dna is a highly polar 
 molecule , evolved to be stable in high - dielectric 
 environments , such as aqueous solution , which is the main environment 
 considered in most experimental and theoretical studies .
however , 
 understanding the properties of dna in low - polarity environments is 
 crucial for many nanotechnological applications of dna and is instrumental in understanding how nucleic acids acting as 
 antigene or antisense drugs pass through the highly hydrophobic cellular 
 membrane , and how dna can be transported in the body by means of liposome 
 carriers .
water and counterions are crucial to screen the electrostatic 
 repulsion 
 among charged phosphates and also favor the apolar stacking of bases . 

accordingly , the large impact of solvent modification on the properties 
 of dna is not surprising .
for example , a subtle change in the neutralizing 
 cation can lead to a dramatic conformational change or 
 even to a complete alteration in the sequence - stability rules of the 
 duplex .
addition of ethanol to an aqueous 
 solution induces drastic changes in the duplex structure , forcing 
 a b  a transition .
some organic solvents , such as methanol , 
 formamide , pyridine , or dimethyl sulfoxide , induce unfolding 
 and strand separation , or formation of toroid - like conformations , while others , such as glycerol or room - temperature 
 ionic liquids , maintain the duplex structure . 
 more surprisingly , a dna duplex does not lose its structure completely , 
 and the two strands remain bound when dna is transferred from aqueous 
 solution to the gas phase .
it is clear that changes in the 
 balance between stabilizing and destabilizing terms controlling dna 
 structure due to alterations in the solvent are complex and still 
 not well understood .
experimental studies in apolar solvents 
 are challenging because 
 of the difficulty of transferring dna from water to such media .
this 
 problem has motivated several theoretical studies , most of them using 
 molecular dynamics ( md ) simulations .
have examined the structure and stability of 
 dna in octane , correlating the observed changes with those required 
 for helicase activity .
lin et al . explored 
 the stability of decorated dnas in interfaces between dna and chloroform 
 and have previously considered the reaction of dna to high concentrations 
 of pyridine .
finally , khalid et al . used coarse - grained models to study the insertion 
 of duplex dna into a lipid bilayer , finding a significant associated 
 free energy barrier , even for small duplexes . here
we use large - scale 
 atomistic md simulations and discrete path 
 sampling to investigate the conformations sampled by a short dna duplex 
 in carbon tetrachloride ( ccl4 ) , a medium comparable to 
 the interior of a biological membrane .
we have also explored the ( free ) 
 energy and structural differences associated with the change of phase 
 of a duplex dna from water to ccl4 .
finally we analyzed 
 for the first time the possibility that dna acts as a proton donor / acceptor 
 when it crosses from a polar to an apolar medium .
our results suggest 
 that dna , at least a short fragment , can maintain its duplex structure 
 for significant periods of time in an apolar solvent and , surprisingly , 
 appears more rigid in such media than when immersed in aqueous solution . 

transfer from aqueous to apolar solvents is , obviously , strongly disfavored , 
 especially if the dna remains fully charged .
typically , the transferred 
 dna is not fully dehydrated but maintains ( irrespective of its formal 
 charge ) a significant amount of water around it .
calculations suggest 
 that a neutral dna state with a compact structure ( not too different 
 from that in aqueous solution ) is the most prevalent form of dna in 
 apolar solvent .
we considered a small oligonucleotide 
 ( d(gcgaagc ) ) as a model system 
 for all our calculations . in aqueous solution , this small dna forms 
 a stable hairpin with a gaa triloop and a two - d(cg)-pair stem , 
 organized in a canonical b - type duplex , as confirmed by nmr .
experiments and theory have shown that this 
 oligonucleotide folds on a ( multi)microsecond time scale in water , and should unfold on a similar time scale in a denaturant 
 solvent such as pyridine .
the small size 
 of the system accelerates calculations , favors sampling , and reduces 
 memory effects , which might be very important for longer oligonucleotides . 

