Salt Induced Structural Collapse, Swelling, and Signature of

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Salt Induced Structural Collapse, Swelling and Signature of Aggregation of Two ssDNA Strands: Insights from Molecular Dynamics Simulation Soham Sarkar, Atanu Maity, Aditya Sarma Phukon, Soumadwip Ghosh, and Rajarshi Chakrabarti J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b09098 • Publication Date (Web): 12 Dec 2018 Downloaded from http://pubs.acs.org on December 12, 2018

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The Journal of Physical Chemistry

Salt Induced Structural Collapse, Swelling and Signature of Aggregation of Two ssDNA Strands: Insights from Molecular Dynamics Simulation Soham Sarkara, Atanu Maitya, Aditya Sarma Phukona, Soumadwip Ghoshb and Rajarshi Chakrabarti*a *Email:

a Department b Beckman

[email protected], Phone: +91-22-25767192

of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai – 400076, India,

Research Institute, Department of Molecular Imaging and Therapy, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, CA-91010, USA

ABSTRACT: Molecular dynamics simulations elucidate the structural collapse shown by two ssDNAs of same base sequence in the presence of either Na+ or Mg2+, starting from in-vivo ionic concentration to higher concentrations. Initially, an increase in ion concentration facilitates the structural distortion of individual ssDNA and helps to bring them close and for this, Mg2+ is better than Na+. However further addition of ions leads to structural re-swelling of the DNA strands and inhibit their proximity. The structural changes are found to be guided by the strong interaction of the cations with the phosphinyloxygen (pn_O). Additionally, a significant difference has been noticed in the interaction of the cations with phosphoester oxygen (pe_O) depending on the nature of the ion. The sequential and nonsequential base-pair stacking is one of the major factors in the structural collapse of individual ssDNA. Overall, the present investigation highlights some of the important aspects of aggregation of two ssDNA with the same base sequence at varying cationic concentration.

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1. Introduction: During transmission of genetic code, nucleic acids undergo structural changes. Dynamic processes related to biological functions (e.g. transcription for DNA and translation for RNA) involve noncoding regions that confer flexibility to the overall structure. To understand how the interaction between nucleic acids is related to their biologically relevant structural adaption, it is important to understand the structural dynamics of these molecules. It is also important to note that cellular processes are also controlled by several physiological conditions. For example, a change in the pH of the soil leads to a structural change in 16S rRNA and amoA genes of both ammonia-oxidizing bacteria and archaea, with distinct populations in acid and neutral soils1. Similarly, results from Hippel et al. indicate a correlation between cation and anion concentration with that of the melting temperature of RNA 2. Smiatek and co-workers incorporated metadynamics to investigate the binding of urea to a seven nucleotide small single-stranded DNA and inferred that the combination of electrostatic and dispersion interactions aids in the accumulation of urea around the DNA, achieved by the replacement of the surrounding water molecules 3. Pati and co-workers showed the calculated optical conductivity about the helical DNA axis and found the origin of the magnetic characteristics while magnetic ions like Cu2+ and Mn2+ are doped in the gap region of the DNA molecule 4. Satpati and group recently looked for the binding of Retinoic acid-inducible gene-I (RIG-I) with dsRNA by means of molecular dynamics simulations 5. So, it is expected that the alteration in physiological conditions effectively influences the structures of nucleic acid leading to change in some biological processes. One way to look into this is to model the physiological condition in-vivo or in-vitro or insilico and to check the structural behavior of the nucleic acids subjected to changes in the environment. Cellular ionic concentration is one of the important factors that have an impact on the structure of DNA/RNA. This is mimicked by introducing ions of different valency under different condition. Here, we briefly mention a few examples of experimental and computational studies on the effect of salt and other factors on the structural property of nucleic acids. Both single-stranded and double-stranded DNAs have been investigated to understand the structural changes they undergo due to a change in environment. The effects of charge and size of ions on structure of the nucleic acid have been studied. Structural collapse of nucleic acid and their condensation have been studied significantly in the past two decades. The word ‘collapse’ usually indicates the structural shrinking of a single, long DNA chain. The collapse plays a pivotal role in the storage and packing of DNA in viral capsids 6. The double-stranded DNA is naturally stable and held together by the interaction between complementary base pairs of the two strands. In single-stranded nucleic acids, due to the lack of hydrogen bonding between complementary base pairs, the strands adopt a range of conformations. Presence of metal ions is expected to influence the conformational dynamics of RNA/DNA since they carry negative charges in their backbone. The ions include simple monovalent ones such as Na+ or multivalent ones like Mg2+ 7, 8 or different types of polyamines like


