Probing the Functional Topology of a pH-Dependent Triple Helix DNA

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Probing the Functional Topology of a pH-Dependent Triple Helix DNA Nanoswitch Family through Gaussian Accelerated MD Simulation Federico Iacovelli, Kevin Cabungcal Hernandez, Alessandro Desideri, and Mattia Falconi J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.9b00133 • Publication Date (Web): 10 May 2019 Downloaded from http://pubs.acs.org on May 10, 2019

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Journal of Chemical Information and Modeling

1 Probing the Functional Topology of a pH-Dependent Triple Helix DNA Nanoswitch Family through Gaussian Accelerated MD Simulation

Federico Iacovelli1‡, Kevin Cabungcal Hernandez1‡, Alessandro Desideri1 and Mattia Falconi1*

1Department

of Biology, Interuniversity Consortium, National Institute Biostructure and

Biosystem (INBB), University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133, Rome, Italy.

‡These

authors contributed equally to this work.

Abstract The topology of a pH-dependent triple helix DNA nanoswitch family has been characterized through simulative analysis to evaluate the efficiency of the switching mechanism varying the length of the loop connecting the two strands forming the double helix portion. In detail, the system is formed by a double helix made by two six base complementary sequences, connected by one loop having an increasing number of thymidines, namely 5, 7 or 9. The triplex-forming sequence made by six base, connected to the double helix through a constant 25 base loop, interacts at pH 5.0 through Hoogsteen hydrogen bonds with one strand of the double helical region. We demonstrate, through molecular dynamics simulation, that the thymidine loop length exerts a fine

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2 regulatory role for the stability of the triple helix structure and is critical in modulating the switching mechanism triggered by the pH increase.

Keywords: DNA triple helix, switching mechanism, pH responsive nano-devices, Gaussian accelerated molecular dynamics simulations.

*Corresponding author: Prof. Mattia Falconi, Department of Biology, University of Rome “Tor Vergata”, Via della Ricerca

Scientifica

1,

00133

Rome,

Italy.

E-mail:

+39.06.72594025.


[email protected],

Phone

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3 Introduction DNA nanotechnology exploits the physical and chemical properties of DNA in order to design and engineer smart nanomaterials and nanodevices using synthetic DNA sequences.1–6 Methods adopted to build these nanostructures include use of DNA tiles, DNA origami approach or supramolecular assembly, allowing the production of complex nanostructures with different shapes and dimensions.7–11 The great versatility and expandability of these methodologies permit an accurate positioning of moleculeresponsive switching elements in specific locations of DNA nanostructures, that allows the assembly of very complex and functional nanodevices.12–16 DNA motifs, which give rise to non-canonical DNA interactions (G-quadruplex, triplex, i-motif, hairpin and aptamers), can be used as dynamic-responsive elements to chemical and environmental stimuli inducing a controlled conformational change.17,18 Triplex nucleic acids have drawn attention for their aptitude to form adaptable mechanisms useful to trigger conformational and functional changes, using pH as a chemical input, through rational incorporation of triplex-forming portions into DNA nanodevices.19–22 A recent review addresses the use of DNA triplexes to assemble sensing platforms and molecular switches, where the use of triplex DNA structures as functional units for the assembly of pH-responsive systems are described.23 Atomistic investigation of the mechanism involved in these responsive systems, and the understanding of their structure/dynamics relationship is of great help for the nanoscience community.17,24 Simulation approaches represent valuable tools to shed light on the structural, thermodynamic and dynamical properties of DNA nanostructures.14,15,25–30 The synergy between experiments and molecular dynamics (MD) simulations has provided