previous studies have demonstrated the ability of current force field 
 and md protocols to simulate this structure properly in a variety 
 of environments .
the 7-mer 
 dna hairpin was first simulated in aqueous solution as a reference 
 for the  native trajectory  .
the nmr structure was neutralized by adding six na ions ( placed at optimal classical molecular interaction potential 
 ( cmip ) positions ) and solvated by 7200 
 water molecules , defining an octahedral box .
the system was optimized , 
 thermalized , and preequilibrated using standard procedures with triplicated 
 simulation windows , and equilibrated for 100 ns prior 
 to 1 ms of production trajectory at constant temperature and pressure 
 ( t  =  300  k ; p = 1 
 atm ) .
long - range electrostatic corrections were represented by means 
 of periodic boundary conditions ( pbc ) and the particle mesh ewald 
 ( pme ) correction with default parameters . starting from the aqueous solution equilibrium structure , 
 we performed unbiased md simulations in ccl4 considering 
 the canonical dna protonation state ( q = 6 ) 
 as well as a fully neutralized system , in which all of the phosphates 
 were protonated ( q = 0 ) following the same procedure 
 used previously for the study of gas - phase structures . the simulation cell contained one dna molecule 
 and around 4288 ccl4 molecules .
the optimization , thermalization , 
 and equilibration procedures were the same as those used in the aqueous 
 simulations .
trajectories were also collected for 1 ms at constant 
 temperature and pressure ( t = 300 k ; p = 1 atm ) using pbc  pme to account for long - range effects . 
 note that the ccl4-simulation systems do not contain counterions , 
 which means that ewald s plasma is responsible for charge balance 
 in these simulations . to discard possible ewald artifacts in this 
 case , we repeated the calculations for the q = 6 
 system using a reaction field correction with a 1.1 nm cutoff to define 
 the boundary between the atomistic system and the exterior dielectric 
 continuum . to 
 explore the conformational space of the hairpin in ccl4 , we performed replica exchange replica exchange molecular dynamics 
 ( rexmd ) simulations for the two charge 
 states ( q = 6 and q = 0 ) 
 starting from ( i ) a fully extended conformation and ( ii ) the aqueous - equilibrated 
 structure .
as an additional test , we extended the rexmd simulations 
 to a minimum - hydration hairpin ( 50 water molecules ) obtained 
 from our simulations of the transfer of the hairpin from water to 
 ccl4 ( see later discussion ) .
a total of 25 temperatures 
 was selected to span the range of 300440 k. the distribution 
 of temperatures was chosen to guarantee an average exchange probability 
 around 25% .
each of the temperature replicas was explored for 150 
 ns ( for a total simulation time of 2  2  
 35  0.150 = 15 ms ) , with exchanges attempted 
 every 1,000  md steps .
disconnectivity 
 graphs provide 
 a simple and powerful way to visualize the dna potential or free energy 
 landscape in terms of local minima and transition states connecting 
 them .
this approach groups minima that are mutually 
 accessible at a given energy threshold into disjoint sets , which are 
 connected by vertical branches .
the shorter the branches linking the 
 minima , the smaller the activation barrier needed to move from one 
 group to the other .
the stationary points ( minima and transition states ) 
 required to construct the disconnectivity graph were obtained with 
 the discrete path sampling ( dps ) method , implemented in the programs pathsample and optim .
dps runs were started from 
 an initial connected path of minima and intervening transition states 
 between two specified end points . to identify suitable end points
, 
 we used 400,000 snapshots obtained from md trajectories at low ( 298 
 k ) and high ( 450 k ) temperatures for the q = 0 system 
 in explicit ccl4 ( that for which rexmd simulations were 
 not conclusive ; see later discussion ) .
the lowest energy structure 
 was chosen as one representative of the  native form  , 
 while an extended structure without any native contacts was designated 
 as the  unfolded form  .
the connections between 
 stationary points were calculated by approximate steepest - descent 
 paths obtained by energy minimization following infinitesimal displacements 
 parallel and antiparallel to the eigenvector corresponding to the 
 unique negative eigenvalue for each transition state .
we define minima 
 and transition states as stationary points on the potential energy 
 surface with zero and one negative hessian eigenvalues , respectively . 

