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/;../
doi:10.1016/j.jmb.2004.07.048
J. Mol. Biol. (2004) 343, 627638
Relative Flexibility of DNA and RNA: a Molecular

Dynamics Study
Agnes Noy1, Alberto Perez1, Filip Lankas2, F. Javier Luque3 and
Modesto Orozco1,4*
1
Molecular Modeling and

Bioinformatics Unit, Institut de
Recerca Biome`dica, Parc
Cientc de Barcelona, Josep
Samitier 1-5, Barcelona 08028
Spain
2
Bernoulli Institute for

Mathematics, EPFL (Swiss
Federal Polytechnical Institute)
1015 Lausanne, Switzerland
State of the art molecular dynamics simulations are used to study the
structure, dynamics, molecular interaction properties and exibility of
DNA and RNA duplexes in aqueous solution. Special attention is paid to
the deformability of both types of structures, revisiting concepts on the
relative exibility of DNA and RNA duplexes. Our simulations strongly
suggest that the concepts of exibility, rigidity and deformability are much
more complex than usually believed, and that it is not always true that
DNA is more exible than RNA.
q 2004 Elsevier Ltd. All rights reserved.
Departament de Fisicoqumica
Facultat de Farma`cia
Universitat de Barcelona
Avgda Diagonal 643, Barcelona
08028, Spain
4
Departament de Bioqumica i
Biologia Molecular, Facultat de
Qumica, Universitat de
Barcelona, Mart i Franque`s 1
Barcelona 08028, Spain
*Corresponding author
Keywords: DNA; RNA; exibility; molecular dynamics; similarity indexes
Introduction
Under physiological conditions DNA exists as an
elongated helix known as B-form, while if possible
RNA forms a double helix more compact than that
of DNA,13 which is named the A-form. The
different puckering of sugars in B (South to SouthEast) and A (North) forms alters the structure of the
grooves,14 which changes completely the ability of
the nucleic acids to interact with other molecules,
particularly with proteins.13 It seems the different
helical structure of DNA and RNA might determine
their different role in the cell. However, structure
itself is not able to explain the incredible ability of
proteins to distinguish between nucleic acid structures. For example, only a fraction of very similar
structural distortions of DNA are recognized by the
Abbreviations used: MD, molecular dynamics; RMSD,
root mean square deviations.
E-mail address of the corresponding author:
modesto@mmb.pcb.ub.es
UvABC excision repair mechanism.13,5 Indeed, it is

known that RNase H recognizes and degrades
DNARNA hybrids, but it is inactive against RNA
RNA duplexes and other RNA-hybrid molecules,
despite the fact that all these duplexes pertain to the
A-family.6,7 More surprisingly, the complex mechanism involving gene silencing by small interference
RNAs is activated by duplex RNA, but not by other
RNA hybrid duplexes of similar structure. 8,9
Clearly, besides the general helical structure, other
properties must be exploited by nature to distinguish between helical structures.
RNA adopts very unique conformations in the
cell, like those of ribozymes, transfer or ribosomal
RNAs. However, it is believed that this is mostly
due to the fact that cellular RNA is single-stranded
and intramolecular RNA duplexes contain
unpaired bases and mismatches which favour
strong twists and kinks in the helix. However, it is
generally accepted in the scientic community that
the DNA double helix is more exible than the RNA
one. This assumption is supported by indirect low
resolution experimental data10,11 like that derived
0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
628
from electron micrography, gel electrophoresis,
hydrodynamic measurements or ber diffraction.12
31
P NMR experiments of highly oriented bers also
suggest that B-DNA is more exible than A-RNA.13,14
Protein NMR experiments also suggest that the
RNA duplex is more rigid than the DNA one for
equivalent sequences,13,14 a nding further supported from the comparison of experimental NMR
J-coupling constants with the values derived from
molecular dynamics (MD) simulations.15 Caution
is, however, necessary, since we cannot ignore that
NMR data provide information about local uctuations in DNA microenvironments, it being less
suitable to ascertain global structural changes of the
duplex.
Crystal data deposited in the Protein Data Bank
have been traditionally used to support the greater
exibility of B-DNA compared to A-RNA. Thus,
depending on sequence, solvent composition or the
presence of certain ions different B-like conformations (C-, D- or T-forms) can exist,13 an effect
that has not been detected for A-RNA. The larger
occurrence of lattice distortions in DNA crystals16,17
and the ability of a given DNA structure to
crystallize in different space groups15 have also
been used to support the higher exibility of
B-DNA compared to A-RNA. However, we cannot
ignore that similar lattice-dependence effects are
detected for A forms.18,19 Furthermore, a recent
extensive comparison of crystal structures of
B-DNA and A-RNA20 have raised doubts on the
hypothesis that DNA is always more exible than
RNA. Thus, not only the number of space groups
found in RNA structures is very similar to that
found in B-DNA, but no relevant differences are
found in both thermal factors and resolution
between B-DNA and A-RNA crystals. In summary,
the analysis of the PDB structures does not support
the view of B-DNA as an intrinsically more
exible entity than A-RNA.
Different theoretical calculations have explored
the structure and exibility of DNA and RNA
duplexes. The landmark work by Cheatham and
Kollman15 provided an exhaustive MD comparison
of DNA and RNA duplexes in aqueous solution.
The analysis of the torsions around a, c and g
dihedral angles and of sugar puckerings during
2 ns trajectories led the authors to conclude that
A-RNA was more rigid than B-DNA.15 The authors
demonstrated the complex nature of the interactions rigidifying the RNA versus the DNA,
suggesting that perhaps high ordered water
molecules around the RNA reduce the exibility
of RNA. Similar calculations were later performed
by Aufnger and Westhof,21,22 who compared
d(CG)6$d(CG)6 and d(TA)6$d(TA)6 DNAs with the
corresponding RNA duplexes, r(CG)6$r(CG)6 and
r(UA)6$r(UA)6. From the analysis of dihedral
changes along the sequence and their time uctuations, DNA was found to be more exible than
RNA. Only one MD simulation, performed recently
by MacKerells group has raised doubts on the
MD Studies of DNA and RNA Flexibility
dominant hypothesis that DNA is more exible

than RNA.23
In summary, despite the lack of denitive
evidence, there is a general consensus that DNA
duplex is more exible than the RNA one under the
same conditions. However, what does exibility
mean? Conceptually, it denotes the ability of a given
structure to be deformed as a result of an external
perturbation. For highly anisotropic macromolecules such as nucleic acids, such a denition
is perhaps too ambiguous, as it would encompass a
wide variety of structural deformations. For
example, a molecule can be locally exible but
globally rigid when exibility motions in different
regions cancel. Here, we used state of art MD
simulations to reinvestigate the relative exibility/
rigidity of B-DNA and A-RNA using a rigorous
denition of these two concepts. Our purpose is
then not only to obtain a qualitative picture of how
rigid are DNA and RNA, but to describe for the
rst time the very complex scenario of the structural
exibility of nucleic acids.
Results and Discussion

