Engineering a responsive DNA triple helix into an octahedral DNA nanostructure for a reversible opening/closing switching mechanism: a computational and experimental integrated study

Abstract

We propose an experimental and simulative approach to study the effect of integrating a DNA functional device into a large-sized DNA nanostructure. We selected, as a test bed, a well-known and characterized pH-dependent clamp-switch, based on a parallel DNA triple helix, to be integrated into a truncated octahedral scaffold. We designed, simulated and experimentally characterized two different functionalized DNA nanostructures, with and without the presence of a spacer between the scaffold and the functional elements. The experimental and simulative data agree in validating the need of a spacer for the occurrence of the pH dependent switching mechanism. The system is fully reversible and the switching can be monitored several times without any perturbation, maintaining the same properties of the isolated clamp switch in solution.

Figure 1.

Modelling of the pH-dependent DNA nanocages. (A) Truncated octahedral DNA cage. Thick lines indicate double helices, thin lines the 5T linkers. (B) Top view of the truncated octahedral cage. (C) Top view of the T-cage, functionalized with two pH-dependent functional elements, at pH 5.0 and at (D) pH 8.0. (E) Model of the LT-cage, functionalized with two pH-dependent functional elements connected to the cage scaffold through seven-base spacers (yellow). The sequence of the clamp-switch triplex functional element is shown at the bottom of the figure. The green and black sequences represent the two strands interacting through the W-C interactions, the underlined sequence represents the 25 bases loop and the red sequence the third strand establishing at pH 5.0 the Hoogsteen hydrogen bonds with the double helix.

Figure 2.

Time-dependent evolution of RMSD values. RMSD of the triple-helix (A) and of the double-helix region (B) evaluated at pH 8.0 for one of the clamp-switch element for the T-cage (black line) and the LT-cage (red line).

Figure 3.

Evolution of the hydrogen bond number between the double helix and the triplex-forming strand (A, Hoogsteen panel) and within the double helix (B, Watson–Crick panel) at pH 8.0 for the T-cage (black line) and the LT-cage (red line).

Figure 4.

Analyses of the MM/GBSA interaction energy and of the buried surface area for the 0–50, 50–100 and 100–150 ns trajectories time windows. (A) MM/GBSA interaction energy at pH 8.0 for the clamp-switch of the T- (blue) and LT-cage (orange), respectively. (B) BSA values at pH 8.0 for the clamp-switch of the T- (blue) and LT-cage (orange).

Figure 5.

Gel-analysis of purified cages at pH 5.0 and 8.0. Lane M: DNA marker. Lane A: non-functionalized octahedral DNA cage, Lane B and D: LT-cage, Lane C: pH-independent cage.

Figure 6.

(A) pH−titration curves of the LT-cage (green curve) and its control duplex cage (black curve) achieved in TAE buffer at 25°C and using a cage concentration of 250 nM. Triplex-to-duplex transition is monitored taking the difference in the fisetin's fluorescence signal in presence and absence of the DNA cage at different pH values. (B) Reversible behaviour of the switching mechanism followed cyclically changing the pH of a solution containing the LT-cage and fisetin from 5.0 to 8.0.