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spermine (spm4+), spermidine (spd3+)

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etc. This is an interesting phenomenon and several

experimental and in-silico approaches have been employed to understand the effect of ions on the structural dynamics of nucleic acids. Spectroscopic techniques have been used to measure the conformational flexibility and structural change of nucleic acids. For example, the flexibility of designed oligodeoxythymidylates ((dT) N, where N varies from 10 to 70) has been measured over a wide range of salt concentrations using fluorescence spectroscopy. This has shown that the persistent length of the ssDNA strand varies with the concentration of NaCl and it increases with the increase in ion concentration

12.

Using fluorescence anisotropy, Sinha et al. showed that the conformational

heterogeneity of the micro RNA increases with an increased salt concentration 13. Chattoraj et al. used electron microscopy and found that the degree of collapse of T7 DNA in the presence of spermidine is dependent on the addition of Na+ ion in the medium

11.

The effect of ion concentration on the

extension of ss-DNA and B form of ds-DNA was studied using a force-measuring laser tweezer 14, 15. One of the most fascinating phenomena that nucleic acids exhibit is their condensation in the presence of multivalent ions. Bloomfield investigated DNA condensation by using differential scanning calorimetry, optical densitometry cobalt (III)

18.

16

and laser Raman spectroscopy

17

in the presence of hexamine

To get an understanding at the microscopic level, mainly two robust theoretical

approaches have been used. In one approach, atomistic details are ignored, instead nucleic acids are modeled as polyelectrolyte chain, and a statistical mechanical model was built to account for the effect of monovalent and divalent cations. This allowed getting an empirical relation between the salt concentrations and the structural folding of nucleic acids

19-21.

The investigation by Post et al.

provides an argument for three different stable DNA conformations under various solvent conditions and explains the solvent effects on the structural collapse and aggregation and subsequent precipitation of DNA using Flory‐Huggins theory

22.

In another approach, molecular dynamics

simulations (atomistic/coarse-grained) were employed to check ion-nucleic acid interactions leading to an alteration of RNA/DNA conformation. Using a coarse-grained model of nucleic acid, Hsiao et al. studied the effect of ions of different size and charge. In the presence of tetravalent ions, the polyelectrolyte monomer shows an extended structure at low and high concentrations of ions whereas at an intermediate salt concentration they form a condensed structure. The behavior was found to be regulated by the overcharging of the nucleic acid at high salt concentrations 23, 24. In a similar kind of study, Wang et al. found that multivalent ions (e.g. Mg2+) are more effective than monovalent ion (Na+) in inducing structural collapse in ssDNA strands

25.

Vemperala and co-workers investigated

polyelectrolyte aggregation owing to like charge attraction by means of MD simulation and numerical analysis

26.

Several other studies used all-atom MD simulation to probe the dynamics of single-

stranded nucleic acid in aqueous solution and also in the presence of ions 27-30. An additional degree of complexity arises when a second DNA is added i.e. when the behavior of one strand gets influenced by the second one. Using small angle x-ray scattering, Qiu, X. et al. showed that with an increasing concentration of Mg2+, the attractive interaction between DNA strands increases

31.

Dialysis in the


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presence of an electric field was used by Musheev et al. to remove the counter ions from ds DNA and this removal of ions resulted in aggregation 32. Saminathan et al. checked the effect of spermine on the aggregation/precipitation of three types of DNA (ssDNA, duplex DNA and triplex DNA) and observed that the ability to provoke precipitation follows the order as triplex > duplex > ss-DNA 33.In the present investigation, using an all-atom molecular dynamics simulation we address how variation of ion concentration (both Na+ and Mg2+) controls the collapse mediated inter-chain aggregation of two ssDNA with the same base sequence, viz 5´-CGCGAATTCGCG-3´ (Dickerson Drew dodecamer). Most of the computational studies focus on dsDNAs 8. To the best of our knowledge, no all-atom simulation-based study exists in the literature that can account for the cation induced collapse mediated initiation of aggregation of ssDNA chains. Our study explores how cations (monovalent and bivalent) facilitate the collapse of the DNA strands onto itself and subsequent initiation of aggregation and in particular how a divalent cation, such as Mg2+ emerges out to be the better glue compared to monovalent Na+.