Global structural descriptors
As it has been largely discussed,24,25 current
force-elds provide stable trajectories for both DNA
and RNA duplexes. This trend is noted in the small
root mean square deviations (RMSD) determined
with regard to the MD-averaged structures (see
Table 1), indicating that despite local oscillations (in
the sub-nanosecond time scale) the two structures
sample well dened regions of the conformational
space centred on their respective averaged structures. In turn, this supports the validity of simple
pseudoharmonic approaches in the analysis of
uctuations detected in MD simulations (see
below).
The RMSD with respect to the corresponding
canonical ber26 and crystal structures (PDB entries
1BNA and 157D for DNA and RNA duplexes,
respectively) are also reasonably small (see Table 1),
which indicates that the sampled structures are
close to the experimental ones. Interestingly, the
RMSD between RNA and the A-form ber structure
is clearly smaller (see Table 1) than that obtained
between DNA and the B-form ber one. This
nding, which demonstrates the suitability of
Parm99 to reproduce RNA duplexes, suggests that
the structure of RNA in the ber and solution does
not change remarkably, while a larger change is
expected for DNA. However, care must taken to
conclude that DNA is more exible than RNA,
because we must stress that the standard deviation
of the RMSD between the sampled conformations in
DNA and RNA trajectories and the corresponding
MD-averaged structures are similar (see Table 1), a
result that does not support the assumption that
RNA is more rigid than DNA.
The distance (C1 0 C1 0 ) between contiguous
629
) between DNA/RNA trajectories and reference structures

Table 1. Average root mean square deviation (RMSD; in A
DNA
RNA
B-forma
A-forma
Crystalb
MD-averagedc
2.22(0.34)
5.16(0.55)
4.43(0.44)
1.63(0.36)
2.05(0.39)
3.54(0.22)
1.16(0.29)
1.23(0.33)
) are given in parentheses.

Standard deviations (in A
a
Canonical structures from Arnotts data.26
b
Crystal structures of DNA (PDB 1BNA) and RNA (157D). Two mismatches of 157D were not considered to compute the RMSD.
c
Structures obtained by averaging the snapshots collected during the last ns of the corresponding MD simulations.
nucleotides is a simple, yet accurate general

descriptor of the helical structure.27 For each
collected snapshot, the sequence-averaged C1 0 C1 0
distance and its associated standard deviation were
computed. This calculation was then repeated
along the entire trajectory to obtain time-averaged
mean C1 0 C1 0 distances, and the associated
standard deviations. As suggested by Hunter and
co-workers 27 C1 0 C1 0 discriminates very well
between DNA and RNA (see Figure 1(top)), since
RNA average distances are always larger than those
of DNA, and no overlap exists between DNA and
RNA C1 0 C1 0 distance distributions. The oscillations (see Figure 1(top)) of the mean C1 0 C1 0
SD)
distance are slightly larger for RNA (0.07 A
than for DNA (0.05 A SD). This suggests that RNA

is slightly more exible than DNA. However, the
oscillations in the standard deviations associated
with the sequence-averaged C1C1 0 distance in
SD) than in
each snapshot are larger in DNA (0.36 A
RNA (0.27 A SD), suggesting the opposite, i.e. that

DNA is more exible than RNA (see Figure
1(bottom)). Thus, the analysis of the C1 0 C1 0
distances and their uctuations do not provide
conclusive evidence about the relative exibility
Figure 1. Top: Variation of the sequence averaged

) along DNA and RNA trajectories.
C1 0 C1 0 distance (in A
)
Bottom: Variation of the standard deviations (in A
associated to the sequence-averaged C1 0 C1 0 distance
) along DNA and RNA trajectories.
(in A
between DNA and RNA. The same lack of

conclusions appears when the distribution of the
groove widths for DNA and RNA is examined (see
Figure 2), since the minor groove uctuates more in
DNA than in RNA; but the reverse trend is found
concerning the uctuations in the major groove.
In summary, the analysis of the global structural
descriptors presented in this section does not
convincingly support the supposed higher exibility of DNA compared to RNA. More precise
analysis needs to be made to analyze the nature of
the differences in exibility of the nucleic acids.
Entropy calculations
The intramolecular entropy can be used as a
direct estimate of the global exibility of DNA and
RNA. As noted above, MD-derived entropy estimates depend on the length of the trajectory.
However, the values obtained for 10 ns trajectories
and those extrapolated to innite time28 are very
close (see Table 2), which suggests a good convergence in the calculations, as expressed in the small
Figure 2. Distributions (tted to normalized Gaussians)

) of the minor (top) and major (bottom)
of the width (in A
grooves of DNA and RNA in the MD simulations.
630
Table 2. Intramolecular entropies (in kcal/mol K) computed using Schlitters (roman font) and Andreocci-Karpluss
methods (italics) for DNA and RNA
DNA (all atoms)

RNA (all atoms)
DNA (nucleobases)
RNA (nucleobases)
DNA (backbone)
RNA (backbone)
S(tZ9 ns)
S(tZN)
S(3)
S(10)
2.0067
1.8321
1.8228
1.6516
0.8946
0.8205
0.8893
0.8145
1.3424
1.2395
1.1429
1.0950
2.14(0.05)
1.93(0.05)
1.90(0.03)
1.71(0.03)
0.91(0.01)
0.84(0.01)
0.91(0.02)
0.83(0.01)
1.43(0.07)
1.34(0.10)
1.18(0.03)
1.09(0.03)
0.0328
0.0327
0.0342
0.0340
0.0286
0.0285
0.0300
0.0299
0.0320
0.0319
0.0333
0.0331
0.0988
0.0983
0.0976
0.0971
0.0849
0.0845
0.0860
0.0858
0.0962
0.0956
0.0944
0.0940
Total intramolecular entropies were determined considering all equivalent atoms. Partial entropies were computed considering only
nucleobases or backbone atoms. In all the cases, values were determined considering all the principal components, as well as only the
rst three and tenth ones. For the total entropy, not only the value directly obtained from the 10 ns trajectory, but also that extrapolated at
innite simulation time is displayed (see Methods for details).
standard error associated to SN. This nding, in

conjunction with the excellent agreement between
the two methods used for the calculation of entropy,
gives strong condence on the quality of the
congurational entropies determined for DNA
and RNA.
The intramolecular entropy of DNA is 1113%
larger (around 0.24 kcal/mol K; see Table 2) than
those of RNA. Therefore, DNA is globally more
disordered and exible that RNA, which supports
the generally accepted picture of the relative DNA/
RNA exibility. However, a partitioning analysis of
the entropy contributions shows that the concept of
exibility is more complex than expected. Thus,
when the entropy is computed considering only the
rst ten principal components, the entropy of DNA

