Structure of Hydrated Na+ Ions Around a Region

of A- or B-DNA Helix

W. K. LEE,* Y. GAO, and E. W. PROHOFSKY, Department of

Physics, Purdue University, West Lafayette, Indiana 47907

Synopsis
A molecular-dynamics simulation was used to carry out an introductory study of the hy-
dration of a section of a rigid single A- or B-DNA helix with one Na+ counterion per nucleotide.
Four Na+ ions and four nucleotides and periodic boundary conditions were used to mimic
an infinite helix. The atoms of the helix and the Na+ ions were assumed to be Lennard-Jones
spheres that also carried charges. Stillinger four-point charge model water molecules were
used. We carried out five calculations, for 26 and 46 water molecules in B-DNA and 20,32,
and 46 in A-DNA fragments. The arrangements of the Na+ ions are found to have some
similarities to those obtained by Clementi and Corongiu. In the calculations with 46 water
molecules, we found that two Na+ ions can be bridged by about two water molecules and form
a hydrated bound pair, which in turn forms a bridge between the guanine N7 and a near
phosphate group. These bound pairs may be important in stabilizing the helix structure of
DNA molecules.

INTRODUCTION
The role of water and ions in stabilizing and determining conformation
of the DNA helix has long been recognized. Experimental evidence for
particular water structures in crystallized structures of DNA fragments1
indicate that very specific chain structures may exist. Monte Carlo cal-
culations2 have indicated that Naf ions do not just neutralize the charge
on the phosphate groups, but form a zigzag pattern with some ions close
to the bases.
We have carried out introductory molecular-dynamics calculations for
water and Na+ ions in the vicinity of a section of poly(dG)-poly(dC)for
relatively small numbers of water molecules. Studies that simulate such
low humidities can be helpful in learning whether specific water-ion-DNA
struc,ures exist that are important in bringing about helical conformation
from random-coil conditions. Some of the calculations are for situations
in which the helix does not have enough water to maintain a stable A con-
formation and others are for water amounts appropriate to a stable A
conformation. The logic here is that the few water molecules for these cases
cannot form particularly large or complicated structures, and a simple
underlying structure, if it exists, would be more apparent. The small

* Present address: Department of Chemistry, Brandeis University, Waltham, Mass.

02254.

Biopolymers, Vol. 23,257-270 (1984)

0 1984 John Wiley & Sons, Inc. CCC 0006-3525/84/020257-14$04.00
258 LEE, GAO, AND PROHOFSKY

number of waters almost guarantees that only small structures can re-
sult.
If the structures are small, then one may be able to observe such struc-
tures by looking a t a small section of helix. In our calculations we include
only one section of backbone along with parts of the base pairs in a helical
stack. Since the distance across a groove between backbones is large
compared to the phosphate-phosphate distance along a backbone, such
an approximation may give a meaningful indication of the small structures
possible when the grooves are mostly empty. The advantage of taking only
one backbone reduces not only the number of atoms on the helix but also
the number of water molecules needed to simulate a given relative humidity
and makes for a much smaller calculation, one that can be run on a mod-
erate size computer. One expects the backbone close to the guanine N7
atom to be the one most likely to contain water structure, since the N7 is
likely the most charged base atom. Poly(dG)-poly(dC)is the simplest helix
that takes on both A and B conformations.
We find that a bridge of Na+-water-Na+ forms between the phosphate
groups and the guanine N7. This quasimolecule bridge does not occur for
small numbers of water molecules, but seems to form when enough water
is present to correspond to conditions of stable A-DNA conformation. We
believe that such a structure could be important in stabilizing the A con-
formation and may also play a role in B conformation, in those cases where
sodium is the primary counterion.

CALCULATIONAL MODEL
Four segments of base pairs and four Na+ ions are used in our calculation.
Suitable periodic boundary conditions are imposed to mimic an infinite
helix. The segment of a DNA helix molecule is assumed to be rigid and
in B- or A-like conformation. The segment consists of a guanine attached
to the single helix backbone and H-bonded to a truncated cytosine. That
is, we have a poly(dG)-poly(dC) homopolymer with the sugar-phosphate
backbone on the cytosine side and a part of the cytosine omitted. (We limit
our study to one backbone because of limited computer resources.) The
only cytosine atoms taken into account are the N3, N4, and C5 (labels of
the atoms are the same as those used by Arnott et aL3),since they are close
to the negatively charged region near the N7 of the guanine, where we ex-
pect the small structure might exist. The C4 of the cytosine is also ignored,
since it is enclosed by the triangle formed by the N3, N4, and C5 and the
Lennard-Jones spheres of the latter three will prevent it from interacting
with the Na+ ions and water directly. The charge of the C4 is then dis-
tributed among the N3, N4, and C5. The charges of the H atoms attached
to the DNA are transferred to the nearest atoms and the H atoms are no
longer considered to be independent elements. The coordinates of the
atoms are taken to be those obtained by x-ray experiment of Arnott et aL3
 SODIUM IONS AROUND DNA HELIX 259