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4 significant information for the rational design of pH-activated nano-switches based on a triple helix motif.31,32 In detail, we have focused our attention on the structural/dynamical behaviour of a wellcharacterized pH-triggered switching mechanism, based on the formation of a parallel DNA triple helix (Fig. 1), which has shown to be adaptable to pH variation simply changing the relative content of TAT/CGC.33 The experimental and simulative analyses of this triple helix switch integrated into a truncated octahedral DNA cage scaffold indicate the need of a spacer between the scaffold and the switch for the occurrence of a pH dependent switching mechanism. However, the minimal spacer length, required to achieve an efficient implementation remains an unresolved question.32 In this work, we have addressed this problem simulating an identical triple helix at pH 5.0 and 8.0, introducing spacer of different length. Figure 1A shows the DNA triple helix switches used in this work, made by an identical double helix formed by two six base complementary sequences (red and pink lines), connected by a spacer, named secondary loop, having a different number of nucleotides (black dashed line). The triplex-forming sequence made by six bases (green line), connected to the double helix strand (red line) through a constant 25 base loop, named primary loop (continuous black line), interacts at pH 5.0 through Hoogsteen hydrogen bonds with the double helical portion (red and pink lines). Using Gaussian accelerated MD simulations, we demonstrate the importance of the length of the secondary loop in exerting a regulatory role on the stability of the entire triple helix structure and on its related pH dependent switching mechanism.


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5 Simulative Methods

DNA nanoswitch modeling The scheme of the three simulated triple helices is shown in figure 1A-B. These sequences have been already tailored and used in a preceding work dealing with a clamp switch triple helix assembled using two oligonucleotides.31 In this work, we have joined the two oligonucleotides introducing a thymidine linker to investigate which is the minimum length required to ensure the occurrence of the switching mechanism. The sequences of the nanoswitches were as follow: 5T: CCTCTT-TCCTTCTCTAGTTTGCTCTCTTCCT-TTCTCC-TTTTT-GGAGAA 7T: CCTCTT-TCCTTCTCTAGTTTGCTCTCTTCCT-TTCTCC-TTTTTTT-GGAGAA 9T: CCTCTT-TCCTTCTCTAGTTTGCTCTCTTCCT-TTCTCC-TTTTTTTTT-GGAGAA where the bases in bold represent the duplex forming regions (red and pink portions in Fig. 1), the italic bases represent the triplex forming region (green portion in Fig.1) and the underlined bases represent the primary 25-base loop and the secondary loop (black portions in Fig. 1). The three systems differ for the length of the secondary loop that is made by 5, 7 or 9 thymidines, respectively. The coordinates of the three structures have been obtained through the fiber and mutate_bases modules of the X3DNA software,34 which have been used to generate a 6 base triple-helix and the single strand loops. The PyMol sculpting module (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC; https://pymol.org) has been used to join these substructures through phosphodiesteric bonds. The 3D models are represented in figure 1B. The nanostructure with a 5, 7 or 9 thymidines secondary loop will be named 5T, 7T and 9T, respectively.



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6

MD and GaMD simulations The topologies and the coordinates files of the structures at pH 5.0 and 8.0 have been obtained through tLeap module of the AmberTools program package,35 parameterizing the structures through the AMBER parmbsc1 force field.36 All the cytosines, composing the triplex-forming oligonucleotide (TFO) at pH 5.0 were modified according to the AMBER nomenclature for protonated nucleotides. Residue name was changed from DC to DCP, corresponding to a cytosine protonated on the N3 atom. The structures have been immersed into a rectangular box of TIP3P37 water molecules and have been neutralized adding Mg+2 ions, imposing a minimum distance between the structure and the box of 13 Å. For each structure, a minimization run of 500 steps using the steepest descent algorithm38 followed by 1500 steps of conjugate gradient was performed to remove any unfavourable interaction. The systems were gradually heated from 0 to 300 K in the NVT ensemble over a period of 500 ps using the Langevin thermostat39 and with a restraint of 0.5 kcalmol-1Å-2 on each nucleotide to relax the solvent. In a 1000 ps long equilibration run the restraint forces were gradually decreased to 0.1 kcalmol-1Å-2. The systems were simulated using an isobaric-isothermal (NPT) ensemble for 10 ns, with a time step of 2.0 fs, using the PME method40 for long-range interactions and a cut-off of 9.0 Å for the short-range interactions. In this phase, the temperature was fixed at 300 K and the pressure at 1.0 atm using the Langevin barostat.41 The SHAKE42 algorithm was used to constrain covalent bonds involving hydrogen atoms. The production phase was run over a period of 500 ns for each structure through a Gaussian Accelerated Molecular Dynamics (GaMD) dual-boost simulation,43 writing the ACS Paragon Plus Environment

