This binding changes the charge, weight, conformation, and flexibility of the DNA molecule. Since DNA molecules are sized by their relative movement through a gel compared to a molecular weight standard, mobility measurements can be critical to size determinations.
After running two identical gels, one without EtBr and one with 0. The cartoon below shows the distortion of the sugar-phosphate backbone when an intercalating agent bind and it also shows that the DNA is lengthened when intercalating agents bind. This changes the properties of DNA considerably. The intercalating agent doesn't bind to closed circular molecules because they can't be lengthened enough to allow insertion of the chemical between the bases.
The structure shown above right is from Reha et al. Cell Reha, D. Nature of stacking interactions between intercalators ethidium, daunomycin, ellipticine, and 4',6-diaminidephenylindole and DNA base pairs. Ab initio quantum chemical, density functional theory, and empirical potential study. Labels: Biochemistry. Anonymous Friday, July 20, PM.
Anonymous Monday, January 12, PM. Anonymous Wednesday, February 17, AM. Unknown Thursday, February 18, AM. Unknown Friday, December 17, AM. Anonymous Tuesday, May 08, AM. In this work, we present extensive MD simulations between ethidium bromide Figure 1 with double stranded DNA in order to detect the intercalation or minor groove binding events without any bias or restraint. Sodium counter ions to neutralize the charge were added and four bromine ions to represent the ethidium bromide salt.
This sequence has been used previously to study the convergence and reproducibility in extended AMBER molecular dynamics simulations 51 , The same minimization, equilibration and production protocols described previously were used. Hydrogen mass repartitioning on the solute atoms allowed the use of a 4 fs time step for the production simulations Biased simulations using the umbrella sampling methodology were additionally performed with the pmemd.
MPI program using distance between the center of mass of the selected base-pairs and the center of mass of the ethidium ligand as the reaction coordinate. In total, windows were computed with 0. A potential of mean force curve was calculated using the weighed histogram analysis methodology 72 as implemented in the WHAM code A. Grossfield, Red is adenine, purple is cytosine, green is guanine and cyan is thymine with the termini gray.
Quantum mechanics calculations and ethidium molecule set-up was performed by the D01 version of Gaussian 16 C. Overall, the DNA duplex remains stable in the B-DNA conformation for the entire length of the sampled trajectories for all of the models.
Fraying events at the termini base-pairs are commonly observed 51 , 52 and the central base pairs show expected DNA breathing dynamics for a Watson—Crick duplex. As depicted in Figure 2 , the simulations started with the DNA duplex surrounded by four EtBr molecules the solvent and ions are not shown.
To study the overall distribution density of the EtBr molecules among the DNA duplex, we employed the curvilinear helicoidal coordinates approach centered on the helical axis of the DNA. Briefly, the positions of the ethidium ligands are determined with respect to the helical axis, which allows us to: i measure the radial distribution of the ligands cylindrical distribution function or angular A plot , ii measure the distribution along the helical axis distance D plot and iii measure the distance from the helical axis towards the bulk of the solvent radial distance R plot, refer to Figure 1 of the original Canion publication This method allows the calculation of time-averaged populations among the DNA duplexes for each of the four EtBr ligands, and this provides insight into the interaction of these two molecules over the extensive amount of sampling data provided by the MD simulations.
The angular A plot top row, Figure 3 depicts the ethidium ligand accumulation within the minor groove region to an extent, and accumulation that explores other values that do not clearly correspond within the minor groove with no discernable trend. When we consider the distance D plot center plot, top row Figure 3 , two regions that show strong molarity peaks at both the terminal base pairs of the DNA duplex with close to zero concentration of the ligands within the central base pair region.
This is only possible if the ligand is either in intercalation between the bases or stacked at the ends of the DNA duplex. Similar behavior is observed for ethidium ligands two to four. Top row: 1D A, D and R plots of the ethidium ligands. The dashed line in the R plot represents the radial position of the phosphorous atoms; values are based on the Canion publication Each line of the top plots represents the analysis performed in each of the four ethidium ligands.
The full trajectory information with an offset of 10 frames was used for the analysis. Middle row: 2D representations for the curvilinear helicoidal information based on the ethidium ligand 1 only.
Bottom row, left: selected representative interaction extracted from one of the simulations. Further refinement of the analysis can be done by 2D representations of the curvilinear helicoidal information middle row, Figure 3. Combining the angular A with the distance D plots DA we can confirm that the ethidium ligand 1 occupies primarily both ends of the DNA duplex with little to no presence in the inner base-pairs.
The RA and DR plots shows the ethidium ligand 1 positioned mainly at the center of the helical axis, consistent with the ligand binding in either intercalation or stacking interactions at the DNA termini. Visual examination of the trajectories confirms the long-lived presence of the ethidium ligands in two main configurations: stacked on the terminal base-pairs or intercalated at the terminal base-pair step. As mentioned, the ethidium ligand stacks in the terminal base-pair and rotates freely with the DNA helical center as the center point.
