An integrated molecular dynamics (MD) and experimental study of higher order human telomeric quadruplexes

Abstract

Structural knowledge of telomeric DNA is critical for understanding telomere biology and for the utilization of telomeric DNA as a therapeutic target. Very little is known about the structure of long human DNA sequences that may form more than one quadruplex unit. Here, we report a combination of molecular dynamics simulations and experimental biophysical studies to explore the structural and dynamic properties of the human telomeric sequence (TTAGGG)(8)TT that folds into two contiguous quadruplexes. Five higher order quadruplex models were built combining known single human telomeric quadruplex structures as unique building blocks. The biophysical properties of this sequence in K(+) solution were experimentally investigated by means of analytical ultracentrifugation and UV spectroscopy. Additionally, the environments of loop adenines were probed by fluorescence studies using systematic single-substitutions of 2-aminopurine for the adenine bases. The comparison of the experimentally determined properties with the corresponding quantities predicted from the models allowed us to test the validity of each of the structural models. One model emerged whose properties are most consistent with the predictions, and which therefore is the most probable structure in solution. This structure features contiguous quadruplex units in an alternating hybrid-1-hybrid-2 conformation with a highly ordered interface composed of loop residues from both quadruplexes.

Figure 1

Representative structures of the five two-quadruplex models. The structures are the time-averaged structures computed over the last 2 ns of the Hybrid-12S, Hybrid-21S, Hybrid-11S, Hybrid-22S and propeller-S trajectories. The G-tetrads bases are shown in green. For clarity the loops bases are not shown. For the Hybrid12, Hybrid-22 and Hybrid-11 models the dT (blue) and dA (red) residues involved in quadruplex-quadruplex interactions are also shown. The strand orientation is shown by the black arrow on the left.

Figure 2

Electrostatic potentials mapped on the solvent-accessible surfaces are shown for all the structures in Figure 1. Red represents negative regions, white represents neutral regions and blue represents positive regions.

Figure 3

Major conformational changes occurring in MD trajectories. A) Hybrid-11S trajectory: initial (left) and after 10 ns of free MD (right). B) Hybrid-12U trajectory: initial structure (left) and after 33 ns of free MD dynamics (right). Residue colors: green for G-tetrads, red for adenine bases and blue for thymine bases. For clarity, loop residues that are not involved in quadruplex-quadruplex interactions are not shown.

Figure 4

Stability of stacking interactions at the quadruplex-quadruplex interface as assessed by the distance between A15 and T38 rings throughout the Hybrid-12S trajectory.

Figure 5

Correlation between the rmsd variation and the relative orientation of the two quadruplex units. The all atom rmsd fluctuations (top) and variations in the angle (θ) between the helical axes of the two quadruplex units (bottom) in the Hybrid-22S1 trajectory.

Figure 6

Dependence of the local environment of each adenine base on the particular model. The different adenine bases are shown in different colors which indicate the position of the 2-AP substitutions as specified in the sequence at the bottom.

Figure 7

Dependence of 2-AP fluorescence on the 2-AP local environment. Steady state fluorescence experiments at 20 °C (left) and 95 °C (right) for all the oligonucleotides containing 2-aminopurine. The colors indicate the position of the 2-aminopurine substitution as shown in Figure 6. The fluorescence spectra at 20 °C (Panel on the left) are from the reference [20].

Figure 8

Stern-Volmer plots of the collisional quenching of 2-AP fluorescence by acrylamide (first panel, top left) and correlation of computed SASA values (black line) for all the models with the experimental collisional quenching results (red line). The data presented in panel A are from the reference [20].

Figure 9

Fluorescence melting curves of AP15 (left) and AP39 (right).