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7 atomic positions every 1000 steps. GaMD represents a biomolecular enhanced sampling method that adds a harmonic boost potential to smoothen the system potential energy surface. By constructing a boost potential that follows Gaussian distribution, accurate reweighting of the GaMD simulations is achieved using cumulant expansion to the second order (Miao et al., 2015). GaMD simulations were performed using the default values described in the method on-line manual (gamd.ucsd.edu/manual.html), since changing the upper and lower limits of the standard deviation of the total potential boost did not produce any significant effect on the conformational sampling of these nanoswitches. All the simulations have been carried out using a NVIDIA TESLA K40C and a TITAN XP GPUs.

Trajectory analysis Root-mean-square deviations (RMSDs), hydrogen bonds percentages, distance and Principal Component Analysis (PCA) have been carried out over the entire 500 ns trajectories using the GROMACS 2016.1 analysis tools.44,45 The hydrogen bond number was evaluated, through the g_hbond module, using an angle cut-off (hydrogen-donoracceptor) of 180  30° and a maximum donor-acceptor distance of 3.5 Å. The gmx cluster module of GROMACS, has been used to perform a clustering analysis using the gromos46 algorithm on all the saved configurations. The reweight of GaMD trajectories, to recover the canonical ensemble and the original free energy profile of the simulated structures, has been executed using PyReweighting,47 a toolkit of python scripts to facilitate the GaMD



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8 simulation reweighting. The DNA geometrical parameters were calculated using the program CURVES+.48


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9 Results and discussion The atomistic behaviour of the triple helices has been investigated at pH 5.0 and 8.0 to understand the role of the secondary loop length in the modulation of the conformational switch.

RMSD analysis Figure 2A and B shows the heavy atoms RMSD values calculated as a function of time at the two pH conditions for the triple and the double helix regions, respectively. The first frame, extracted from each GaMD trajectory, has been used as a reference for the calculation. The triplex RMSD values at pH 5.0 are constant for all the structures, indicating a full stability of the triple helix (Fig. 2A, black lines). At pH 8.0 the values undergo a quite large deviation in the 7T and 9T structures, indicating a destabilization of the triple helix (Fig. 2A, red lines), not detectable in the 5T structure. A corresponding analysis, carried out on the double helix region (Fig. 2B), shows at pH 5.0 a constant value for all the structure. At pH 8.0 the RMSD values of the 5T and 7T structures do not show any significant variations and a quite large deviation, indicative of a disarrangement of this region, is observed only for the 9T structure (Fig. 2B).

Hydrogen bond analysis Hydrogen bonds (HBs) analysis has been carried out for each structure to estimate the Hoogsteen HBs, between the TFO and the duplex region, and the Watson-Crick HBs within the double helix. Table 1 shows the percentage of the HBs number, initially present in the duplex (15 HBs) and in the TFO-duplex (12 HBs), analysed in five 100 ns time ACS Paragon Plus Environment


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10 windows, at pH 5.0 and 8.0, for the three structures. The distances between the centers of mass of the bases involved in the HBs interactions have been reported in Figure S1, as a signature of the HBs displacement. In the 5T switch, at both pHs, the percentage of Watson-Crick and Hoogsteen HBs is quite low (Table 1) suggesting the occurrence of a deformed triplex, although the constant distance between the Hoogsteen and Watson-Crick bases (Fig. S1, 5T) indicates the presence of a compact structure. Inspection of the three-dimensional structure observed in the MD trajectories shows the presence of a distorted triplex at both pHs, which is maintained for all the simulation time (Fig. S2). This is due to the short secondary loop that, due to chemical constraints, cannot permit the correct formation of both the duplex and triplex, thus invalidating the pH dependent switching mechanism. In the 7T structure the percentage of the Watson-Crick HBs is preserved at both pH conditions while that of the Hoogsteen HBs strongly decreases at pH 8.0 (Table 1), indicating the maintenance of a stable double helix at both pH and a destabilization of the third strand at pH 8.0. Distance analysis validates these results showing a constant short distance for the double helix portion at both pHs (Fig. S1, 7T, right panel), while at pH 8.0 the Hoogsteen pairs distances are much longer than those at pH 5.0 (Fig. S1, 7T, left panel). The 9T structure shows at pH 5.0 a high percentage of Watson-Crick HBs that is strongly reduced at pH 8.0. At the same time the percentage of the Hoogsteen HBs is maintained at pH 5.0 and is almost completely lost increasing the pH to 8.0 (Table 1). Distance analysis of the 9T switch shows a stable constant distance of the triplex region at pH 5.0 and a large increase at pH 8.0 (Fig. S1, 9T, left panel), whilst in the duplex, almost ACS Paragon Plus Environment