is only around 1% larger than that of RNA, and
when only the rst three principal components are
considered (see Table 2) the RNA appears 4% more
disordered than DNA. This nding suggests that
disorder in RNA stems from a relatively small
number of very soft motions, while for DNA is
the result of many soft motions.
Diagonalization of mass-weighted covariance
matrices built up by considering only certain
groups of atoms allows us to examine their
contribution to the global disorder in DNA and
RNA. For our purposes here, we have analyzed
separately the contributions of atoms in the backbone and in the nucleobases. The results in Table 2
clearly demonstrate that the larger intramolecular
entropy of DNA is due to the greater disorder in the
backbone. This nding is further supported from
inspection of the distributions of selected backbone
angles sampled along the trajectory (see Figure 3).
However, the uctuations in the backbone do not
affect the nucleobases, whose entropy in DNA and
RNA is not very different. Finally, it is worth noting
that the addition of backbone and nucleobase
entropies approaches the total entropy, conrming
the relatively small coupling between backbone and
nucleobases movements.
Essential dynamics analysis
Figure 3. Distributions (tted to normalized Gaussians)

of selected backbone angles (g,c,d,3) in DNA and RNA
trajectories.
Similarity analysis (see equations (1) and (2))

shows that, in general, the easiest essential motions
in DNA and RNA are similar, they being associated
with twisting and bending of the polymers. When
similarity is computed using 500 essential movements, absolute all-atoms similarity indexes
gDNA/DNA and gRNA/RNA are around 0.87, which
are close to similarity indexes obtained considering
only the backbones (see Table 3). This demonstrates
the larger complexity of the essential movements of
backbones compared to those of the nucleobases.
Furthermore, the all-atom cross similarity index
gRNA/DNA is around 0.74 (yielding a relative
631

Table 3. Absolute (g) and relative (k) similarity indexes
for DNA and RNA essential movements
Ten eigenvectors
gRNA/DNA
gRNA/RNA
gDNA/DNA
kRNA/DNA
500 eigenvectors
gRNA/DNA
gRNA/RNA
gDNA/DNA
kRNA/DNA
All atoms
Nucleobases
Backbone
0.564
0.860
0.794
0.682
0.616
0.926
0.829
0.701
0.566
0.865
0.770
0.692
0.744
0.873
0.878
0.850
0.938
0.967
0.967
0.970
0.799
0.919
0.916
0.870
Calculation is repeated considering the rst ten and 500 essential

movements.
kRNA/DNA of 0.85; see Table 3), reecting the

different nature of the essential movements in
DNA and RNA backbones (gRNA/DNAZ0.799 and
kRNA/DNAZ0.87 in the backbone; see Table 3).
Absolute self-similarity indexes obtained considering only the rst ten eigenvectors are much
higher for RNA than for DNA (see Table 3). This
nding demonstrates that more essential movements are necessary to explain the exibility of
DNA than that of RNA, which reects the higher
complexity of DNA exibility compared to that
of RNA.
In summary, despite a general similarity differences exist in the dynamics of DNA and RNA.
These differences are mostly related to the higher
complexity of movements in DNA, and to the
nature of the essential movements of DNA and
RNA backbones.
Stiffness analysis
The stiffness analysis provides useful information
on the force necessary to deform a molecule along a
given geometrical variable. Thus, the analysis
performed using the eigenvectors obtained by
diagonalization of the covariance matrix as deformation variables can be used to measure the
deformability of DNA/RNA along the axis dened
by their essential motions. Elastic force constants
associated with the rst ve essential movements of
2) associated to
Figure 4. Force constants (in cal/mol A
different essential movements of DNA and RNA. Essential movements are labelled in decreasing order of impact
in the explanation of trajectory variance.
RNA are similar or even weaker than those of DNA,

but the situation dramatically changes when further
essential movements are considered (see Figure 4),
and the RNA becomes stiffer than DNA. Therefore,
RNA is very deformable along a small set of
essential motions, but very rigid along all the
others, whereas DNA has a more degenerated
pattern of deformability (see Figure 4). Note that
this agrees with the picture of DNA as a molecule
with a much more complex exibility pattern than
RNA.
The stiffness analysis performed using essential
motions as deformation variables provides information on the natural deformability of nucleic
acids. However, it is difcult to interpret in term
of canonical motions (see Methods). Thus, we
extended the stiffness analysis using helical parameters as deformation variables. First, we used a
reduced set of global parameters (tilt, roll, twist and
stretch) for the 10mer central portion of DNA and
RNA duplexes. The global parameters are dened
essentially,29 and also we describe the geometry of
the fragment using the X3DNA analyzer to maintain consistency with other parts of this study. The
global stretch is the sum of distances between
neighbouring base-pairs along the fragment, global
twist is the sum of base-pair step local twists. We
dene a middle frame by taking its z-axis as the
average of the normal vectors of the rst and last
base-pair in the fragment, and its x-axis as the
projection of the x-axis of the central base-pair onto
the plane perpendicular to the z-axis. The y-axis
complements the set to form a right-handed triad.
The global roll and tilt are then dened exactly as
roll and tilt in the X3DNA algorithm, using the
middle frame just described. Since the x-axis of a
base-pair (as dened by X3DNA) is close to the
dyadic vector pointing to the major groove, our
global roll measures the bending of the fragment
towards the major groove in its center, and global
tilt describes the bending in the perpendicular
direction. If the fragment has even number of
pairs, we rst average the x-axes of the two central
base-pairs.
The stiffness at the global level can be described
by the stretch modulus and by twisting and
bending persistence lengths. Since we measure
bending in two perpendicular directions, we have
two bending persistence lengths corresponding to
the global roll (bending towards the grooves) and
global tilt. In this way, we can capture a possible
anisotropy in the elastic behaviour. For longer
fragments (several helical turns) the anisotropy is
in general supposed to disappear and the bending
stiffness is then fully described by one, isotropic
bending persistence length, which can be estimated
as the harmonic average of the two anisotropic
ones.29
Table 4 indicates that deformation of the global
twist of DNA is easier than that of RNA, but the
stiffness of DNA and RNA with respect to roll is
similar, and quite surprisingly DNA is stiffer than
RNA in terms of global tilt and stretch. Caution is
632
Table 4. Elastic force constants for deformations along a reduced set of global helical parameters (angular force constants
2) computed for the central 10mer portion of DNA
in kcal/mol degree2 and displacement force constants in kcal/mol A
and RNA duplexes (top); RNA and DNA length independent persistence lengths for tilt, roll and twist deformations (in
nanometers) and stretch modulus (in picoNewtons) (bottom)
Top
DNA
RNA
Bottom
DNA
RNA
a
b
Global tilta
Global rolla
Global twista
Global stretchb
0.0043
0.0030
0.0059
0.0058
0.0056
0.0129
1.50
0.80
73.85
56.01
101.85
107.80
96.15
242.00
3269
1891
In kcal/mol deg2.
2.
In kcal/mol A
then necessary when general global concepts like