TABLE I
Charges of Atoms of the Helix in Electronic Charge Units
Phosphate Guanine
P 1.70 C2 0.418
01 -0.64 c4 0.000
0 2 -0.71 c5 0.227
Oe -0.71 C6 0.408
0 4 -0.64 C8 0.214
Sugar N1 0.000
C1’ 0.196 N2 -0.370
C2‘ 0.128 N3 -0.667
C3’ 0.000 N7 -0.568
C4’ 0.000 N9 0.000
c5’ 0.000 06 -0.493
04‘ -0.267
Cytosine
N3 -0.184
N4 10.100
C5 -0.164

Each atom of the partial helix has been assigned a Lennard-Jones 12-6
potential (our work to be published later). This defines the region of the
helix from which the water and ions are excluded. Atoms with small ex-
pected charges, ie., charges less than O.le have been assumed to be neutral
and their contribution to the Coulomb interaction ignored. Those with
expected charges greater than O.le have their charges included in the cal-
culations of the Coulomb interactions. The charges are listed in Table I
and are taken from calculations of Pullman and Pullman4 and Renugopa-
lakrishnan et aL5 In retrospect, we find that the Na+ ions and water do
not accumulate in regions that would be occupied by H atoms and the ap-
proximation of not retaining the H atoms as independent variables is
probably not as poor as might be inferred initially. The Na+ and water
molecules are also characterized by charges and Lennard-Jones potentials
as discussed in a previous work.6 See Fig. 1.
The Coulomb interactions are truncated when the two atoms are farther
than 6.6 A apart, except for the Na-Na interaction, which is truncated when

TABLE I1
Lennard-Jones Parameters for the (Helix Atom)-(Water Oxygen) and (Helix Atom)-
(Sodium Ion) Interactions

Water Oxygen Sodium Ion

Helix f t
Atom a (A) (kcal/mol) (i) (kcal/mol)
P 3.079 0.134 3.00 0.072
0 3.100 0.072 2.85 0.072
C 3.100 0.072 3.0C 0.072
N 3.100 0.072 3.00 0.072
260 LEE, GAO, AND PROHOFSKY

b-
f
b+ b-

H
Fig. 1. Ben-Nain-Stillinger four-point-charge water model.

their separation is larger than 15 A. Using the forces derived from the
interaction potentials described above, the equations of motion are solved
by a molecular-dynamics method that consists of two parts in each time
step. In the first part, all the atoms of the water molecules are assumed
to be unconstrained and the equations of motions are solved by using the
Verlet a l g ~ r i t h m . ~
The positions of the atoms obtained are then, in the
second part, corrected by using the SHAKE method invented by Ryckaert
et al.? so that the rigidity of the molecules is restored. The time-step used
is 0.978 X s.
We simulate the hydration of an infinite helix by using periodic boundary
conditions applied to a cell along the helix. In this case, our cell consists
of eight base pairs plus backbone, which are treated as a rigid array of fixed
force centers. The free ions and water molecules used for the simulation
are restricted to the region of the four central base pairs and the extra two
base pairs on each side extend the potential as seen by the ions and water.
The periodic boundary conditions are then applied at the ends of the central
4-base-pair region. Two screw symmetric images of each entity inside the
central region are included, i.e., one above and the other below the central
cell, which interact with the elements inside. However, there is no cylin-
drical confinement around the helix. Once in a while, a water molecule
may move far away from the helix, but it comes back after a long time. We
have tried two calculations in B-DNA using 26 and 46 water molecules, and
three in A-DNA using 20,32, and 46 water molecules. We find that half
of the Na+ ions are closer to the N7 atoms than the phosphate groups, which
was also found by Clementi and Corongiu.* Detailed discussions and the
velocity correlation functions will be covered in our paper to be published
later.
T o start the calculation, we put water and Na+ ions randomly in the re-
gion of the central calculation cell. The temperature, which is measured
by the average kinetic energy of the water molecules, and the Na+ ions are
adjusted by scaling their velocities until the ambient temperature of about
300 K is reached. For convenience in describing our results, we introduce
the following symbols: The four Na+ ions are labeled as Nai, i = 1, 2,3,
+
4, and the water molecules are labeled as W j ,j = 5,6, . . . , n, 4, where
n,,, is the total number of water molecules in the system.
 SODIUM IONS AROUND DNA HELIX 261

-1

Fig. 2. Arrangement of the Na+ ions found in B-DNA and nu, = 26 calculation projected
on (A) the X - Y plane and (B) the X - Z plane. The shaded regions represent the regions oc-
cupied by the Na+ ions during a molecular-dynamics run.