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11 all the pairs show a similar distance at both pH values (Fig. S1, 9T, right panel). The apparent conflicting results at pH 8.0 between the decrease of the Watson-Crick HBs percentage and the short distance observed between the bases are explained analysing the corresponding three-dimensional structures. The bases are close to each other but they are not correctly oriented for the formation of hydrogen bonds (see Fig. S3, as an example), indicating that in the 9T structure the switching mechanism occurring at pH 8.0 involves the breaking of both the duplex and the TFO regions.

Free energy projection of the principal components of the motion The different conformational behaviour generated by the secondary loop length, representing the only variable portion of the switch, has been investigated through free energy plots reporting the three-dimensional structures corresponding to the lowest energy basins (Fig. 3). The plots of the 5T structure show a relatively low number of sampled conformations at both pH values, indicative of the presence of a limited number of stable conformations in both conditions (Fig. 3, 5T). Moderate energy barriers values characterize these energy landscapes, each with two shallow energy basins. All the extracted low-energy conformations indicate a stable, compact, although deformed triple-helix conformation close to the starting structure and a flexible primary loop that permits the structure to explore several conformations. The free energy landscape of the 7T structure at pH 5.0 (Fig. 3, 7T), shows a restricted number of sampled conformations characterized by moderate energy barriers values with a single low energy basin, indicative of the presence of a single stable well-formed tripleACS Paragon Plus Environment


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12 helix. The corresponding plot at pH 8.0 reports three low energy basins, separated by low energetic barriers. Conformations 1 and 2 are characterized by a fully formed duplex with a detached triple forming strand. Conformation 3 shows a structure characterized by a hook-like fold of the TFO at the 5' end. Free energy plots of the 9T structure well depict its pH dependent switching mechanism (Fig. 3, 9T). At pH 5.0 the only extracted conformation is characterized by a stable triplehelix, as observed for the 7T switch. At pH 8.0 the conformations corresponding to the two lowest energy basins represent two different steps of the switching mechanism. Conformation 1 shows the triple-helix opening, with the TFO 5' end starting its detachment from the duplex portion, conformation 2 is characterized by a strongly altered structure corresponding to a complete unfolding of the switch, where also the double helix looses its structure. Inspection of the conformations belonging to the two basins revealed the presence of transient hydrogen bonds established between different partners during the simulation time. These HBs involve the 9T loop and the triple helix bases and may accelerate the unfolding process.

Double helix geometrical analysis All the geometrical parameters distinctive of the standard B-DNA have been monitored and averaged along the trajectories to accurately analyze the geometrical deformations of the 6 base pairs forming the DNA double helix in the switches. The average values with their standard deviations are shown in Table S1A and B of Supporting Information, in comparison with the standard B-DNA geometrical parameters. In the 5T structure the average parameters indicate an evident deviation from the standard double helix at both ACS Paragon Plus Environment

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13 pH values, due to a destabilizing effect induced by a short secondary loop, which compromises the correct formation of double helix and accordingly abolishes the pH dependent switching mechanism. In the 7T structure, the geometrical parameters are close to those of the standard B-DNA double helix, indicating a regular geometry maintained over all the simulation time at both pH values. In the 9T structure at pH 5.0 the geometrical parameters are compliant with those of a standard B-DNA helix, while at pH 8.0 they show a large deviation confirming the breaking of both the double and triple region. These data confirm that the 5T secondary loop is too short and does not allow the formation of a correct double and triple helix structure at both pH values and so the switch does not undergo a pH dependent conformational change. The presence of a 7T loop secondary loop permits the occurrence of a stable triple helix at pH 5.0 and the occurrence of a pH dependent conformational change that destabilizes the third strand but does not involve the opening of the double helix portion. Finally, the 9T secondary loop permits at pH 5.0 the formation of a stable triple helix that undergoes a large conformational change at pH 8.0 involving a destabilization of both the triple and double regions of the triplex.