This rotational variation is confirmed by the A plot Figure 3 , top left which represents the angular position with respect to the helical axis. The intercalation process that occurs on both sides of the terminal base-pairs is depicted with molecular graphics at the bottom of Figure 3. The frayed nucleotides interact with solvent and ions step 4 until it reforms the WC pairing trapping the ethidium ligand in an intercalated position step 5.
Binding energy for the representative interaction of the ethidium stacked in the terminal base-pairs corresponds to a value of — Both values were extracted from an average of the MM-GBSA calculations on frames, noting that this method tends to over-estimate the binding affinity somewhat and is typically better for relative free energy comparisons.
Although ethidium can in principle bind to sites every 4—5 base pairs at saturation, the simulations do not show intercalation at the central AT step. This could be due to the kinetics and enhanced thermal stability or simply this was not observed because the simulations were not long enough. However, as the intercalation at the ends of the helix occurs more rapidly than with internal base pairs, and these interactions stabilize and rigidify the helix, this could in principle inhibit intercalation at the central steps.
Although our speculation is that the end base pair step inhibits internal base pair intercalation, it could possibly also relate to sequence in the AATT A-track which tends towards a narrower minor groove. Even though we used an older version of the force field for the DDD example bsc0 , we do not expect to observe any significant differences with regards to the interactions between the ethidium molecule and the DNA due to the update in the force field.
Given the extensive amount of data generated, as a first approach, a grid-based atomic density population histogram for each of the four ethidium ligands was computed and visualized Figure 4. This enables a quick visual assessment of the average interactions of the ethidium molecules within the GAAC sequence. As observed with the DDD, there is an accumulation of the ligand at both ends of the duplex, although in this case, it is clear that additional binding modes appear.
Accumulation data of the ethidium ligands is observed throughout the minor groove and intercalation events both in the base-pairs at the end of the GAAC and in the central regions are observed. Remembering that we have a simulation with four free binders, ethidium ligand 2, for example, shows a clear intercalation event in the central base-pairs of the GAAC duplex and ligands 1, 3 and 4 present a similar intercalation density close to the terminal base-pairs, noting that since the shown reference DNA is an average structure without ethidium present, the DNA shown does not have the characteristic DNA deformations seen in previous studies of ethidium interaction with duplexes Atomic density population histograms for each of the ethidium ligands taking into account every 10th frame from all of the trajectory information using the 20 independent copies.
The curvilinear helicoidal coordinate analysis provides further detail on the coarse image rendered by the density histograms. The angular A plot top row, Figure 5 shows elevated concentration of the ethidium ligands within the minor groove for all four molecules. These two modes of interaction are observed also in the umbrella sampling simulations see next section, Figure 7. In contrast with the same plot from the DDD sequence simulations, which show little to no concentration within the central inner base-pairs, the GAAC simulations shows multiple contact points that translates to the ethidium ligands exploring the minor groove.
We know that the ligands are within the minor grove since the A plot shows the majority of the molarity within that region. The dashed line in the R plot represents the radial position of the phosphorous atoms. Bottom row: 2D representations for the ethidium ligand 2 set of trajectories red line on the 1D plots. Molarity increases from blue to yellow.
The 2D plots complement the 1D data bottom row, Figure 5. In this case, the data is from the ethidium ligand 2 that presents the sharp intercalation event in the C8pG9 region as can be seen from the DA and DR plots. High accumulation within the minor groove is visible from the data presented in the DA and RA plots and to some extent the interaction with the terminal base-pairs.
All the trajectories were visually inspected to extract the most representative binding modes found between the ethidium molecule and the GAAC sequence. As expected with the amount of sampling calculated in this work, the observed binding modes can be broadly classified in five states: stacking on the terminal base pair, minor groove binding, intercalation through base-pair eversion, intercalation from the minor groove and intercalation from the major groove.
A representative structure of each binding mode is depicted in Figure 5. After stacking interactions at both ends of the GAAC sequence, the most commonly observed binding mode corresponds to the phenanthridine moiety of the ethidium ligand in direct contact within the minor groove of the DNA, with the 6-phenyl ring towards the solvent.
In this binding mode, the ligand freely explores the length of the minor groove interacting with the ApA regions as observed in the 2D RA plot Figure 5 , notice the three regions of high molarity that correspond to the three GAAC repeats and the population density plot. The base-pair eversion mechanism has been reported earlier in related works 80— Using the dynamic breathing motions of DNA, the ethidium molecule pushes both adenine and thymine nucleobases towards the major groove until the nucleotides flip open towards the major groove.
The ethidium proceeds to move into the resulting cavity, forming stacking interactions with both base-pairs within the cavity. Minor groove intercalation Figure 6c was found in three of the 20 sampled copies; two were found to be in an ApA base step and one in the ApC step. Intercalation always started within the minor groove with the phenanthridine side of the ethidium towards the floor of the minor groove which starts the eversion mechanism and it was an irreversible event whereas if the ethidium only produces base-pair eversion, we found that this event was reversible in all 20 independent copies.
Selected frames of the above process showing structural details are depicted in Supplementary Figure S1 and the included molecular graphics movie file in the supporting information.
Intercalation of ethidium shifted the helical twist of the GAAC sequence to a value of In comparison, the twist value of a GAAC simulation with no ethidium molecule has an average of The last binding mode found was indeed unexpected and was observed only in one case.
