## 4 Conclusions  
In this manuscript, we analyzed the effect of increasing the length of the variable loop integrated on three diprotic and three tetraprotic triple helix nanoswitches, using the same sequences experimentally characterized in a recent work [29]. The atomistic investigation, carried out through ABMD simulations, permitted us to finely describe the unbinding process of the TFO from the double helical portion of the nanoswitch. In detail, the analysis of the six trajectories identified a different TFO unbinding path for the diprotic switch, depending on the length of the variable loop, both in terms of the energy required to switch from the bound to unbound states (Figures 2 and 4) and in terms of the per-base unbinding profile (Figure 5A). In fact, the DIPRO5 switch requires, for the TFO unbinding, more energy than the DIPRO15 and DIPRO25 ones (Figure 5A). Moreover, for the DIPRO5, the unbinding is incomplete because the bases directly connected to the variable loop continue to interact with the DH even in the final state (Figure 4A). Finally, the per-base unbinding profile is different following a 3\({}^{\prime}\) to 5\({}^{\prime}\) direction for the DIPRO5 and a 5\({}^{\prime}\) to 3\({}^{\prime}\) for the DIPRO25 one, while it concomitantly occurs at both the 3\({}^{\prime}\) and 5\({}^{\prime}\) ends of the TFO for the DIPRO15, demonstrating the importance to the loop length in modulating the mechanism. Analyzing the results obtained for the tetraprotic switches, we did not find differences in the per-base unbinding profiles (Figure 4B), and we observed only slight energy differences in the free energy profiles (Figure 5B), confirming that linker-dependent modulation is dissipated upon increasing the number of protonation centers in the TFO. These results provide an atomistic explanation to the experimental results showing, for the TFO unbinding in the diprotic switches, a decrease of more than two units of the pKa increasing the linker length from 5 to 25 bases, whilst the pH-dependent switching mechanism--dependent on the linker length--is lost when the protonation centers are four. Interestingly, our data indicate that the presence of a longer variable loop does not destabilize the DH, as can be observed in Figure S2A,B and in Tables S1 and S2 of Supplementary Materials. This is an important point to ensure a correct functioning of the switch as a pH-sensing device, because a loss of the DH stability would not permit the reversibility of the pH-dependent switching process. Computational approaches, such as enhanced sampling MD simulations, proved to be of fundamental importance for the design and tuning of the functional properties of DNA nanoswitches. Software like mfold [48] or NUPACK [49], although allowing the prediction of the thermodynamics of Watson-Crick interactions, are unable to predict the behaviour of DNA nanodevices characterized by the presence of non-canonical interactions or sequence-independent effects. Therefore, the use of complex and advanced simulation techniques has become an essential tool in the field of DNA nanotechnology, which is also thanks to the reduced computational time required to perform MD simulations due to the improvement of the available workstations. The following are available online at [[https://www.mdpi.com/article/10.3390/app11094052/s1](https://www.mdpi.com/article/10.3390/app11094052/s1)]([https://www.mdpi.com/article/10.3390/app11094052/s1](https://www.mdpi.com/article/10.3390/app11094052/s1)). Figure S1: Schematic and cartoon representations of the simulated tetraprotic DNA nanoswitches; Figure S2: Per-nucleotide RMSF values calculated for the DH of the six DNA nanoswitches; Table S1: DNA DH geometrical parameters calculated for the diprotic switches; Table S2: DNA DH geometrical parameters calculated for the tetraprotic switches. Conceptualization, F.I. and A.D.; methodology, A.R. and F.I. ; validation, F.I., A.D. and M.F. ; formal analysis, A.R. and F.I. ; writing--original draft preparation, F.I., A.D. and M.F.