local minimization of minima and transition states was carried out 
 using a modified limited - memory broyden 
the doubly nudged elastic band ( dneb ) algorithm was used to identify transition - state candidates , which 
 were accurately refined using a hybrid eigenvector - following approach . once an initial connected path was found ,
the 
 database was enlarged by adding all of the minima and transition states 
 found during successive connection - making attempts for existing pairs 
 of minima .
finally , to 
 construct disconnectivity graphs on the basis of free energy , we estimated 
 the relative free energies of minima and transition states from their 
 potential energy and normal - mode frequencies using the harmonic superposition 
 approximation for the density of states .
the energy and analytical first derivatives of the energy , 
 required 
 for geometry optimization , were computed through an interface for 
 the amber9 package available for optim using the parm99+parmbsc0 force field . for disconnectivity 

graph calculations we represented the ccl4 solvent with 
 the generalized born model implemented in amber and without distance cutoffs for the nonbonded interactions . to obtain an atomistic representation of the transfer of the hairpin 
 from aqueous solution to ccl4
, we performed a steered md 
 simulation , where we moved the hairpin 
 across the major axis of an orthorhombic box ( size 5.85    5.85    19.4  nm ) containing 9,460 ccl4 molecules ( placed in the 
 center of the long axis ) and 34,935 water molecules ( placed at both 
 sides of the ccl4 phase ; see supporting 
 information figure s1 ) .
simulations for the q = 6 dna included six na counterions ( see previous 
 discussion for neutralization protocol ) , while no explicit counterions 
 were included for the q = 0 simulations .
the steered 
 md simulations either move the hairpin from one water box to the other 
 passing through the ccl4 phase or from the center of the 
 ccl4 phase to one of the neighboring water boxes .
a slow 
 steering velocity [ 0.05  nm / ns ; with force 1,000 kj/(mol  nm ) ] was used to reduce hysteresis effects .
the steered md trajectory 
 was then used to extract 75 starting configurations ( spaced 0.2 nm 
 along the major axis of the simulation system ; see supporting information figure s1 ) from which umbrella sampling 
 ( us ) calculations were performed . prior to the us calculations all 
 configurations selected from the steered md simulations were relaxed 
 for 20 ns . for the us procedure , we used the position of the dna hairpin 
 in the transfer direction ( z ; see supporting information figure s1 ) as the reaction coordinate , 
 and a harmonic umbrella potential ( k = 1,000 kj/(mol nm ) ) with 75 overlapping 
 windows , centered at consecutive values of the reaction coordinate 
 that span the complete waterccl4water transfer 
 path .
we define the central window as that centered at the center 
 of mass ( com ) of the ccl4 box ( z  =  0 
 nm ) and centered the first and last windows at points located at z = 7.3 and z = + 7.3 nm with respect 
 to this com .
individual trajectories for each of the 75  us windows 
 extend for 100 ns ( for a total simulation time of 9  s    system ) . 

the potential of mean force ( pmf ) was calculated using the cyclic 
 implementation of the weighted histogram analysis method ( wham ) in 
 gromacs 4.5.5 .
because iact is 
 subject to uncertainties due to limited sampling , we have smoothed 
 the iact along the reaction coordinate using a gaussian filter of 
 width of 0.5 nm .
boltzmann 
 ( pb ) equation 
 with a very fine grid resolution as implemented 
 in our cmip code , to determine the change in solvation 
 free energy when the dna moves from regions of low ( ccl4 ) and high ( h2o ) dielectrics .
dielectric boundaries were 
 defined from the placement of the interfaces between phases in the 
 us calculations .
for each of the 75 windows considered in the us calculations 
 we selected 50 individual dna conformations ( for q = 6 and q = 0 states ) performing then a 
 total of 15,300 pb calculations from which an average free energy 
 of solvation for each point of the transfer process was determined . 
 to solve the pb equation we used a regular grid ( spacing 0.05 nm ) 
 with two solvent regions ( ccl4  = 2 and h2o  = 80 ) .
the d(gcgaagc ) oligonucleotide 
 forms a very stable dna hairpin in aqueous solution , with a well - organized 
 and stable triloop and a short , but stable , b - gpc dna stem .
the native nmr conformation 
 is stabilized by eight base - pairing 
 hydrogen bonds ( h - bonds)six d(gc ) bonds plus two d(ga ) bonds  that we term 
  canonical h - bonds  . despite 
 backbone 
 fluctuations , around 98% of these canonical h - bonds 