The ethidium molecule moved throughout the solvent towards the major groove until it reached a CpG step at the middle of the GAAC sequence. The phenanthridine side interacts with N4 of the dC28 then moves toward N4 of the dC8 on the complementary strand which produces an increase in helical bend and roll of the base-pairs. This space allows the phenanthridine to slide within the base-pairs, which causes that particular base-step to unwind Figure 6D.
Top panel: selected representative binding modes of the ethidium ligand with the GAAC sequence. A Minor groove binding, B intercalation interaction between the ApC step and a base-pair eversion insertion mode in the A6:T31 step, C stacking on the terminal base-pairs and insertion mode on the A3:T34 position and D major groove intercalation between the ApA step and minor groove binding.
Ethidium molecule is colored green for clarity. We have presented unbiased simulations of two different double-stranded DNA sequences and their interaction with the ethidium molecule. Both tested systems included four of the ethidium molecules. In addition, all the sampled trajectories show the ethidium exploring the minor groove, which lead to either intercalation or base-pair eversion.
This latter binding mode was observed to start only when an ethidium molecule is within the minor groove, whereas the intercalation interaction was observed to start both from the major and the minor groove. In order to study the energetic preference of this process, we conducted a series of biased simulations using the umbrella sampling approach. The reaction coordinate used consisted in a distance restraint between the center of mass of the base pairs before and after a manually intercalated ethidium molecule and the mentioned ethidium.
One set of simulations started with the ethidium from the major groove and another from the minor groove. Both ApA and GpG steps were employed in both situations to provide some insight regardless of sequence preference.
Free energy profiles for the umbrella sampling experiments show a preference of the ethidium molecule to bind within ApA steps — Binding values for ethidium intercalating from the major groove correspond to —4. The global minima represent the ethidium molecule interacting with the minor groove of the DNA. In both cases, the 5-ethylphenyl substituents of the phenanthridinium form a robust network of hydrogen bonds that increase the stabilization of the ligand when intercalating within the minor groove.
When in the intercalation pocket, the phenyl ring interacts with O5 from thymine and O2 from cytosine. When in the minor groove, the ethidium molecule forms extensive hydrogen bonds with the atoms present in the floor of the minor groove and with the back-bone of the DNA Top row: Free energy profiles from the umbrella sampling simulations using a distance restraint as the reaction coordinate between the center of mass of the DNA either an ApA or a GpG step and the center of mass of an ethidium molecule.
The profile is the average of five independent runs per window, standard deviation is represented by the error bars. Major and minor groove represents at what side the intercalation was started. Ethidium molecules are depicted in green for clarity. Ethidium-DNA interaction through the major groove using every 10th frame from all three independent copies and quasi-harmonic entropy approximation show a binding free energy of — Interaction through the minor groove show binding energy values of — The curvilinear helicoidal coordinates analysis for these four systems is depicted in Supplementary Table S1 , which confirms the location of the ethidium ligand within the major and minor grooves for the duration of the simulations.
The binding energies from the unbiased simulations with the ethidium manually intercalated into DNA can be compared with the umbrella sampling simulations that show the minor groove interaction being more stable than intercalation through the major groove. One possible explanation to the stability of the DNA—ethidium complex when the ligand is in the minor groove is an increased number of hydrogen-bonds between ethidium and the backbone We tested this hypothesis with a hydrogen-bond population analysis see Supplementary Table S1 in the Supporting Information between the DNA and the ethidium ligand using the exact same number of frames for each system corresponding to 1.
As can be seen from the data, the mentioned hydrogen-bond is the most populated interaction for all the cases, regardless of the intercalation side. This is due to the position of the amino groups forming the H-bond donors, positioned on each side in a linear manner across the phenanthridinium rings.
These allows a close contact with the oxygen from the deoxyribose from both sides of the DNA duplex. It has been reported that the main stabilization interaction between intercalated ethidium and the DNA nucleobases is provided by aromatic stacking interactions, mainly through hydrophobic interactions and dispersion energy 85— The short simulations presented by Monaco and collaborators support the idea of ethidium binding to any GXXG site, forming hydrogen bonds with guanine's O6 and N7 centers 40 , Even though our data with the GAAC sequence do detect the formation of these hydrogen bonds and support the idea of the ethidium molecule exploring the major groove of the DNA, any binding event observed was short lived, with no discernable sequence preference, as seen, for example, when the ethidium ligand is bound within the minor groove high density population close to the ApA steps as observed in Figure 4.
The simulations presented in this work allowed us to observe naturally occurring intercalation events without any bias or restraint. The minor groove binding showed a two-step mechanistic pathway of interaction which involves the base-pair eversion mechanism as a meta-stable step with the reforming of the Watson—Crick pairing to complete the intercalation whereas our single major groove binding event was driven by the present breathing and dynamics of the DNA duplex.
The ethidium molecule co-exists in both an intercalative binding mode and an electrostatic binding mode within the minor groove. This work also stresses the relevance of extended sampling time in molecular dynamics simulations and the use of multiple replicas to increase statistical significance, especially for biomolecular processes.
0コメント