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14 Conclusions In this work we have analysed the importance of the length of the secondary loop integrated on an identical triple helix framework having a primary loop of 25 bases. An atomistic investigation, carried out through GaMD simulations has permitted to analyse the structural effects of the secondary loop on the stability of the triple helix portion in modulating the pH dependent switching mechanism. In detail, our simulations identify the minimal length of the secondary loop required to induce a switching mechanism at pH 8.0. The secondary loop composed of 5 thymidines is not long enough to guarantee the correct formation of a triple helix structure which, being largely altered, does not undergo a pH dependent switching mechanism. The secondary loop composed of 7 thymidines is sufficiently long to induce, at pH 8.0, a destabilizing effect of the Hoogsteen interactions and generates the detachment of the TFO, maintaining the stability of the double helix portion. The secondary loop with 9 thymidines is long enough to generate a pH dependent conformational change involving the unfolding of the triple and double helix structures. This systematic analysis demonstrates the importance of the secondary loop length in modulating the stability of the triple helix. Interestingly, the switching mechanism of 9T structure resembles that observed for the triple helix clamp switch, composed of two independent oligonucleotides, analyzed in our previous work.31 Our analysis suggests that 9 bases represent the optimal cut-off length permitting the transition from folded to unfolded state of the nanoswitch. This work demonstrates the importance of MD simulations in defining the optimal sequences to build a DNA nanoswitch, avoiding timeconsuming trial and error experimental approach. Indeed, the addition or the removal of ACS Paragon Plus Environment

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15 two bases in the secondary loop largely alters the switching mechanism of the nanodevice, indicating that a simulative screening permits a fine selection of the dynamical, stimuli responsive, DNA nanodevices. As an example, engineering a nanoswitch able to bind and release triple helix specific ligands, such as fisetin,49 would require only a reversible triplex-to-duplex transition mechanism, as ensured by a triple helix with a secondary loop composed of seven bases as predicted by simulation. DNA-based nanodevices are usually designed using software like mfold,50 UNAfold51 or NUPACK,52 which allow to predict the thermodynamics of Watson-Crick interactions. However, these tools are unable to predict the behaviour of DNA nanodevices where noncanonical interactions or sequence-independent effects are involved. Applying simulation approaches before carrying out experiments can circumvent these limitations.

Associated content Plot indicating averages and standard deviations of the distances between the centers of mass of the bases involved in Hoogsteen and Watson-Crick interactions (Fig. S1). Ribbon representation of two average conformations obtained for the 5T switch at pH 5.0 and 8.0 (Fig. S2). Ribbon representation of a typical 9T DNA conformation observed at pH 8.0 (Fig. S3). Average values and standard deviations of the DNA geometrical parameters of the 6 base pairs forming the DNA double helix in the switches (Table S1A and B). This information is available free of charge via the Internet at http://pubs.acs.org

Author information ACS Paragon Plus Environment


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16 Corresponding Author e-mail: [email protected]; phone +39.06.72594025; fax.+39.06.2022798 (M.F.).

Author Contributions: M.F. and A.D. conceived and designed the paper; F.I. and K.C.H. performed the calculations and analysed the data; the manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡F.I. and K.C.H. contributed equally.

Notes: The authors declare no competing financial interest.

Acknowledgments: The authors thank NVIDIA Corporation for the donation of a TESLA K40C and of a TITAN XP to M.F. and A.D., respectively, on which the simulations have been carried out. This work was partly supported by the Progetto Ateneo FunDNA to A.D.