2. Simulation Details: Molecular dynamics simulations have been performed using GROMACS 4.5.6 CHARMM model

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force field, which is routinely used in other studies on DNA

27, 36

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with all-atom

and TIP3P water

is used to solvate the ssDNA (PDB ID 436D) 38. The required sequence of the bases of the

ssDNA is obtained by extracting the complementary chain A from the double helix and the 5´ and 3´ Phosphate groups are capped. The freely available package 3DNA 39 is used to insert hydrogen atoms into the initial DNA structure. Initially, two ssDNAs of the same base sequence are kept inside a box of volume 203 nm3 at 4 nm Center of mass (COM) separation from each other (Fig S1 of ESI†). We designate these two chains as Chain_A and Chain_B in our study. The net charge of the native system is found to be -22. We explore five concentrations of salts, i.e. 0.18M, 0.3M, 0.5M, 0.8M, 1.0M, where 0.18 M refers to the charge neutralized system. The number of cations, anions, water molecules required for each system is provided in Table S1 of ESI†. Distributions of ions in the systems are governed by the total simulation time (200 ns in the present study) and how they are introduced in the simulation box 40. Using ‘genion’ tool of GROMACS 4.5.6 34, the monovalent and divalent ions are introduced to the system randomly which resembles in-vivo condition, rather than constraining the ions in some specific region in the simulation box. Steepest descent algorithm

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is used to converge

the potential energy of the system and to remove bad contacts and steric clashes. The system is equilibrated in NVT ensemble from 0 K to 310 K for 300 ps using V-rescale

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thermostat to avoid

void formation in the box and further equilibration in isobaric-isothermal ensemble (NPT) for another 5 ns at this temperature to reach the pressure 1 bar using Parrinello-Rahman barostat

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(Table S2 of

ESI†). The configurations of the systems are updated by GROMACS using the leap-frog integrator 44. The production run of 200 ns with 2 fs time step is performed and the trajectory is saved at a frequency of 2 ps. The average temperature of the equilibrated trajectory is found to be 309.993 K, in


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consistency with the physiological temperature 310K (Table S2 of ESI†). The minimum image convention

45

is taken into account to calculate the short-ranged Lennard–Jones interactions. The

spherical cut-off distance of electrostatic as well as van der Waals forces is kept at 1 nm. The SHAKE 46

algorithm is applied to impose constraints on the equilibrium bond distance of the TIP3P water

molecules. The long-ranged electrostatic interactions are calculated using the particle mesh Ewald

47

method. The specific base pairs of interest are tagged as energy groups while starting each MD run. VMD1.9.2

48

is used to render the trajectories and capture snapshots of the ssDNAs. Simulations for

each system are performed twice for estimating statistical uncertainties and testing the convergence of the results obtained. The sampling efficiency is checked by monitoring the root mean square deviation (RMSD)s of the chain backbone from two independent runs. The time evolution of RMSD and its cumulative values for the last 100 ns of the total trajectory are provided in the ESI† for Na+ (Fig S2, Fig S3) and Mg2+ (Fig S4, Fig S5) respectively. This stretch of the trajectory was used to check the other properties from the simulation. Different structural parameters have been calculated using the utilities available in GROMACS and in-house scripts.

3. Results and Discussions: 3.1. Initial Signature of Collapse induced initiation of aggregation of two ssDNAs To monitor the extent of intramolecular and intermolecular interactions within a strand and between the two DNA strands, we choose two distance parameters which can describe the conformational space effectively. These two distance parameters are the COM distance between the two chains (Chain_A and Chain_B) (Fig S6 and Fig S7 of ESI†) and the end-to-end distance of each strand (Fig S8 and Fig S9 of ESI†). We compute the free energy landscape corresponding to the aforesaid distance parameters. First, the probability of the points within (0.1 nm x 0.1 nm) grids is calculated from the COM vs end-to-end distance plots as –

where i and j represent the COM and the end-to-end distance axes respectively in the plot. Free energy was then calculated using the formula

For this calculation, the last 100 ns of the simulated trajectory is considered to minimize fluctuation during the equilibration phase.