our long md simulations in aqueous 
 solvent suggest that the dna native hairpin conformation is stable 
 at room temperature ( supporting information figure s1 ) , in agreement with previous experimental ( root mean square 
 deviation ( rmsd ) = 0.08  0.01 nm with 
 respect to the nmr average structure ) and md studies .
unbiased microsecond 
 md simulations for the dna hairpin in ccl4 reveal that 
 for both q  =  6 and q = 0 charge states the aqueous conformation can survive in ccl4 for a significant amount of time , undergoing only small structural 
 changes ( supporting information figure 
 s1 ) .
in fact , unbiased md simulations show rmsd fluctuations smaller 
 for trajectories in ccl4 than for equivalent trajectories 
 ( at the same temperature ) in water ( see supporting 
 information figure s2 ) . clearly , the ability of water to establish 
 h - bond interactions with the dna favors flexibility , since in aqueous 
 solution temporarily lost intra - dna interactions ( due to thermal fluctuations ) 
 are compensated with dna  water contacts , while this is impossible 
 in ccl4 , or any other apolar environments . to verify that
the , perhaps surprising , microsecond - scale stability 
 of the q = 6 hairpin was not related to a 
 pme artifact , we repeated the simulation using a reaction field to 
 correct for distant interactions , instead of the default pbc 
as shown in supporting information figure s3 and figure s1 , reaction field and ewald simulations give 
 ( as expected ) nearly equivalent results , 
 confirming that aqueous solution structure is indeed a microsecond - stable 
 structure of the hairpin in ccl4 , even when no neutralization 
 of the phosphates occurs .
it is , however , unclear whether or not the 
 solution structure corresponds to the real free energy minima in ccl4 or it is simply a metastable conformation that becomes trapped 
 due to the stiffness of the structure . to clarify this point , we performed 
 large - scale rexmd simulations with a wide range of temperatures and 
 an aggregated simulation time of more than 15 s ( see methods and figure 1a  d ) . 

as previously described , to check for memory artifacts , we started 
 simulations from both an  extended  and a  native - like  

irrespective 
 of the starting conformation , both rexmd simulations of the q = 6 hairpin lead to quite extended configurations 
 at the end of the simulations ( figure 1b ) .
summary 
 of rexmd results for the dry hairpin in ccl4 solution ( q = 6 and q = 
 0 ) started from the native conformation ( the folded state in water ) 
 and a fully extended structure .
the results correspond to the last 
 50 ns of the replicas at room temperature ( extracted from an aggregated 
 simulation time of 3.75 s ; see methods ) .
the structures sampled have a 
 significant number of h - bonds , but 
 few of them are native ( figure 1a , b , d ) .
the 
 loop and general hairpin structure is also destroyed , and it is difficult 
 to find any signal of the original hairpin structure in the sampled 
 structures at room temperature ( figure 1 ) . 
 from these rexmd and unbiased md simulations , we conclude that the 
 native structure is a metastable ( on the microsecond time scale ) conformation 
 for a hypothetical fully charged hairpin , but it is not the most favorable 
 conformation for this oligonucleotide in ccl4 .
the 
 results of the rexmd simulations are less clear for the q  =  0 state . by comparing the independent simulations , 

we conclude that the conformational space is dominated by compact 
 structures ( see figure 1 ) , but it is not clear 
 whether or not these compact structures are close to the native one . 