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17 Table 1 Time window

5T (HBs residual %)

7T (HBs residual %)

9T (HBs residual %)

Watson-Crick HBs

pH 5.0

pH 8.0

pH 5.0

pH 8.0

pH 5.0

pH 8.0

0-100 ns

62

30

81

80%

75

30%

100-200 ns

69

37

86

75

74

31

200-300 ns

64

37

85

83

86

25

300-400 ns

55

38

86

84

86

28

400-500 ns

48

38

85

84

86

25

Hoogsteen HBs

pH 5.0

pH 8.0

pH 5.0

pH 8.0

pH 5.0

pH 8.0

0-100 ns

52

39

34

23

65

18

100-200 ns

56

58

57

17

65

5

200-300 ns

54

48

56

18

66

3

300-400 ns

44

56

56

17

66

7

400-500 ns

38

40

53

5

70

6

Table 1: Hydrogen bonds percentages for the 5T, 7T and 9T structures, observed in five 100 ns time windows at pH 5.0 and 8.0, within the double helix (Watson-Crick HBs) and the double helix and the TFO (Hoogsteen HBs).

Figure legends

Figure 1: Schematic (A) and cartoon (B) and sequence (C) representations of the simulated DNA structures. In A the red and the pink lines indicate the two strands interacting through the W-C hydrogen bonds, the continuous black line a constant 25 base loop, named primary loop, the green line the TFO, establishing at pH 5.0 Hoogsteen hydrogen bonds with the double helix. The black dashed line represents the 5, 7 and 9T loop, named secondary loop. In B is used the same colour code, with the constant primary ACS Paragon Plus Environment


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18 loop in the upper part of the structure and the variable secondary loop in the lower part of the structure. In C the sequences of the three DNA nanoswitch are depicted using the same colour code.

Figure 2: Time-dependent evolution of RMSD calculated at pH 5.0 (black line) and at pH 8.0 (red line), for the 5T (upper box), 7T (middle box) and 9T (lower box) DNA structures. (A) RMSD analysis of the triple helix region. (B) RMSD analysis of the double helix region.

Figure 3: Free energy principal component projection of the three systems simulated at pH 5.0 (left plot) and at pH 8.0 (right plot). The numbered boxes identify representative threedimensional conformations corresponding to the lowest energy basins.


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Probing the Functional Topology of a pH-Dependent Triple Helix DNA Nanoswitch Family through Gaussian Accelerated MD Simulation

Federico Iacovelli1‡, Kevin Cabungcal Hernandez1‡, Alessandro Desideri1 and Mattia Falconi1*

1Department

of Biology, Interuniversity Consortium, National Institute Biostructure and

Biosystem (INBB), University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133, Rome, Italy.


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Figure 1: Schematic (A) and cartoon (B) and sequence (C) representations of the simulated DNA structures. In A the red and the pink lines indicate the two strands interacting through the W-C hydrogen bonds, the continuous black line a constant 25 base loop, named primary loop, the green line the TFO, establishing at pH 5.0 Hoogsteen hydrogen bonds with the double helix. The black dashed line represents the 5, 7 and 9T loop, named secondary loop. In B is used the same colour code, with the constant primary loop in the upper part of the structure and the variable secondary loop in the lower part of the structure. In C the sequences of the three DNA nanoswitch are depicted using the same colour code. 508x1057mm (72 x 72 DPI)



Figure 2: Time-dependent evolution of RMSD calculated at pH 5.0 (black line) and at pH 8.0 (red line), for the 5T (upper box), 7T (middle box) and 9T (lower box) DNA structures. (A) RMSD analysis of the triple helix region. (B) RMSD analysis of the double helix region. 189x221mm (300 x 300 DPI)


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Figure 3: Free energy principal component projection of the three systems simulated at pH 5.0 (left plot) and at pH 8.0 (right plot). The numbered boxes identify representative three-dimensional conformations corresponding to the lowest energy basins. 60x126mm (300 x 300 DPI)



FOR TABLE OF CONTENTS USE ONLY 82x45mm (300 x 300 DPI)


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Probing the Functional Topology of a pH-Dependent Triple Helix DNA

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