-pairs with their H6cis amino proton and N1 nitrogen pointing towards each other

Terminal Base Pairs of Oligodeoxynucleotides: Imino Proton Exchange and Fraying

International audienc

Junctions between i-motif tetramers in supramolecular structures

The symmetry of i-motif tetramers gives to cytidine-rich oligonucleotides the capacity to associate into supramolecular structures (sms). In order to determine how the tetramers are linked together in such structures, we have measured by gel filtration chromatography and NMR the formation and dissociation kinetics of sms built by oligonucleotides containing two short C stretches separated by a non-cytidine-base. We show that a stretch of only two cytidines either at the 3′- or 5′-end is long enough to link the tetramers into sms. The analysis of the properties of sms formed by oligonucleotides differing by the length of the oligo-C stretches, the sequence orientation and the nature of the non-C base provides a model of the junction connecting the tetramers in sms

The formation pathway of i-motif tetramers

The i-motif is a four-stranded structure formed by two intercalated parallel duplexes containing hemiprotonated C•C+ pairs. In order to describe the sequence of reactions by which four C-rich strands associate, we measured the formation and dissociation rates of three [TCn]4 tetramers (n = 3, 4 and 5), their dissociation constant and the reaction order for tetramer formation by NMR. We find that TCn association results in the formation of several tetramers differing by the number of intercalated C•C+ pairs. The formation rates of the fully and partially intercalated species are comparable but their lifetimes increase strongly with the number of intercalated C•C+ pairs, and for this reason the single tetramer detected at equilibrium is that with optimal intercalation. The tetramer half formation times vary as the power −2 of the oligonucleotide concentration indicating that the reaction order for i-motif formation is 3. This observation is inconsistent with a model supposing association of two preformed duplex and suggests that quadruplex formation proceeds via sequential strand association into duplex and triplex intermediate species and that triplex formation is rate limiting