exibility or rigidity are applied to DNA and RNA.
Stiffness analysis performed using local helical
parameters (tilt, roll, twist, shift, slide and rise)
computed using X3DNA 30 provides a direct
measure of the average local deformability of
each base-pair in DNA and RNA. Fortunately for
our purposes, the local helical values sampled in
our MD simulations follow a normal distribution
(r2O0.998 between the real distribution and the
Gaussians predicted one (equation (7)), which
supports the goodness of the harmonic analysis
described below). RNA is around 190%, 75%, 35%
and 12% stiffer than DNA regarding twist, slide,
shift and rise deformations (see Table 5). On the
contrary, DNA is stiffer than RNA in terms of
roll and tilt deformations, but the differences are
small (between 10% and 20% increment in forceconstants; see Table 5). When cross-terms are
considered (equation (8)), qualitative results remain

mostly unaltered (see Table 6), reecting the fact
that stiffness matrix is dominated by diagonal
terms. The only signicant difference between
force constants in Table 5 and diagonal forceconstants in Table 6 appears for rise, as a consequence of the strong (especially for RNA) positive
coupling of rise with slide and the negative one
with twist and roll.
As a whole, results in Tables 5 and 6 show that for
most local motions DNA is more exible than RNA,
but it is difcult to evaluate the implication of the
different force constants in the local deformability.
To obtain additional information in this point we
computed the local deformation energy (see
equation (9)) of DNA and RNA in front of random
perturbation of helical parameters (to obtain meaningful results random changes are limited to a
maximum of two times the largest standard
Table 5. Elastic force constants for deformations along local helical parameters of DNA and RNA
DNA
RNA
Tilta
Rolla
Twista
Shiftb
Slideb
Riseb
0.031
0.028
0.016
0.013
0.016
0.046
1.094
1.484
1.564
2.709
5.174
5.814
Couplings between different parameters are neglected.

a
In kcal/mol deg2.
b
2.
In kcal/mol A
Table 6. Stiffness matrix for deformations along local helical parameters of DNA and RNA
DNA
Tilt
Roll
Twist
Shift
Slide
Rise
RNA
Tilt
Roll
Twist
Shift
Slide
Rise
Tilt
Roll
Twist
Shift
Slide
Rise
0.031
0.001
0.023
0.000
0.004
0.014
K0.043
K0.016
K0.003
1.228
K0.001
K0.011
K0.060
0.044
1.886
0.004
K0.021
K0.186
K0.001
0.951
7.217
0.026
0.000
0.015
K0.001
0.001
0.052
K0.180
0.001
K0.020
1.369
K0.007
K0.007
K0.141
K0.030
3.193
0.004
K0.111
K0.156
K0.013
1.077
6.183
) are included. Since the Tables are symmetric, only the upper part is shown.
All couplings (in kcal/mol deg. A
633
deformations the opposite situation is found (see

also Tables 5 and 6). On the contrary, when the same
analysis is performed using global elastic constants,
RNA is not found to be more rigid than DNA (see
Figure 5), in line with the results found in essential
dynamics and entropy analysis.
In summary, stiffness analysis using either essential motions or helical coordinates demonstrates
that the concepts of exibility/rigidity in DNA and
RNA are complex, and that the ability of a polymer
to be perturbed by an external force depends on the
type of deformation. Overall, DNA is more susceptible than RNA to local deformations, but the
resulting local geometrical changes are more
efciently propagated to the global level in RNA.
Analysis of interaction proles
Figure 5. Elastic energy (in kcal/mol) associated to
local (top) or global (bottom) distortions of DNA and
RNA. In order to reduce noise, values represented here
are averaged in blocks of 100 congurations.
deviation for each helical parameter). The elastic

energy proles (see Figure 5) clearly demonstrate
that in terms of local deformations RNA is stiffer
than DNA, despite that for a few types of special
As noted by other authors,15,21,22 the relative

exibility of DNA and RNA is probably not only a
direct consequence of its covalent structure, but also
of its solvent environment (water and counterions).
Solvation maps (see Figure 6) are clearly different
for DNA and RNA because of the different groove
distribution and the presence of an extra hydroxyl
group, which alters the electrostatic potential
around the polymer (see Figure 7). The total
number of water molecules that solvate the RNA
duplex is slightly larger (by around three per
Figure 6. Solvation maps for DNA (left) and RNA (right). Water density contours correspond to an apparent density of
2 g/ml.
634
Figure 7. Classical molecular interaction potentials (in kcal/mol) for DNA (left) and RNA (right). Contour plots
correspond to K4 kcal/mol for RNA and K3 kcal/mol for DNA.
base-pair in average; see Table 7) than for the DNA,

the difference being mostly located in the backbone
(see Table 7). However, longer residence times are
found in general for DNA than for RNA, including
those in the backbone (see Table 8), which is more
exible for DNA than for RNA (see above). These
results strongly suggest that polymer exibility is
not closely related to specic solvation, and that the
DNA/RNA motions are slow enough to allow a fast
reorganization of the solvent.
The different electrostatic potential around DNA
and RNA generates a different pattern of interaction
with NaC (see Figure 7), thus justifying a different
counterion distribution around DNA and RNA.
Not only are NaC located closer to RNA than to
DNA, but they have larger residence time for RNA
when bound to the nucleic acid structure (up to 2 ns
residence times are found for RNA in our simulations; see Table 7). Most of these extra bound-
NaC are located in the major groove of the RNA,

which seems to be a better cationic binding site than
the minor groove of DNA. It is worth to note that
similar results were previously obtained by Westhof
and co-workers21,22 and Cheatham & Kollman.15
Thus, though much caution is necessary in the
analysis of counterion distribution due to the slow
NaC equilibration,25 MD simulations suggest that
the ability of RNA to tightly capture NaC is larger
than that of DNA. However, we must emphasize
that, as shown in Figure 2, the major groove is more
exible in RNA than in DNA, which indicates that
NaC binding has not a direct relationship with
differential exibility.
Conclusions
Essential dynamics analysis strongly suggests
Table 7. Average number of water molecules located at each base-pair of DNA and RNA
DNA
RNA
Backbone
m-groove
M-groove
Grooves (both)
Total
10.4
12.8
6.5
6.1
8.4
9.3
14.9
15.4
25.3
28.2
from a heteroatom of DNA or RNA.