thus , simulations starting from the native state remain very close 
 to the aqueous structure , maintaining the pattern of native h - bonds 
 ( figure 1 ) , while simulations starting from 
 the extended state collapse to a variety of different compact , but 
 nonnative , structures .
clearly , replica exchange seems to be nonoptimal 
 to explore low - lying minima in this complex conformational equilibrium .
thus , for the hairpin ( q = 0 ) we decided to complement 
 rexmd simulations with dps calculations to build disconnectivity graphs 
 ( see methods ) . the results in figure 2 show that the lowest free energy structures correspond 
 to folded hairpins with a high content of native base - pairing ( native 
 in blue and near - native in green in this figure ) .
the vertical axis represents the free energy difference with respect 
 to the global minimum , and the spacing of the branches on the horizontal 
 axis was chosen to reveal the structure as clearly as possible .
the 
 branches terminate at the energies defined by the individual free 
 energy minima and are joined together at energy thresholds where the 
 barriers separating different sets can be overcome .
branches are colored 
 according to the fraction of native base - pairing h - bond contacts ( from 
 no contacts in red to all native contacts in blue ) and highlight the 
 diversity of conformations adopted at different energies ( unfolded , 
 misfolded , near - native , and native ) .
the graph reveals that 
 the lowest free energy funnel corresponds to folded structures with 
 a high content of native contacts ( blue ) , and the existence of a funnel 
 of misfolded structures with no native contacts ( red ) separated from 
 the global minimal by a high free energy barrier .
the long branches ( > 30 kj / mol on the vertical axis ) reflect 
 the 
 high - energy barriers associated with rearrangements between native 
 and misfolded or unfolded conformations .
extended ( unfolded ) states 
 ( no native contacts and radius of gyration greater than 0.8 nm , red ) 
 are sparse ( see supporting information figure 
 s4 ) , and are separated by high - energy barriers from the native state 
 ( see figure 3 and supporting 
 information figure s4 ) .
in fact , starting from the unfolded 
 state , we observe that the pathway to the native state involves a 
 potential energy barrier of around 130 kj / mol ( figure 3 ) .
overall , the disconnectivity graphs confirm that the conformational 
 landscape of the hairpin in ccl4 is very frustrated with 
 several nonnative conformations separated by high - energy barriers . 