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Fig 1: Color maps of COM distance between two ssDNAs (nm) and End-to-end distance of (i) Chain_A and (ii) Chain_B in the presence of (a) 0.18M, (b) 0.3M, (c) 0.5M, (d) 0.8M, (e) 1M NaCl salt solution.

The free energy surface has been presented in Fig 1 and Fig 2 for Na+ and Mg2+ ions respectively. These free energy plots are obtained from the 2nd independent simulation, using the same protocol and are provided in Fig S10 and Fig S11 of ESI†. The darker shade in the contour represents the densely


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populated region in the conformational space whereas the lighter ones represent a region with a lower population. At a low concentration of NaCl, viz. 0.18M, DNA chains prefer to remain stretched owing to the repulsion between phosphate groups along the chain backbone. This is reflected in the contour plot of free energy variation with COM and end-to-end distance of either of the chains. For example, as can be seen from Fig 1a(i) and Fig 1a(ii) (Chain_A and Chain_B respectively), the conformation of Chain_A (Chain_B) with the lowest free energy has an end-to-end distance ~3nm (2.5nm) and the COM distance is ~2nm. Interestingly, there is a difference in the end-to-end distance of the two chains. Chain_A shows a narrower distribution of the end-to-end distance (2.5-3.5 nm) whereas Chain_B samples a wide range of end-to-end distance (0.5-4nm). This describes a situation where one of the chains remains in a partially collapsed state and the other explores varying conformations (in terms of end-to-end distance) (Fig 2a).

Fig 2: Snapshot of interaction between DNA strands in the presence of a) 0.18 M NaCl, b) 1.0 M NaCl, c) 0.18 M MgCl2 and c) 1.0 M MgCl2. The phosphinyl oxygen (pn_O) and the positive ions (Na+ or Mg2+) present at a distance of 5 Å from the phosphinyl oxygen are shown in red and blue sphere respectively.

A similar result was found from an independent trajectory (Fig S4). Now on increasing NaCl concentration of the system to 0.3M, as represented in Fig 1b(i) and Fig 1b(ii), it is found that the lowest free energy conformation corresponds to Chain_A (Chain_B) with an end-to-end distance of ~1nm and the COM separation ~0.75nm. This infers that, with the increase in NaCl concentration


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from 0.18M to 0.3M, the strands come close to each other and also their end-to-end distances decrease. Hence, this concentration regime results in structural collapse owing to the higher degree of negative charge neutralization and initiates aggregation. On further increasing the NaCl concentration to 0.5M, one could see that the individual chain starts swelling. This can be seen from Fig 1c(i) and Fig 1c(ii), as the end-to-end distance of the chains is in the range 1-1.5 nm. In this concentration window, the minimum free energy corresponds roughly to 1nm COM separation between the strands which is greater than that of the COM distance obtained for lowest free energy at 0.3M concentration. At 0.8M concentration of NaCl (Fig 1d(i) and Fig 1d(ii)), the lowest energy corresponds to a situation where Chain_A and Chain_B are at a separation of 2 nm. The end-to-end distance observed for the single strands are found to be in the range of 2-2.5 nm. Further addition of NaCl in the system increases the concentration of Na+ ion to 1(M) (Fig 1e(i) and Fig 1e(ii)). We observe two energy minima while the COM distance between the strands is in the range of 1.5-2.5 nm. The strands are stretched (Fig 2b), as can be seen from the value of end-to-end distance, which is about 4 nm. Similar trends for the two properties with increasing concentration were also observed for an independent trajectory (Fig S8 of ESI†).


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Fig 3: Color maps of COM distance between two ssDNAs (nm) and End-to-end distance of (i) Chain_A and (ii) Chain_B in the presence of (a) 0.18M, (b) 0.3M, (c) 0.5M, (d) 0.8M, (e) 1M MgCl2 salt solution.