A contact is detected when there is a water oxygen at less than 3.5 A
635

Table 8. Maximum residence times (in ps) for water
molecules (roman font) or sodium (in italics) bound to
polar groups of DNA/RNA
Atom
DNA
RNA
O2 0
O4 0
O3 0
O1P
O2P
O5 0
617
270
518/668
410/298
266
244
171
113
234/723
510/600
388
Cyt O2
Gua N3
Gua N2
Thy/Ura O2
Ade/Ura N3
600/353
438
534
521
571
200/273
145
244
245
175
Cyt N4
Gua O6
Gua N7
Thy/Ura O4
Ade N6
Ade N7
297
537
352/121
402/351
563
558/415
332
544
465/655
374/449
442
500/2006
First block, backbone atoms; second block, atoms in the minor

groove; third block, atoms in the major groove.
that the pattern of exibility of DNA and RNA

duplexes is different. In the rst case there are many
possible deformations of low energetic cost, while
for RNA there are a few very soft deformations, but
the others are very stiff.
Entropy and stiffness analysis suggest that overall the DNA duplex is more disordered and can
uctuate more than the RNA one. However, this
greater exibility of DNA is mostly local and due to
backbone uctuations that do not introduce large
global changes in the helix.
Stiffness analysis using local helical parameters
shows that DNA is much more exible than RNA in
terms of local twist, slide and shift and slightly
more exible for local rise. RNA is slightly more
exible in terms of local roll and tilt deformations.
Considering global helical deformations the DNA is
more deformable in terms of global twist, but stiffer
for global tilt and stretch.
Overall, molecular dynamics simulations demonstrate that the concept of exibility needs to be
applied with caution to duplexes of RNA and DNA,
since one duplex can be more exible for some
perturbations and more rigid for others. Clearly, a
new, more complex view of nucleic acids dynamics
seems necessary.
Methods
were then optimized, heated and pre-equilibrated using

our standard multistage protocol,31,32 followed by 1 ns
unrestrained MD simulation for equilibration. Finally, a
10 ns trajectory was collected for each structure. All MD
simulations were performed in the isothermic-isobaric
ensemble (1 atm, 298 K). Periodic boundary conditions
and the Particle Mesh Ewald (PME33) technique were
used to account for long-range effects. All bonds were
constrained using SHAKE,34 which allowed us to use 2 fs
time step for integration of Newtons equations.
Parm9924,35 and TIP3P 36 force-elds were used to
describe molecular interactions. Global translations
were removed every 0.5 ns to remove erroneous partitions of the kinetic energy of the system. The analysis
(see below) was performed using the last 9 ns of each
trajectory and removing the base-pairs at both 5 0 and 3 0
ends. All the trajectories were obtained using the
SANDER module of AMBER6.1 computer program.
Structural and energetic analysis
The helical parameters of the structures sampled along
the 10 ns MD simulations were analyzed using the
X3DNA program.30 Additional geometrical parameters
were derived with analysis modules in AMBER and with
in house software. Classical molecular interaction potentials were computed using the CMIP program37 with NaC
as probe particle, and the electrostatic potentials were
computed by solving numerically the Poisson-Boltzman
equation.38. Water densities around DNA and RNA
duplexes were determined by integrating water population around polar atoms in DNA/RNA (cutoff distance
). Maximum water residence times were comof 3.5 A
puted by tracing all water molecules around polar groups
of the nucleic acids.
Essential dynamics
This powerful analysis was used to determine which
type of motions represents the most important structural
uctuations in DNA and RNA.25,37,38 Essential motions
were determined from a principal component analysis. To
this end, covariance matrices for common atoms of DNA
and RNA (i.e., by excluding 5-methyl/H groups of T/U
and 2 0 -OH/H groups in sugar moieties) were built and
diagonalized. The resulting eigenvectors dene the type
of essential motions, and the associated eigenvalues
determine how much of variance in the trajectory is
explained by each eigenvector. Note that for two
molecules of the same size the number of eigenvectors
necessary to explain a given positional variance indicates
the complexity of the molecular motions: the larger the
number of essential motions, the greater the complexity.
The similarity between the essential motions of two
molecules (dened by covariance matrices of the same
size) can be computed using absolute (g) and relative (k)
similarity indexes,25,41,42 as shown in equations (1) and
(2):
gAB Z
Molecular dynamics simulations

Two dodecamers, d(CGCGAATTCGCG)2 and
r(CGCGAAUUCGCG)2, were built up using standard
helical parameters26 for B-DNA and A-RNA conformations. The structures were surrounded by around
4400 water molecules and 22 NaC placed in the regions of
more electronegative potential. The hydrated systems
n X
n
1 X
nA $nB 2
n jZ1 iZ1 i j
(1)
where nA
i stands for the unit eigenvector i of molecule A, n
is the minimum number of essential motions that account
for a given variance in the trajectory and the dot denotes a
scalar product:
g
kAB Z 2 T AB T
(2)
gAA C gBB
636
where the self-similarity indexes gTAA are calculated by

comparing eigenvectors obtained with the rst and
second parts of the same trajectory.
orthogonal (independent), harmonic (elastic) force constants KX can be easily derived from the variance of the
variable X during the trajectory using equation (6):
Entropy calculation
KX Z kT
Since the intramolecular entropy is the best single

parameter to describe the intrinsic order of a molecule, it
provides a good estimate of the overall exibility of DNA
and RNA. The entropy of DNA and RNA was determined
using Schiltters43 and Andreocci-Karplus44 methods. The
two models are based on the diagonalization of the massweighted covariance matrix, and on the use of a simple
harmonic oscillator (hybrid classical-quantum (S) or
purely quantum (AK)) to link eigenvalues and entropy
(see equations (3) and (4)). As noted before, intramolecular entropies were computed considering only
atoms common to DNA and RNA:

X
e2
S z0:5k
(3)
ln 1 C 2
a
i
S Zk
X
i
ai
K ln1 K eKai
eai K 1
(4)
where ai Z Zui =kT, u denotes the eigenvalues obtained by

diagonalization of the mass-weighted covariance matrix,
and the sum extends to all the non-trivial vibrations.
The entropy estimates obtained by equations (3) and (4)
depend on the length of the trajectory (the larger the
simulation, the greater the number of visited microstates),
which generates uncertainties in the calculation. To
accelerate convergence, we used the extrapolation
method,28 which allows us to estimate the entropy at
innite simulation times from the time-evolution of the
entropy estimated along the trajectory.25,28
Ki Z kT=li
(5)
2
) associated with the
where li is the eigenvalue (in A
essential movement ni determined by diagonalization of
the covariance matrix, T is the temperature (in Kelvin), k is
the Boltzman constant and Ki is a force constant
associated to the essential motion.
Stiffness analysis applied to essential motions provides
useful 3-D information on the deformability of nucleic
acids. Unfortunately, it is not always simple to interpret
the nature of eigenvectors, thus making it desirable to
compute stiffness in a helical coordinate system closer to
chemical intuition.45,47 Under the assumptions that the
distribution of values adopted by a given variable X is
fully Gaussian and that the deformation variables are
(6)
where Xo is the equilibrium value of variable X and the

brackets mean that uctuations are averaged over
thermodynamic ensemble.
An alternative way to compute force constants for a
given deformable variable can be obtained by tting the
normalized histogram of values sampled for a given
parameter along the trajectory, =X, to a Gaussian
function (equation (7)). The exponent of the tted
Gaussian provides directly the force constant, and the
goodness of the tting (determined for example by the c2
test) gives an indication of the harmonicity of the
deformation:

1
KX
2
=X K p exp
X K X0 K errorX Z 0
2kT
2p
(7)
The non-orthogonality of the helical parameters can be

taken specically into account by means of coupling
terms, which requires the inclusion of force constants, Kij,
to account for contributions to the deformation energy
arising from coupling interactions. After simple matrix
algebra, equation (7) can be generalized by dening the
stiffness matrix K with entries Kij, which can be easily
derived by inverting the covariance matrix C with entries
Cij Z hXi K Xi0 Xj K Xj0 i as shown in equation (8).
Diagonal elements in K represent contributions to
deformation arising from individual helical variables,
while off-diagonal components account for coupling
terms:
K Z kTCK1
Stiffness analysis
The positional uctuations of atoms along the trajectory
were used to derive force-constants to describe the
stiffness of DNA or RNA with respect to certain types
of elastic deformations. This analysis provides then a
unique tool for the description of the exibility of nucleic
acids.25,29,4547
We rst computed the stiffness associated with the
deformation of DNA and RNA along their essential
modes. This analysis provides quantitative information
on the deformability of both helices along the easiest
motional pathways. Taking advantage of the orthogonality of eigenvectors and assuming a harmonic behaviour,
force constants are easily determined from the eigenvalues by using equation (5):
1
hX K Xo 2 i
(8)
Note that when the stiffness matrix is known, the

deformation energy of a given conguration can be
calculated by equation (9), where the subindex 0 stands
for the equilibrium value:
Edef Z
X Kii
i
Xi K Xi0 2
X Kij
isj
Xi K Xi0 Xj K Xj0
(9)
Acknowledgements
This work has been supported by the Spanish
Ministry of Science and Technology (BIO2003-06848
and SAF2002-04282), the Fundacion BBVA and the
VIth Framework Program of the European Union
(UE project QLG1-CT-1999-00008). The computer
facilities of the CIRI-CEPBA computer centre are
also acknowledged. F.L. thanks for support from the
Swiss National Science Foundation.
637
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for the description of the solvent effect in biomolecular systems. Chem. Rev. 100, 41874226.
39. Sherer, E. C., Harris, S. A., Soliva, R., Orozco, H. &
Laughton, C. A. (1999). Molecular dynamics studies
of DNA A-tract structure and exibility. J. Am. Chem.
Soc. 121, 59815991.
40. Wlodek, S. T., Clark, T. W., Scott, L. R. & McCammon,
J. A. (1997). Molecular dynamics of acetylcholinesterase dimer complexed with tacrine. J. Am. Chem. Soc.
119, 95139522.
41. Cubero, E., Abrescia, N. G. A., Subirana, J. A., Luque,
F. J. & Orozco, M. (2003). Theoretical study of a new
DNA structure: the antiparallel Hoogsteen duplex.
J. Am. Chem. Soc. 125, 1460314612.
42. Rueda, M., Kalko, S. G., Luque, F. J. & Orozco, M.
(2003). The structure and dynamics of DNA in the gas
phase. J. Am. Chem. Soc. 125, 80078014.
43. Schlitter, J. (1993). Estimation of absolute and relative

entropies of macromolecules using the covariancematrix. Chem. Phys. Letters, 215, 617621.
44. Andricioaei, I. & Karplus, M. (2001). On the calculation of entropy from covariance matrices of the
atomic uctuations. J. Chem. Phys. 115, 62896292.
45. Lankas, F., Sponer, J., Langowski, J. & Cheatham, T. E.
(2003). DNA basepair step deformability inferred
from molecular dynamics simulations. Biophys. J. 85,
28722883.
46. Matsumoto, A. & Olson, W. K. (2002). Sequencedependent motions of DNA: a normal mode analysis
at the base-pair level. Biophys. J. 83, 2241.
47. Olson, W. K., Gorin, A. A., Lu, X. J., Hock, L. M. &
Zhurkin, V. B. (1998). DNA sequence-dependent
deformability deduced from protein-DNA crystal
complexes. Proc. Natl Acad. Sci. USA, 95, 1116311168.
Edited by P. J. Hagerman
(Received 8 March 2004; received in revised form 30 June 2004; accepted 6 July 2004)
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Published on Web 03/03/2005
Structure, Recognition Properties, and Flexibility of the

DNARNA Hybrid
Agnes Noy,, Alberto Pe rez,, Manuel Ma rquez, F. Javier Luque,*, and
Modesto Orozco*,,
Contribution from the Molecular Modeling and Bioinformatics Unit, Parc Cientific de
Barcelona, Josep Samitier 1-5, Barcelona 08028, Spain, Departament de Bioqumica i Biologa
Molecular, Facultat de Qumica, UniVersitat de Barcelona, Mart i Franque` s 1, Barcelona
08028, Spain, Departament de Farma` cia, Unitat de Fisicoqumica, Facultat de Farmacia,
UniVersitat de Barcelona, AVgda Diagnonal 643, Barcelona 08028, Spain, and
Los Alamos National Laboratory, Chemistry DiVision, Los Alamos, New Mexico 87545
Received November 8, 2004; E-mail: modesto@mmb.pcb.ub.es; javier@far1.far.ub.es
Abstract: Molecular dynamics is used to investigate the properties of the DNARNA hybrid in aqueous
solution at room temperature. The structure of the hybrid is intermediate between A and B forms but, in
general, closer to the canonical A-type helix. All the riboses exhibit North puckerings, while 2-deoxyriboses
exist in North, East, and South puckerings, the latter being the most populated one. The molecular recognition
pattern of the DNARNA hybrid is a unique combination of those of normal DNA and RNA duplexes. Finally,
the results obtained from essential dynamics and stiffness analysis demonstrate the large and very
asymmetric flexibility of the hybrid and the strong predilection that each strand (DNA or RNA) has on the
nature of their intrinsic motions in the corresponding homoduplexes. The implications of the unique structural
and dynamic properties of the DNARNA hybrid on the mechanism of cleavage by RNase H are discussed.
Introduction
There are two major types of heterogeneous nucleic acids:

hybrids and chimeras. In the former, not all the strands are of
the same type (DNA or RNA), while in the latter, both DNA
and RNA coexist in at least one of the strands. Hybrids and
chimeras are minor species but play a key role in the cell life.
Thus, transient DNARNA hybrids are formed as the RNA
strand is created using the DNA template. Moreover, DNA
replication relies on the existence of Okazakis fragments, which
are hybrid chimeras, where one strand is pure DNA and the
other is an RNA-DNA chimera.1 Furthermore, RNA viruses
create DNARNA hybrids during retrotranscription, and the
stability of these hybrid sequences is crucial in the replication
cycle of these viruses.2
The large research effort spent in the past decade on the study
of DNARNA hybrids is due not only to its biological relevance
but also to its potential therapeutic application in antisense
therapy. This new pharmacological strategy fights diseases by
inactivation of the pathological messenger RNA (mRNA)
following three main possible mechanisms:3-8 (i) the degrada
Parc Cientific de Barcelona.

