Duplex-tetraplex equilibrium between a hairpin and two interacting hairpins of d(A-G)_(10) at neutral pH

摘要

d(A-G)_(10) forms two helical structures at neutrality, at low ionic strength a single-hairpin duplex, and at higher ionic strength a double-hairpin tetraplex. An ionic strength-dependent equilibrium between these forms is indicated by native PAGE, which also reveals additional single-stranded species below 0.3 M Na~+, probably corresponding to partially denatured states. The equilibrium also depends upon oligomer concentration: at very low concentrations, d(A-G)_(10) migrates faster than the randomcoil d(C-T)_(10), probably because it is a more compact single hairpin; at high concentrations, it co-migrates with the linear duplex d(A-G)_(10)-d(C-T)_(10), probably because it is a two-hairpin tetraplex. Molecular weights measured by equilibrium sedimentation in 0.1 M Na~+, pH 7, reveal a mixture of monomer and dimer species at 1C. but only a monomer at 40°C; in 0.6 M Na~+, pH 7, only a dimer species is observed at 4 C. That the single- and double-stranded species are hairpin helices, is indicated by preferential S1 nuclease cleavage at the center of the oligomer(s), i.e., the loop of the hairpin(s). The UV melting transition below 0.3 M Na~+ or K~+, exhibits a dT_m/dlog[Na~+/K~+] of 33 or 36°C, respectively, consistent with conversion of a two-hairpin tetraplex to a single-hairpin duplex with extrahelical residues. When [Na~+/K~+] >= 0.3 M, dT_m/dlog [Na~+/K~+] is 19 or 17°C, respectively, consistent with conversion of a two-hairpin tetraplex directly to single strands. A two-hairpin structurestabilized by G-tetrads is indicated by differential scanning calorimetry in 0.15 M Na~+/5 mM Mg~(2+), with AH of formation per mole of the two-hairpin tetraplex of -116.9 kcal or -29.2 kcal/mol of G-tetrad.