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When the concentration of MgCl2 is low, viz. 0.18M, the DNA chains prefer to remain stretched due to repulsion between phosphate groups along the chain backbone. This is evident from the contour plot of free energy. As can be seen from Fig 3a(i) and Fig 3a(ii), the conformation of Chain_B has an end-to-end distance ~2.5 nm whereas Chain_A can have a wide range of end-to-end distance. Thus, in this condition, one of the chains remains in a partially collapsed state whereas the other explores varying conformations (in terms of end-to-end distance) (Fig 2c). A stark structural difference between Chain_A and Chain_B, in terms of end-to-end distance, is observed owing to the presence of the divalent cation. Now on increasing MgCl2 concentration to 0.3M, as represented in Fig 3b(i) and Fig 3b(ii), the lowest possible free energy corresponds to a COM separation of roughly below ~1nm. This infers that with an increase in the concentration of MgCl2 to 0.3M, the strands approach each other. Under this condition, Chain_A acquires an end-to-end distance of roughly ~2 nm, while Chain_B shows an end-to-end distance just below 1nm. This concentration regime is more prone towards structural collapse due to the higher degree of negative charge neutralization and subsequent initiation of aggregation. On further increase in MgCl2 concentration to 0.5M (Fig 3c(i) and Fig 3c(ii)), the strands start swelling and it is evident from the contour plot where the free energy minimum corresponds to COM separation of ~1.5 nm with an end-to-end distance of roughly 1.5 nm for each strand. The COM separation for this case is considerably higher than that observed for free energy minimum at 0.3 M MgCl2. At 0.8M concentration of MgCl2 (Fig 3d(i) and Fig 3d(ii)), the minimum free energy corresponds to a COM separation of ~2nm between Chain_A and Chain_B. We find that the end-to-end distance of Chain_A is in the range 3-3.5 nm but for Chain_B, it is around 3 nm. On further increase in MgCl2 concentration to 1M, represented in Fig 3e(i) and Fig 3e(ii), the free energy is found to be minimum for the strands separated by 2 nm COM distance, with a corresponding end-to-end distance of ~3.5 nm for both the chain. The stretching of the strands can be seen from the snapshot (Fig 2d). Thus, with the initial increase in ion concentration starting from 0.18 (M) to 0.3(M), two ss-DNA approaches each other (the distance between COMs decreases) accompanied by a signature of collapse i.e. the two ends of the DNA started interacting with each other (the end-to-end distance decreases). Whereas with a further increase in concentration, the effect is gradually getting reversed i.e. the two ssDNAs get separated from each other and the end-to-end distance increases. A similar trend has been observed from the second independent run (Fig S9 of ESI†).

3.2. Radial distribution Function: To check how ions exert such impact on ssDNA, the distribution of the metal ions around the ssDNAs was quantified from their radial distribution functions (RDFs). Since the positively charged ions mostly interact with the negatively charged parts of the DNA, the RDFs were computed for the cation and two types of oxygen i) phosphinyl oxygen (oxygen connected to phosphorus) and ii) phosphoester


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oxygen (connecting the phosphorus atom with the sugar unit) (Fig 4a(ii)). These will be referred to as pn_O and pe_O respectively throughout the manuscript. We represent the radial distribution functions between phosphoester oxygen (pe_O) and phosphinyl oxygen (pn_O) with (a) Na+ and (b) Mg2+ in Fig 4 and Fig 5 respectively. Both Chain_A and Chain_B have been considered together while calculating the RDFs. The result from another independent set of simulation has been provided in supporting information (Fig S12 and Fig S13). The radial distribution between the metal ion and different types of oxygen are calculated using the RDF construction tool of VMD 1.9.2 48. It is evident from the plots that in the presence of NaCl and MgCl2, the cations essentially accumulate surrounding the phosphinyl oxygen more than that of the phosphoester oxygen.

Fig 4: Radial distribution functions (RDFs) between Na+ and the (a) (i) phosphoester (pe_O) oxygen and (b) (i) phosphinyl (pn_O). Different regions of the plots are separately shown in (a) (ii), (iii) and (b)(ii), (iii) for further clarification.

Fig 4 represents the variation of g(r) at different concentration of Na+. There is a sharp peak at around 2.5Å for both the pe_O (Fig 4a(i)) and pn_O (Fig 4b(i)) type of oxygen representing the first hydration shell, although the height of the peak differs in each case. Na+ ions are more concentrated at the first hydration shell around the pn_O type of oxygen compared to pe_O type of oxygen. This characteristic difference in RDF around the two different types of oxygen is probably due to the fact that the phosphoester oxygens (pe_O) are buried within the backbone of the nucleic acid, while the phosphinyl oxygens (pn_O) are exposed. Accumulation of large concentration of Na+ around the phosphinyl oxygen was observed earlier by Martinez et al.