Facultat de Qumica, Universitat de Barcelona.
Facultat de Farmacia, Universitat de Barcelona.
Los Alamos National Laboratory.
(1) Meselson, M.; Stahl, F. W. Proc. Natl. Acad. Sci. U.S.A. 1958, 44, 671.
(2) Alberts, B.; Bray, D.; Lewis, J.; Raff, M.; Roberts, K.; Watson, J. D.
Molecular Biology of the Cell, 3rd ed.; Garland Publishing Inc.: New York,
1994.
(3) Biroccio, A.; Leonetti, C.; Zupi, G. Oncogene 2003, 22, 6579.
(4) Dean, N. M.; Bennett, C. F. Oncogene 2003, 22, 9087.
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Nat. Biotechnol. 1996, 20, 384.
4910
J. AM. CHEM. SOC. 2005, 127, 4910-4920
tion of the mRNA by means of the RISC-DICER mechanism,

(ii) the sterical interference of translation or splicing by a
complementary oligonucleotide (typically a DNA derivative)
bound to the target mRNA, and (iii) the degradation of the
mRNA bound to a complementary DNA analogue by RNase
H, which is a specific enzyme that recognizes in a catalytically
active manner only DNARNA hybrids and degrades the RNA
strand of the hybrid. At the present time, most antisense
treatments in clinical trials are based on the activation of the
RNase H mechanism,3-6 and then the design of stable and
RNase H-susceptible hybrids is crucial.
The structure of DNARNA hybrids has been largely studied
by means of experimental techniques. Early fiber diffraction
data9,10 suggested that the structure of the hybrid was very close
to the A-form. This finding was also supported by many highresolution X-ray data. Thus, the structure of an Okazakis
fragment, r(gcg)d(TATACGC), solved by Richs group11
showed that it was very close to a canonical A-helix, with all
sugars in the North conformation. The same conclusions were
found by different crystallographers in other hybrid-chimeras.12,13 Several pure DNARNA hybrids solved by X-ray
(6) Taylor, M. F.; Wiederholt, K.; Sverdrup, F. Drug DiscoVery Today 1999,
4, 562.
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A.; Diber, A.; Biton, S.; Tamir, Y.; Khosravi, R.; Nemzer, S.; Pinner, E.;
Walach, S.; Bernstein, J.; Savitsky, K.; Rotman, G. Nat. Biotechnol 2003,
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1967, 57, 1804.
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S. A.; Rich, A. Nature 1982, 299, 601.
10.1021/ja043293v CCC: $30.25 2005 American Chemical Society
Characteristics of the DNARNA Hybrid
crystallography exhibit a typical A-form, with all riboses in the

North conformation and all14 or almost all15-17 2-deoxyriboses
with North puckerings. Surprisingly, data provided by lowresolution NMR, CD, and Raman18-21 spectroscopies raise
doubts on the X-ray structural picture of the hybrid; since all
riboses are in the North conformation, a sizable portion of 2deoxyriboses exist in the South form, which is traditionally
linked to the B-DNA. The same is found in recent NMR studies,
which suggest a general structure of the hybrid intermediate
between A and B canonical helices, with many characteristics
of the A-form, but with important alterations in the grooves
and with a large percentage of 2-deoxyriboses in South
conformations.22-33
The discrepancy between high-resolution NMR and X-ray
studies cannot be fully explained from the differences in base
sequence between hybrids solved by X-ray (typically with
polypyrimidine in the DNA strand) and by NMR (generally
more heterogeneous), as demonstrated by Gyi et al. and Fedoroff
et al.24,29,30 Clearly, experimental conditions are driving the
structure toward the A and A/B conformations, suggesting a
unique intrinsic structural plasticity in the DNARNA hybrid.
Though the discrepancy between NMR and X-ray results
makes it difficult to define the major conformation of the hybrid
in physiological conditions, it seems that NMR data provide
an easier explanation of the fact that while DNARNA hybrids
are degraded by RNase H, pure RNA duplexes are inhibitors.34
Thus, according to NMR results, the smaller width of the minor
groove in the hybrid compared to that in the pure RNA duplex
has been considered to be the main structural feature to explain
the different enzymatic susceptibility to RNase H.16,21,22,24,33,34
However, recent studies have pointed out that factors other than
the shape of the minor groove must also be involved,32,33 thus
raising doubts on the structural determinants that justify the
degradation of the hybrid by RNase H.
Compared with the large amount of high-level experimental
data, very few theoretical studies have examined the structure
of the DNARNA hybrid. In a seminal paper, Cheatham and
(12) Egli, M.; Usman, N.; Zhang, S. G.; Rich, A. Proc. Natl. Acad. Sci. U.S.A.
1992, 89, 534.
(13) Egli, M.; Usman, N.; Rich, A. Biochemistry 1993, 32, 3221.
(14) Xiong, Y.; Sundaralingam, M. Nucleic Acids Res. 2000, 28, 2171.
(15) Conn, G. L.; Brown, T.; Leonard, G. A. Nucleic Acids Res. 1999, 27, 555.
(16) Horton, N. C.; Finzel, B. C. J. Mol. Biol. 1996, 264, 521.
(17) Katahira, M.; Lee, S. J.; Kobayashi, Y.; Sujeta, H.; Kyogoku, Y.; Iwai, S.;
Ohtsuka, E.; Benevides, J. M.; Thomas, G. J. J. Am. Chem. Soc. 1990,
112, 4508.
(18) Chou, S. H.; Flynn, P.; Reid, B. Biochemistry 1989, 28, 2422.
(19) Chou, S. H.; Flynn, P.; Reid, B. Biochemistry 1989, 28, 2435.
(20) Hall, K. B.; McLaughlin, L. W. Biochemistry 1991, 30, 10606.
(21) Fedoroff, O. Y.; Salazar, M.; Reid, B. R. J. Mol. Biol. 1993, 233, 509.
(22) Fedoroff, O. Y.; Ge, Y.; Reid, B. R. J. Mol. Biol. 1997, 269, 225.
(23) Lane, A. N.; Ebel, S.; Brown, T. Eur. J. Biochem. 1993, 215, 297.
(24) Salazar, M.; Fedoroff, O. Y.; Miller, J. M.; Ribeiro, N. S.; Reid, B. R.
Biochemistry 1993, 32, 4297.
(25) Gao, X.; Jeffs, P. W. J. Biomol. NMR 1994, 4, 367.
(26) Gonzalez, C.; Stec, W.; Kobylanska, A.; Hogrefe, R. I.; Reynolds, M.;
James, T. L. Biochemistry 1994, 33, 1062.
(27) Gonzalez, C.; Stec, W.; Reynolds, M.; James, T. L. Biochemistry 1995,
34, 4969.
(28) Gyi, J. I.; Conn, G. L.; Lane, A. N.; Brown, T. Biochemistry 1996, 35,
12538.
(29) Gyi, J. I.; Lane, A. N.; Conn, G. L.; Brown, T. Biochemistry 1998, 37, 73.
(30) Gyi, J. I.; Gao, D.; Conn, G. L.; Trent, J. O.; Brown, T.; Lane, A. N. Nucleic
Acids Res. 2003, 31, 2683.
(31) Cross, C. W.; Rice, J. S.; Gao, X. Biochemistry 1997, 36, 4096.
(32) Nishizaki, T.; Iwai, S.; Ohkubo, T.; Kojima, C.; Nakamura, H.; Kyogoku,
Y.; Ohtsuka, E. Biochemistry 1996, 35, 4016.
(33) Szyperski, T.; Gotte, M.; Billeter, M.; Perola, E.; Cellai, L.; Heumann, H.;
Wuthrich, K. J. Biomol. NMR 1999, 13, 343.
(34) Salazar, M.; Fedoroff, O. Y.; Zhu, L.; Reid, B. R. J. Mol. Biol. 1994, 241,
440.
ARTICLES
Kollman35 reported 2 ns molecular dynamics (MD) trajectories

