Structure and stability of higher-order human telomeric quadruplexes

Abstract

G-quadruplex formation in the sequences 5'-(TTAGGG)(n) and 5'(TTAGGG)(n)TT (n = 4, 8, 12) was studied using circular dichroism, sedimentation velocity, differential scanning calorimetry, and molecular dynamics simulations. Sequences containing 8 and 12 repeats formed higher-order structures with two and three contiguous quadruplexes, respectively. Plausible structures for these sequences were determined by molecular dynamics simulations followed by experimental testing of predicted hydrodynamic properties by sedimentation velocity. These structures featured folding of the strand into contiguous quadruplexes with mixed hybrid conformations. Thermodynamic studies showed the strands folded spontaneous to contain the maximum number contiguous quadruplexes. For the sequence 5'(TTAGGG)(12)TT, more than 90% of the strands contained completely folded structures with three quadruplexes. Statistical mechanical-based deconvolution of thermograms for three quadruplex structures showed that each quadruplex melted independently with unique thermodynamic parmameters. Thermodynamic analysis revealed further that quadruplexes in higher-ordered structures were destabilized relative to their monomeric counterparts, with unfavorable coupling free energies. Quadruplex stability thus depends critically on the sequence and structural context.

Figure 1

Sedimentation coefficient distributions of (TTAGGG)₄ and (TTAGGG)₄TT (A), of (TTAGGG)₈ and (TTAGGG)₈TT (B), (TTAGGG)₁₂ and (TTAGGG)₁₂TT (C). The sequence with the two thymine residues at 3′end is shown in red.. Each distribution was normalized with respect to the highest c(s) value.

Figure 2

The average structure of the Hybrid-121, all-propeller and dt12 models are shown on the (top). The colors of the residues are: green for dG, blue for dT and red for dA residues. On the bottom of the figure, the sedimentation coefficient distributions obtained from the MD trajectories of the model (indicated by the colored arrows) are superimposed on the experimental distribution (black line) obtained for the (TTAGGG)₁₂ telomeric sequence in K+ solution.

Figure 3

CD spectra of the (TTAGGG)₄ and (TTAGGG)₄TT (A), (TTAGGG)₈ and (TTAGGG)₈TT (B), (TTAGGG)₁₂ and (TTAGGG)₁₂TT C).The sequence with the two thymine residues at 3′end are shown in red. In the panel D the CD spectrum of the (TTAGGG)₄TT (black line) is compared to the CD spectrum of (TTAGGG)₈TT (red line) and (TTAGGG)₁₂TT (green line). The CD spectrum of (TTAGGG)₈TT and (TTAGGG)₁₂TT were divided by factors of two and three , respectively.

Figure 4

CD (top) and DSC (bottom) melting profiles of (TTAGGG)₄ and (TTAGGG)₄TT (A,B), (TTAGGG)₈ and (TTAGGG)₈TT (C,D), (TTAGGG)₁₂ and (TTAGGG)₁₂TT (E,F). The sequences with the two thymine residues at 3′end are shown in red.

Figure 5

Deconvolution of DSC profiles for the (TTAGGG)_nTT (n=4,8,12) series. Panels A, C and E show the species plots for the monomer, dimer and trimer structures, respectively. Panels B, D, and F show the corresponding best fits to experimental thermograms.

Figure 6

Free energy diagram illustrating the coupling free energy for folding of three contiguous quadruplexes compared to folding of three individual quadruplex structures.