49.

Both types of oxygen are associated

with a second hump at 4.5 Å. From the variation of RDF in the first and the second hydration shell at


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different concentrations, it is evident that the g(r) decreases with an increase in ion concentration in almost all cases (Fig 4a(ii) and Fig 4b(ii)). This decrease is compensated by the increase in RDF at the bulk water for a higher concentration of Na+ (Fig 4a(iii) and Fig 4b(iii)). The RDF of Na+ ion for the other independent trajectory also reflects the same observation (Fig S11). The nature of RDF around pe_O is similar for Mg2+ and Na+ with the exception that the peaks have been shifted to higher values of r (Fig 5a(i) and Fig 4a(i)). There is a peak around 2Å around phosphinyl oxygen (Fig 5b(i)) whereas the phosphoester oxygen lacks that peak (Fig 5a(i)). One probable reason for that could be the larger size of magnesium ion that prevents its accommodation near buried pe_O (Fig 5a).

Fig 5: Radial distribution functions (RDFs) between Mg2+ and the (a) (i) phosphoester (pe_O) oxygen and (b) (i) phosphinyl (pn_O). Different regions of the plots are separately shown in (a) (ii), (iii) and (b)(ii), (iii) for further clarification.

The change in distribution with increasing ion concentration is similar to that of Na+ i.e. radial distribution decreases as the ion concentration increases. One explanation of this could be the fact that with an increase in ion concentration the repulsion between the ions of first hydration shell with the ions at a larger distance (e. g. in the second hydration shell) increases. The RDF plots, obtained from a 2nd independent run for both Na+ and Mg2+ are provided in the ESI† (Fig S12 & Fig S13).

4. Intra- and inter-strand interactions:


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In this section, we depict the intra- and inter-strand interaction of the ssDNA chains using contacts maps. The contact map was constructed by using the ‘g_mdmat’ utility of GROMACS considering 1 nm cut-off distance for both inter and intra-strand contacts. In the contact map, red color represents the maximum distance cut-off (i.e. 1nm), whereas the minimum distance between residues within the cut-off is represented by blue color as indicated in the color bar in Fig 6 and Fig 7. The residue index in the axes of the maps represents the number of residues of Chain_A and Chain_B. Residue 1-12 stands for DNA Chain_A (in green box and represented with red backbone holding the green color base) and residue 13-24 stands for DNA Chain_B (in purple box and represented with blue backbone holding the purple color base).

Fig 6: Intra- and inter-strand interactions of the DNA chains at different concentrations of NaCl

Here in Fig 6, we represent intra- and inter-strand contacts of the ssDNA chains for five different concentrations of NaCl, considered for our investigation. It is clearly visible from the contact map that for NaCl at low concentration such as 0.18M, inter-strand contacts are fewer than that of intra-strand contacts, which shows that the strands are apart from each other. While increasing the concentration from 0.18M to 0.30M, single DNA strands (chains) come close to each other and the individual chains are structurally collapsed, which is evident from the contact map that shows an increased number of intra- and inter-strand contacts. On further addition of NaCl that corresponds to concentration 0.5 M,


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an interesting behavior is observed. The contact map depicts that the single strands have started to swell and move away from each other, which can be seen from the fewer number of inter and intrachain contacts in comparison to 0.30 M concentration of NaCl. The number of inter-chain contacts even decreases, while NaCl concentration reaches 0.80 M. At a higher concentration of NaCl (i.e. 1M) it can be seen that the number of inter-chain contacts have reduced, which is in accordance with the free energy contour map generated for COM distance and the end-to-end distance of individual chains.

Fig 7: Intra and inter-strand interactions of the DNA chains at different concentrations of MgCl2

Fig 7 represents the similar contact map for five different concentrations of MgCl2, considered in our investigation. It is seen from the contact map that for MgCl2 at 0.18M, inter-strand contacts are fewer than that of intra-strand contacts, which depicts that the strands (chains) remain separated. While increasing the concentration from 0.18M to 0.30M, the single DNA strands approach each other in addition to the structural collapse of the individual chains. This is clear from the contact map that shows an increased number of intra- and inter-strand contacts. While upon increasing the concentration of MgCl2 to 0.50M it is seen that the single strands start swelling and retreat from each other as seen from the fewer inter- and intra-chain contacts compared to 0.30M MgCl2. The number of inter-chain contacts even decreases, while MgCl2 concentration reaches 0.80M. This signifies that the


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chains are moving apart. At the highest concentration of MgCl2 examined in this work (1.0M) we see a further decrease in inter-chain contacts. This is in accordance with the free energy contour map generated for COM distance and end-to-end distance of the individual chains.