B-DNA Fragment with 26 Water Molecules (n, = 26)

The locations of the four Na+ ions are shown in Fig. 2(a) and (b), which
are the projections on the X - Y and X - Z planes, respectively. For
clarity, only the phosphate groups along the helix backbone are shown.
The locations of the N7 atoms of the guanine bases are shown by the open
triangles. The open circles represent oxygen atoms. The oxygen atoms
connected by polygonal lines to the N7 atoms are the 0 6 of the guanine
bases. The intersections of the polygonal lines represent carbon atoms of
262 LEE, GAO, AND PROHOFSKY

Fig. 3. Arrangement of the Na+ ions found in B-DNA and n, = 46 calculation projected
on (A) the X - Y plane and (B) the X - 2 plane. The shaded regions represent the regions oc-
cupied by the Na+ ions during a molecular-dynamicsrun. The Na+ ions in regions 3 and 4
are bridged by the water molecules W9 and W14.

the same bases. The shaded regions are regions occupied by the Na+ ions
during a molecular-dynamics run. These regions are labeled in the same
manner as the Na+ ions. That is, Nai is in region i, i = 1 4 . Regions 1and
3 and regions 2 and 4 are sometimes referred to as inner and outer regions
of the inner and outer Na+ ions, respectively. The finite sizes of the regions
are due to thermal motions. A convenient measure of the size of a region
 SODIUM IONS AROUND DNA HELIX 263

%
&aUNa+
(33
Fig. 4. Two Na+ ions bridged by the four water molecules between them.

is the mean-square displacement of the Na+ ions, which is defined as

Si(t)= ( ( r , ( t )- 1;(0))2)
where ri(t)is the position of the Nai a t time t and ( 1, denotes the average
over time origins. Si ( t )is clearly a measure of how far a Na+ ion 'moves
from its original position. Since the Na+ ions are found to be localized, the

ymptotic values by Sp. We found that 5': S! 0.2 A and s'$ - -

S,( t )are independent o f t for large enough t , and we will denote these as-
- - S i 0.4
A;that is, the size of the outer regions are larger than those of the inner
regions. The inner Na+ ions are bonded to the N7 of the guanines and the
outer ones associate with the phosphate groups of different base pairs. The
zigzag arrangement of the Na+ ions is somewhat similar to that obtained
by Clementi and Corongiu (CC).2 The X - 2 projection of our Na+ ion ar-
rangement is different from the helical arrangement found by CC. For
completeness, we mention that the total energy of this hydrated DNA
fragment is -1970 kcaI/mol.

B-DNA Fragment with 46 Water Molecules ( nw = 46)

The arrangement of the Na+ ions is shown in Fig. 3(A) and (B). Again,
these are the projections on the X - Y plane and X - Z planes, respectively.
The total energy of the arrangement is -2188 kcal/mol. The new feature
in this calculation is the fact that the Na3 and Na4 ions are only about 3.7
A apart. The reason is that the Na3 and Na4 are bridged by two (some-
times three) water molecules, as shown schematically in Fig. 3. Compared
264 LEE, GAO, AND PROHOFSKY

(51

-1

Fig. 5. Arrangement of the Na+ ions found in A-DNA and n, = 20 calculation projected
on (A) the X - Y plane and (B) the X - Z plane. The shaded regions represent the regions oc-
cupied by the NaC ions during a molecular-dynamics run.

with that in n, = 26, the inner Na+ ion is pulled from the vicinity of the
lower N7 to the upper one, and a Na+-water-Na+ bridge is built between
the N7 of the guanine and the phosphate group to which the base pair is
connected. The attractive forces between each Na+ ion and the lone-pair
electrons of the bridging water molecules apparently balance the repulsion
between the two Na+ ions and hence stabilize the bridge structure.
To test for the stability of this configuration, we removed one of the two
bridging water molecules and placed it far from its original position. The
Na+ ions returned in 200 steps (0.2 ps) to their positions closer to the
 SODIUM IONS AROUND DNA HELIX 265

pinning N7 and phosphate positions in which the Na+ ions were about 4.4
A apart. This separation lasted for 600 steps (0.6 ps) before the bridge was
reformed by the capture of another water molecule.

