Structural dynamics of possible late-stage intermediates in folding of quadruplex DNA studied by molecular simulations

Abstract

Explicit solvent molecular dynamics simulations have been used to complement preceding experimental and computational studies of folding of guanine quadruplexes (G-DNA). We initiate early stages of unfolding of several G-DNAs by simulating them under no-salt conditions and then try to fold them back using standard excess salt simulations. There is a significant difference between G-DNAs with all-anti parallel stranded stems and those with stems containing mixtures of syn and anti guanosines. The most natural rearrangement for all-anti stems is a vertical mutual slippage of the strands. This leads to stems with reduced numbers of tetrads during unfolding and a reduction of strand slippage during refolding. The presence of syn nucleotides prevents mutual strand slippage; therefore, the antiparallel and hybrid quadruplexes initiate unfolding via separation of the individual strands. The simulations confirm the capability of G-DNA molecules to adopt numerous stable locally and globally misfolded structures. The key point for a proper individual folding attempt appears to be correct prior distribution of syn and anti nucleotides in all four G-strands. The results suggest that at the level of individual molecules, G-DNA folding is an extremely multi-pathway process that is slowed by numerous misfolding arrangements stabilized on highly variable timescales.

Figure 1.

Schemes of experimental structures used in our simulations. (a) 2GKU, (b) 1KF1, (c) 1JPQ and (d) 352D and 1J8G (3TVB has the first tetrad in syn). (Deoxy)guanosine residues are depicted by rectangles. Yellow and orange indicate anti and syn conformation, respectively; darker residues are at the back. Red lines represent G-DNA WC/H hydrogen bonding. Black arrows show sugar-phosphate backbone in 5′→3′ direction. Loops are depicted by thin black curves while flanking residues are not shown.

Figure 2.

Graph (left) of simulations performed on the d[A(G₃T₂A)₃G₃] parallel-stranded quadruplex. Boxes in the graph mark important conformations and show their labels. The vertical line indicates the unfolding no-salt simulation and the horizontal lines re-folding simulations with excess-salt conditions. Branches from the horizontal lines indicate further simulations from the corresponding structures. The numbers in boxes show the times (ns) when the corresponding structures were observed from the beginning of either the no-salt simulation or excess-salt simulation (except the last numbers in green boxes, which show the end of the simulation). The cyan box corresponds to the starting experimental structure. Green boxes represent structures that were successfully refolded (see the text), whereas yellow boxes mark unsuccessful refolding attempts. Structural schemes A-G are visualized as follows: deoxyguanosine residues are depicted by rectangles, yellow indicates anti conformation, orange syn conformation and darker residues are at the back. Solid red lines represent standard WC/H hydrogen bonding, and dashed red lines represent any other hydrogen bonding. Black arrows show sugar-phosphate backbone in 5′→3′ orientation. Loops are depicted by thin black curves. Flanking residues and ions are not shown. The coloring of the edges of the rectangles in the structures indicates residues with approximately the same normal vector of their respective base plane; the letters below the structures correspond to the labels used in the vertical (no-salt) graph. For full structural details, see Supplementary Figures S7–S14 and Supplementary Table S4.

Figure 3.

(a) Final structure of the parallel all-anti tetrameric [d(G₄)]₄ G-DNA stem forming the cross-like structure in the no-salt unfolding simulation. (b) The structure at the end of the standard re-folding simulation, which has a stem with three tetrads and mutual slippage of the diagonally placed strand pairs, leading to formation of GG base pairs above and below the stem. Strands forming the cross-structure in part (a) are colored red and yellow, the Na⁺ ions are blue and the backbone brown. (c) Structural scheme of the molecule depicted in (a). (d) Structural scheme of the molecule depicted in (b). For further explanation of the schemes, see the legend of Figure 2.

Figure 4.

Terminal structure of the 190 ns long no-salt simulation of the RNA quadruplex [r(UG₄U)]₄, in which base pairing is maintained between red strands and between yellow strands. Yellow strands are those (strands c and d) that consecutively slipped in the simulation. Uridines are colored green.

Figure 5.

(a) Structure after 40 ns of the no-salt simulation of [r(UG₄U)]₄. (b) The structure resulting after addition of ions, which is fully restored to its typical conformation. Guanosines forming the individual tetrads in the native G-stem are colored mauve, red, yellow and green; Na⁺ ions are colored blue, and the backbone and uridines brown.

Figure 6.

Two types of movements observed during the 300 ns no-salt simulation of the hybrid 2GKU quadruplex. (a) Modest and reversible opening of the groove between the first and last strands, with local loss of direct base pairing. (b) Large opening of the quadruplex from the upper part downward. The schemes are visualized as in Figure 2. The red arrows show directions of the movements.

Figure 7.

Two graphs (left) showing changes with time of the 2GKU quadruplex in the single no-salt simulation. The colors of the boxes indicate whether the corresponding structure was successfully refolded (green) or not (yellow) in the subsequent KCl and NaCl simulations. The conformations (right) occurring in this simulation that were used as starts for subsequent NaCl and KCl simulations (except for the ZZ molecule). For further details, see Supplementary Table S5, and for further explanation, see the legend of Figure 2.

Figure 8.

Substantial temporary unwinding (unbinding) of one strand from the DNA antiparallel [d(G₄T₄G₄)]₂ quadruplex structure in the no-salt simulation. Guanosines forming the consecutive tetrads in the native G-stem are in blue, red, yellow and green, respectively. The sugar-phosphate backbone and thymidines are in brown. Black dashed lines represent C1′-C1′ distances of nucleotides between the unwound strand and the adjacent strand. The sketch on the right is shown from a different angle for clarity; see the legend of schemes in Figure 2 for further explanation.

Figure 9.

Final structure of the [d(G₄T₄G₄)]₂ quadruplex in the 1.5 μs no-salt simulation. Guanosines forming the consecutive tetrads in the native G-stem are in blue, red, yellow and green, respectively. The sugar-phosphate backbone and thymidines are in brown. Five base triads and their respective locations in the structure are highlighted. Black dots mark nucleotides in syn conformation. The structure remains compact, but the native interactions have been visibly perturbed.