however , the combination of these results with rexmd suggests that 
 compact structures , not far from the aqueous native state , are the 
 dominant ones for the neutral state of the hairpin .
potential energy difference 
 with respect to the native state as 
 a function of the integrated path length for a discrete path connecting 
 an unfolded structure with the native structure for q = 0 .
the integrated path length is the integrated euclidean distance 
 in cartesian coordinates high energy .
snapshots for selected intervening 
 structures are also shown and colored according to the fraction of 
 native h - bond base - pairing interactions as described in figure 2 up to this point we have 
 explored the structure of dna in dry ccl4 , but dna is naturally 
 found in water , and it is therefore 
 interesting to study the transfer process and how it affects the structure 
 and energetics of the hairpin . to study the unlikely transfer process 
 ( see methods ) , we first performed a very slow 
 velocity steered md simulation to obtain starting conformations for 
 umbrella sampling free energy calculations .
we first pulled the hairpin 
 from one water box to the other , passing through the ccl4 phase ( see schematic representation in supporting 
 information figure s5 ) .
as seen in figure 4 , even for very small pulling velocities , the hairpin crosses 
 the apolar phase hydrated and maintains the general hairpin structure 
 ( see supporting information figures s5 
 and s6 ) , confirming that the structure of dna in solution is a metastable 
 conformation in ccl4 .
representation of different snapshots 
 obtained in steered md simulations 
 forcing the phase transfer of dna hairpin : ( a ) from water to ccl4 
 and back to water for q = 6 dna hairpin ; 
 ( b ) from water to ccl4 and back to water for q = 0 dna hairpin ; ( c ) from ccl4 to water for the q = 6 dna hairpin ; ( d ) from ccl4 to water for the q = 0 dna hairpin .
the number of water molecules in the microhydration 
 drop depends 
 on the charge state of the hairpin ( see supporting 
 information figure s5 ) , but even for the neutral hairpin there 
 is a small , but nonnegligible , number of water molecules protecting 
 the hairpin from the apolar solvent ( see supporting 
 information figure s6 ) .
although displacements of the hairpin 
 within the box were as slow as possible , they are still much faster 
 than the relevant experimental time scale , and we can not rule out 
 potential problems in the relaxation of the solvent environment around 
 the dna . to avoid these potential relaxation artifacts as far as possible 
 and to confirm that even a neutral hairpin is hydrated in ccl4 , we repeated the pulling procedure but started with a dry 
 hairpin placed in the middle of the ccl4 phase .
these complementary 
 simulations confirm that as soon as the hairpin moves from the center 
 of the apolar phase , a stream of water molecules flows in ( especially 
 dramatic for the q = 6 charge state ) , spontaneously 
 generating a microhydration environment ( figure 4 ) .
this result indicates that the entrance of water molecules in 
 the apolar phase is not a relaxation artifact but a real physical 
 effect .
hence we predict that under normal conditions a hairpin ( especially 
 a fully charged one ) would cross a hydrophobic phase surrounded by 
 a small number of water molecules .
the discussion in the previous 
 paragraph suggests that if transferred 
 directly from water , the dna in ccl4 will probably be surrounded 
 by several water molecules .
thus , we repeated the rexmd calculations 
 but used microhydrated species ( q = 0 and q = 6 ) obtained from the pulling experiments when 
 the center of mass of the hairpin is placed at the middle of the apolar 
 phase .
we found that the impact of the microhydration environment 
 helps to stabilize the folded state ( see supporting 
 information figures s7 and s8 ) , shielding the electrostatic 
 repulsion between the charged phosphate groups . as a result ,
a microhydrated 
 dna placed in the middle of a ccl4 phase maintains a conformation 
 that is not far away for the native structure in water , even when 
 we consider that phase transfer does not imply change in the ionization 
 state of the oligonucleotide . in summary
, our simulations strongly 
 suggest that the neutral hairpin maintains well the solution structure 
 when transferred to either dry or wet ccl4 . on the contrary , 
 the fully charged hairpin , while remaining close to the aqueous structure 
 in wet ccl4 ,
we used wham and umbrella sampling ( from steered - md snapshots ; 
 see methods ) to compute the free energy change 
 associated with the transfer of the hairpin across polar and apolar 
 phases .
the results displayed in figure 5 ( see methods and supporting information figure s9 for details ) clearly illustrate the large free energy 
 barrier associated with crossing a hydrophobic phase : around 650 kj / mol 
 for a fully charged hairpin and 150200 kj / mol for the neutral . 