Model System Calculation

In order to examine the stability of the Na+-water-Na+ bridge, we car-
ried out the following model calculation. We picked a configuration from
a molecular-dynamicsrun and retained only Na3 and Na4 and the 12 water
molecules closest to the two Na+ ions. We then removed the DNA frag-
ment to see whether the Na+ ions separated. After the system achieved
equilibrium, we found that the two Na+ ions were bridged by four water
molecules (Fig. 4). The average separation between the two Na+ ions is
3.6 A. The energy of this cluster at 300 K is -161 kcal/mol (the total energy
of two Na+ ions infinitely far apart and each surrounded by six water
molecules is -214 kcal/mol). Apparently, activation energy is required
to break the water bridges.

A-DNA Fragment with 20 Water Molecules (n, = 20)

The zigzag arrangement of Na+ ions is also found for A-DNA. In Fig.
5(a) and (b), we see that the Na+ near the N7 are closer to the helix axis than
in B-DNA. This may be due to the fact that in A-DNA more space near
the N7 is exposed than in B-DNA, and the Na+ ions can therefore move
into the helix to reach the energy minimum. The exposed space allows the
0 6 of the guanine, which is also highly charged, to participate in attracting
the Na+ ion. The outer Na+ oscillates with the considerable amplitude
of 0.6 A about its equilibrium position, which is between the phosphate
group of the same base pair as the inner Na+ and the lower phosphate
group. The distance between the inner and outer Na+ ions is about 7.5 A.
The total energy of the arrangement is -1824 kcal/mol.

A-DNA Fragment with 32 Water Molecules (n, = 32)

The positions of the Na+ ions and some nearby water molecules are
shown in Fig. 6(A) and (B). The positions do not change much from those
with n, = 20. Occasionally,an outer Na+ ion moves closer to the inner one
with the formation of a bridge of water molecules and a Na+ ion separation
of about 4.5 A. This bridge is relatively unstable and breaks up within 300
steps (or 0.3 ps). The total energy is -1988 kcal/mol.

A-DNA Fragment with 46 Water Molecules (n, = 46)

The stable bridges appear in A-DNA with 46 water molecules. The ar-
rangement is shown in Fig. 7(A) and (B). Compared with that a t n, = 32,
266 LEE, GAO, AND PROHOFSKY

IFll
Y

-1

-1

Fig. 6. Arrangement of the Na+ ions found in A-DNA and n, = 32 calculation projected
on (A) the X - Y plane and (B) the X - Y plane. The shaded regions represent the regions oc-
cupied by the Na+ ions during a molecular-dynamics run.

Na+l and Na+3 are displaced slightly, while the Na+ 2 and Na+ 4 are pulled
by the bridging water molecules from outside the phosphate groups to the
inside. The Na+3 and Na+4 are about 3.8 8,apart, which is compatible with
that in B-DNA. Also, the relative positions of the inner Na+ ions to the
N7 and 0 6 atoms are similar to those in B-DNA. The outer Na+ ion is
bound to both the phosphate group of the same base pair as the inner Na+
ions and the lower one, which makes the ends of the bridges slightly dif-
ferent in position from those in B-DNA. The total energy is -2115 kcal/
mol.
 SODIUM IONS AROUND DNA HELIX 267

I 18)
Y

-12.w
-1 -8.00 Y .m 1JL
I 8;m

L-12.m

Fig. 7. Arrangement of the Na+ ions found in A-DNA and n, = 46 calculation projected
on (A) the X - Y plane and (B) the X - 2 plane. The shaded regions represent the regions oc-
cupied by the Na+ ions during a molecular-dynamics run. The Na+ ions in regions 3 and 4
are bridged by the water molecules W38 and W40.

