Book of Abstracts: Albany 2007
June 19-23 2007
The Formation Pathway of Tetrameric Oligonucleotide Structures
Oligonucleotides containing cytidine or guanosine stretches can form four stranded structures. In slightly acidic solutions, the C-rich oligonucleotides adopt a structure, the i-motif, built by two hemiprotonated parallel duplexes intercalated into each other in a head to tail orientation. Association of G-rich oligonucleotides in four stranded structures forms stacked coplanar G-quartets that are stabilized by monovalent ions. Although atomic resolution data exist for many guanine quadruplexes and i-motif tetramers, the formation pathway of theses structures is not clear.
Using NMR methods, we investigated the formation-dissociation kinetics of the i-motif tetramers formed by the TCn series (n=2 to 5) and of two G-quadruplexes: [(TG4T)]4 and [(TG3T)]4
The salient result of the kinetics studies is the observation that the formation rate of the i-motif and of the G-quartets increases as the square of the stand concentration and thus indicates third-order reactions.
i-motif: At 0 °C, the half association time of TC3 measured at the strand concentration of 10-4 M is about one hour. As it must occur for a bimolecular process involving association of neutral and protonated residues, the formation rate varies with pH as the product of the fractions of protonated and neutral cytidine. In the early stage of the association kinetics of TC4 and TC5 one observes the accumulation of incompletely intercalated tetramers that are formed more rapidly than the fully intercalated species. Their lifetimes are much shorter than that of the fully intercalated tetramer which is the single species observed at equilibrium. The lifetimes at 10 °C of the fully intercalated tetramers formed by TC4, TC3, and TC2 are 97 days, two hours, and about 4s, respectively.
G-quadruplexes: The formation rates of [TG4T]4 and [TG3T]4 are comparable at 30 °C. Typically, in the presence of 0.125 M monovalent ion, the half association time measured at the strand concentration of 10-4 M is about 15 hours at 30 °C. The formation rates decrease as a function temperature with activation energies of -73 kJ/mol and -25 kJ/mol for TG4T and TG3T respectively. They increase approximately as the square of the monovalent concentration but they are weakly dependent on the nature of the monovalent cation (K+ or Na+). By contrast, the G-quartet lifetimes are strongly dependent on the nature of the monovalent ion. At 60 °C, the lifetime of [TG4T]4 is 16 minutes in the presence of Na+ 0.125 M and about 1.5 years at the same K+ concentration.
Quadruplex formation may occur via the stepwise addition of four strands or by association of two duplexes. Duplex association that should exhibit a second or a fourth-order kinetics vs. the strand concentration is inconsistent with our kinetics data. The third-order dependence of the association rates of oligo-C and oligo-G strands into tetrameric structures suggests that the formation of both tetramers is limited by the emergence of elusive three-stranded species in fast exchange equilibrium with the monomer and a minor and undetectable duplex species. This model entails that the association of the fourth strand, that locks the structure into a long-lived arrangement, is not limiting. The negative activation energy of the G-quartet formation and the strong dependence on the ionic strength might reflect the destabilization of the three stranded structure at high temperature and low ionic strength.
Institut de Chimie des Substances Naturelles