these numbers are in qualitative agreement with those obtained from 
 a poisson  boltzmann calculation ( see figure 6 ) which demonstrated that transfer of a hairpin from water 
 to an apolar solvent , even if microhydrated , is largely disfavored . 
 these results reinforce the notion that decoration of the dna with 
 apolar moieties is desirable to improve permeation .
potential of mean force associated with 
 the transfer of the hairpin 
 ( q = 6 and q = 0 ) across 
 a polar / apolar / polar simulation box ( see methods and supporting information figure s1 ) .
change in the electrostatic solvation energy 
 associated with the 
 transfer waterccl4water of a fully charged 
 ( q = 6 ) and neutral ( q = 
 0 ) hairpin .
finally , we studied the 
 relative weight of neutral and charged 
 states of a hairpin in ccl4 . using standard thermodynamic 
 cycles ( see supporting information figure 
 s10 )
, we computed the free energy cost of transforming a neutral to 
 a charged state in ccl4 , the cost of removing the hairpin 
 charge in water , and the cost associated with the phase transfer of 
 the neutral and charged hairpins .
the free energy change associated 
 with the annihilation of the hairpin charge can be determined at neutral 
 ph from the experimental pka in water 
 of a dna phosphate ( pka around 2 ; g around 160 kj / mol assuming that all phosphate groups protonate 
 independently of each other ) .
thus , transfer of a hairpin ( q = 6 ) in water into a ( q = 0 ) 
 hairpin in ccl4 is overall disfavored by around 350 kj / mol , 
 while the same transfer assuming q = 6 in 
 both solvents has an associated free energy change around 450500 
 kj / mol higher . in other words , our simulations strongly suggest that 
 dna in apolar media should have a reduced charge and could even be 
 completely neutral . under these conditions dna
dna 
 is a highly polar molecule , which has evolved over millions 
 of years to be stable and functional in aqueous solution .
dna will 
 not spontaneously transfer to an apolar phase , such as a biological 
 membrane , which represents a clear advantage for confinement in a 
 given cellular compartment .
however , biotechnological and biomedical 
 applications often require the dna to cross biological membranes , 
 be encapsulated in a hydrophobic , environment , or just act as a chemical 
 reactant in a nonaqueous solvent .
it is then necessary to study the 
 behavior of nucleic acids in apolar solvents as a preliminary step 
 to design modifications that can favor transfer from water . for this 
 purpose ,
theoretical approaches are ideal , since they allow us to 
 study dna in such hostile environments .
our large - scale simulation 
 effort , described in the present report , illustrates the significant 
 changes occurring in the conformational landscape of a small model 
 system of dna when transferred from water to ccl4 .
the 
 dna becomes stiffer and the landscape is more frustrated in the apolar 
 phases , which might generate kinetic traps .
compact states , probably 
 not far from the aqueous structure , are sampled if the dna is neutralized , 
 while more extended conformations are expected if the dna maintains 
 its ionic state in water .
the transfer of dna from aqueous solution 
 to ccl4 is 
 energetically disfavored , especially if the dna maintains its aqueous 
 ionic state .
the size of the water droplet increases with the net charge 
 of the dna , but even for a neutral dna a significant number of water 
 molecules appears in our simulations for the apolar phase .
a combination 
 of the umbrella sampling potential of mean force simulations ( supported 
 by poisson  boltzmann calculations ) with experimental pka estimates demonstrates that the neutral state 
 is favored , suggesting that transfer of dna could generate a net proton 
 flux across a membrane .
overall , neutralization of dna charges seems 
 a key mechanism to improve transfer properties of dna and either changing 
 phosphoric by another acid with higher pkas or neutralizing decorations neutralizing dna charge seems a promising 
 approach to improve dna bioavailability .
 the 
 study of nucleic acids in low - polarity environments paves the 
 way for novel biotechnological applications of dna . here 
 , we use a 
 repertoire of atomistic molecular simulation tools to study the nature 
 of dna when placed in a highly apolar environment and when transferred 
 from aqueous to apolar solvent . 
 our results show that dna becomes 
 stiffer in apolar solvents and suggest that highly negatively charged 
 states , which are the most prevalent in water , are strongly disfavored 
 in apolar solvents and neutral states with conformations not far from 
 the aqueous ones are the dominant forms . 
 transfer from water to an 
 apolar solvent such as ccl4 is unlikely to occur , but our 
 results suggest that if forced , the dna would migrate surrounded by 
 a small shell of water ( the higher the dna charge , the larger the 
 number of water molecules in this shell ) . even the neutral form ( 
 predicted 
 to be the dominant one in apolar solvents ) would surround itself by 
 a small number of highly stable water molecules when moved from water 
 to a highly apolar environment . 
 neutralization of dna charges seems 
 a crucial requirement for transfer of dna to apolar media , and the 
 most likely mechanism to achieve good transfer properties . 
Introduction
Methods
Results and Discussion
Conclusion