DISCUSSION AND CONCLUSIONS

The Na+ ions repel each other strongly and therefore stay as far apart
as possible. They are also strongly attracted by the negative charges of
the helix, especially the oxygen atoms of the phosphate groups and the N7
atoms of the guanine bases. The result is the zigzag arrangement as shown
in Figs. 2(A), 5(A), and 6(A), which minimize the repulsion between the Na+
ions. Nal and Na3 are attracted to the N7 atoms, while the Na2 and Na4
268 LEE, GAO, AND PROHOFSKY

E
10‘ kcol / rnol

Fig. 8. Absolute values of the binding energy versus nu for A-DNA (-) and B-DNA (-.-),
The binding energy in B-DNA is diminished by 36 kcal/mol, since the finite truncate range
is such that the Na+l ( 3 ) cannot “see” the image of Na+2 (4) in B-DNA, whereas it can in
A-DNA.

are attracted to the oxygen atoms of the phosphate groups. Using the same
reasoning, it is easy to see that the arrangements of the Na+ in Figs. 2(B),
5(B),and 6(B) are also a natural consequence of the Coulomb interaction.
Although our charge model is an approximation to the true charge distri-
bution of the DNA helix, we believe that it does display the gross features
of the DNA potential and that the results may be quite similar to what one
would find using a more complex potential.
The first two calculations described above were carried out for a section
of DNA having atoms in B-conformation positions. The number of water
molecules are too few for B-conformation stability. The DNA would likely
be in A-conformation for the nu:= 46 calculation. This is because the
number of water molecules per base pair would occur a t roughly 70% r.h.
according to the experimental measure of the amount of water per base
versus relative humidity carried out by Beekg We have 10 water molecules
per base pair, which would correspond to 40% r.h. (according to the results
of Beetz) if the water molecules are evenly distributed around both bases.
But since all the water molecules are concentrated on the guanine-backbone
side, we estimate that the actual relative humidity of our system is about
70%. The calculation at n, = 26 would correspond to very low relative
humidity, and the helix at this low humidity would likely be in a random-
coil configuration. We then would expect the Naf bridges we found at
about 70% r.h. to occur in A-conformation DNA.
We have calculated the equilibrium binding energy for the water sodium
helix systems described. This is not very significant, since only the in-
teraction between a part of the helix and the water and sodium is included.
The results for both conformations as a function of the number of water
 SODIUM IONS AROUND DNA HELIX 269

molecules (nu)) is plotted in Fig. 8. From the curves plotted, we see that
the absolute value of the binding energy (BE) in B-DNA is greater than in
A-DNA, which is contrary to our expectation that A-DNA is the preferred
form in this range of relative humidity. The interaction within the helix
can alter this result. The variation of the BE is such that the difference
in BE is increasing with nu,,indicating that A-DNA may be the more stable
form a t lower nw when all factors have been included, and that B-DNA
would be more stable at higher nu). The reason the energy rather than free
energy is used is that the entropy is difficult to calculate in molecular dy-
namics.
These bridges, if they exist, can be important in the sense that they can
reverse the behavior of the largest contributions to the nonbonded inter-
actions as they are normally calculated. The highly negatively charged
regions of DNA are usually calculated to repel each other by electrostatic
effects. The formation of these bridges will induce a net attractive effect
between such regions and could considerably alter a number of models used
in studying the behavior of the DNA helix.
The results here are limited by the assumed rigidity of the DNA helix.
The approximations used allowed T = 300 K for the water and sodium, but
the helix was at 0 K. The stability of the bridges could be greatly affected
by the vibration motion of the helix. Such effects are yet to be considered
in calculations of this kind. The vibrational motion of the DNA helix may
destabilize the formation of these bridges. We do not know of any exper-
imental data that could determine if such bridges do, in fact, form. Cer-
tainly, the helix motion would increase the size of the region occupied by
the Na+ ions, making it more difficult to locate in x-ray analyses. Further
effort may indicate whether such bridges have a significant probability to
form and whether they exist in a time-average sense. That such bridges
are probable may be important in DNA studies.
These Na+ water bridges are less likely to be as stable for adenine bases
in the A-conformation, since the hydrogen associated with the nitrogen
attached to the 0 6 reduces the attraction to the N7; this may relate to the
reduced tendency for A-conformation in adenine bases.
This work was supported in part by NIH Grant GM 24443 and NSF Grant DMR 7820602.
W.K.L. would like to thank Mr. Devi Prasad for his help.

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Biol., Vol. 2: Nucleic Acids, 3rd ed., Fasman, G. D., Ed., pp. 411-422.
4. Pullman, B. & Pullman, A. (1969) Prog. Nucleic Acids Res. 9,327-353.
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7. Verlet, L. (1967) Phys. Reu. 159,98-103.
270 LEE, GAO, AND PROHOFSKY

8. Ryckaert, J.-P., Ciccotti, G. & Berendsen, H. C. (1977) J . Comput. Phys. 23, 327-
338.
9. Beetz, C. (1978) Ph.D. thesis, Department of Physics, Purdue University.

Received December 7,1982

Accepted May 30,1983