5. Conclusions: In this work, using all-atom MD simulations, we monitor the dynamics of structural collapse and subsequent initiation of aggregation of two ssDNAs of same base sequence in the presence of a monovalent (NaCl) or divalent (MgCl2) salt at different concentrations. The pivotal evidence of structural collapse and ssDNA aggregation come from the free energy landscape of end-to-end distance of the chains and COM separation of a particular chain from the other one. It seems from the color map that the DNA strands undergo structural collapse to a large extent at 0.3 M concentration under the influence of either bivalent or monovalent cations owing to the higher degree of negative charge neutralization and subsequently the strands approach to each other. The collapsed coil-like conformation of the ssDNA molecule correlates significantly with the formation of stable sequential and non-sequential stacking of base pairs at 0.3M NaCl and MgCl2 concentration (Fig S14 & Fig S15 of ESI†). Our work is more reasonably supported by the experimental findings by Pollack et al. in which a similar extent of structural compaction of ss-DNA and ss-RNA are observed at a lower concentration of Na+ and Mg2+. Caliskan et al.

51

observe a dramatic change in persistence length, as

RNA folds over a smaller concentration range of Mg2+ compared to Na+. Our simulations establish that the extent of the ssDNA collapse is greater while increasing the concentration of either NaCl or MgCl2 from 0.18M to 0.3M and further addition of ion (NaCl and MgCl2) leads to a monotonic swelling of the strands. This is evident from the time evolution of the number of contacts between Chain_A and Chain_B in the presence of NaCl and MgCl2 (Fig S16(a) Fig S16(b) respectively, ESI†). In a nutshell, it is visible from our simulations that though these two ssDNAs have a similar qualitative response to the ionic environment, they differ in microscopic details such as average endto-end distance, accumulation of cations around Phosphates oxygens etc. especially in the presence of the divalent cation. This can be seen from the detailed analysis of the radial distribution function of the cations surrounding the DNA backbone oxygen. Association of cations (Na+ and Mg2+), especially around the phosphinyl oxygen (pn_O) governs the structural distortion of the DNA strands. Our simulations also suggest that Mg2+ can bring two ssDNA chains more efficiently close to each other than Na+ at the low concentration and help in the initiation of the aggregation process. In future, we would like to extend our study to in-vivo conditions involving longer DNA strands.


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Supporting information A brief overview of the simulated systems of different concentrations, box volume, density, temperature and pressure, Lennard-Jones parameters for the ions used in the simulation, initial box dimensions and position of ss-DNAs, RMSD plots of ssDNA strands at different concentration of Na+ and Mg2+, Time evolution of ‘Centre of mass distance (COM)’ and ‘End-to-end distance’ between two ss-DNAs in presence of Na+ and Mg2+, color maps of COM distance between two ssDNAs (nm) and End-to-end distance of Chain_A and Chain_B in the presence of Na+ and Mg2+ from trajectory2, radial distribution of Na+ and Mg2+ around the phosphoester and phosphinyl oxygen. Time evolution of sequential and non-sequential base pair stacking in the presence of Na+ and Mg2+, time evolution of number of contacts between Chain_A and Chain_B in the presence of Na+ and Mg2+.

Author Information Corresponding Author *Email:

[email protected]

Mailing Address: Indian Institute of Technology Bombay, Department of Chemistry, Mumbai400076, India Phone: + 91-022-2576 7192. Fax: + 91-022-2576 7152.

Disclosure Statement The authors declare no competing financial interest.

Acknowledgments R.C acknowledges SERB (SB/SI/PC-55/2013) for financial support. S.S thanks IIT Bombay for a fellowship. S.S acknowledges Mr. Jaladhar Mahato for stimulating discussions.

Dedication We would like to dedicate this research article to Professor Biman Bagchi, Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore, on the occasion of his 65th Birthday.


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Salt Induced Structural Collapse, Swelling, and Signature of

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