of a 10-mer DNARNA duplex with Cornells force field.36 They
found that, irrespective of the starting structure (pure A- or
B-forms), the hybrid adopts a mixed A/B conformation, whose
general conformation resembles that of the A-form, but with
2-deoxyribose puckerings in the South region, thus supporting
NMR data and previous JUNMA results by Laverys group.37
Identical conclusions were reached by Venkateswarlu et al. from
1 ns MD samplings38 and more recently by Lane and coworkers30 in a 2 ns MD study.
In this paper, we will present extended MD simulations of
DNARNA hybrids starting from two different X-ray structures.
Present trajectories (which expand to 10 times longer simulation
time than previous ones) analyzed with the help of a powerful
set of datamining algorithms allowed us to obtain a complete
picture of the structure and dynamics of DNARNA hybrids.
Our trajectories clearly converge to a conformation close to that
suggested by NMR techniques. The structure, molecular recognition, and especially flexibility characteristics of the hybrid
are determined and compared with those obtained for pure DNA
and RNA duplexes built up with the equivalent base sequence.
Finally, a tentative explanation of the specificity of RNase H
for the DNARNA hybrid is provided.
Methods
Molecular Dynamics Simulations. Dodecamers of sequence
r(cgcgaauucgcg)d(CGCGAATTCGCG) were built using as template
(i) the PDB crystal structure 1FIX39 and (ii) an A-form conformation
taken from Arnotts fiber diffraction data as implemented in the
Biopolymer module of InsightII.40 Choice of the Dickersons sequence
allowed us to compare the hybrid trajectories with those recently
collected for the same sequence in DNA and RNA duplexes41 and which
will be used as reference for pure duplexes. Moreover, the selection of
these two starting structures allows us to determine whether MD
simulations36,42 are consistent with crystal A-like structures or whether
the trajectories spontaneously jump to the A/B NMR-like conformation.
The structures were first partially optimized (2000 cycles with
restraints in the backbone) to avoid bad contacts emerging from changes
in the sequence of the bases used in MD simulations with regard to
that present in the X-ray crystallographic structure. The hybrids were
surrounded by approximately 4400 water molecules and 22 Na+
molecules placed in the regions of more electronegative potential (this
generates rectangular boxes, 62 62 57 (in angstroms)), with greater
than 12 of waters from the DNA to the faces of the box. The hydrated
systems were then optimized, heated, and equilibrated using our
standard multistage protocol with length double than usual43,44 to prevent
any equilibration problem arising from the fact that we are studying
hybrids and not homoduplexes. Then, two (11 and 5 ns) unrestrained
MD simulations were run (the first nanosecond was considered extra
equilibration in both cases). All MD simulations were performed in
(35) Cheatham, T. E.; Kollman, P. A. J. Am. Chem. Soc. 1997, 119, 4805.
(36) Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K. M.;
Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell, J. W.; Kollman, P.
A. J. Am. Chem. Soc. 1995, 117, 5179.
(37) Sanghani, S. R.; Lavery, R. Nucleic Acids Res. 1994, 22, 1444.
(38) Venkateswarlu, D.; Lind, K. E.; Mohan, V.; Manoharan, M.; Fergurson,
D. M. Nucleic Acids Res. 1999, 27, 2189.
(39) Horton, N. C.; Finzel, B. C. J. Mol. Biol. 1996, 264, 521.
(40) (a) Arnott, S.; Bond, P. J.; Selsung, E.; Smith, P. J. Nucleic Acids Res.
1976, 3, 2459. (b) Insight II; Accelrys Co.: San Diego, CA, 2004.
(41) Noy, A.; Perez, A.; Lankas, F.; Luque, F. J.; Orozco, M. J. Mol. Biol.
2004, 343, 627.
(42) Cheatham, T. E.; Cieplak, P.; Kollman, P. A. J. Biomol. Struct. Dyn. 1999,
16, 845.
(43) Shields, G. C.; Laughton, C. A.; Orozco, M. J. Am. Chem. Soc. 1997, 119,
7463.
(44) Shields, G. C.; Laughton, C. A.; Orozco, M. J. Am. Chem. Soc. 1998, 120,
5895.
J. AM. CHEM. SOC.
VOL. 127, NO. 13, 2005 4911
Noy et al.
ARTICLES
the isothermic-isobaric ensemble (1 atm, 298 K). Periodic boundary
conditions and the Particle Mesh Ewald (PME45) technique were used
to treat long-range effects. All bonds were constrained using SHAKE,46
which allowed us to use an integration time step of 2 fs. Parm9936,42
and TIP3P47 force fields were used to describe molecular interactions.
Global translations were removed every 0.5 ns to remove erroneous
partitions of the kinetic energy of the system. Since both trajectories
converged to a similar region of the configurational space (see below),
the structural and flexibility analyses were performed by using only
the last 10 ns of the longest trajectory. Moreover, the base pairs at
both 5 and 3 ends were not considered in the analysis. All trajectories
were obtained using the SANDER module of the AMBER6.1 computer
program.48
Structural and Energetic Analysis. Geometrical parameters sampled
along the trajectories were studied using analysis modules in AMBER,
the X3DNA program,49 and in house software. Classical molecular
interaction potentials were computed using the CMIP program50 with
Na+ as a classical probe particle. Water densities around duplexes were
determined by integrating the water population around polar atoms
(cutoff distance of 3.5 ) of the nucleic acids. Water residence times
were computed by tracing all water molecules around polar groups
following the standard procedure in the PTRAJ module of AMBER.
Essential Dynamics. Essential motions41,51-53 were determined from
principal component analysis (PCA) using covariance matrixes for
common atoms of DNA and RNA (i.e., by excluding 5-methyl/H groups
of T/U and 2-OH/H groups in sugars). The diagonalization of the
covariance matrix provided eigenvectors, which describe the nature of
the essential movements, and eigenvalues, which determine the
contribution of each essential movement to the positional variance of
the trajectory. For two molecules of the same size, the number of
eigenvectors necessary to explain a given positional variance indicates
the complexity of the molecular motions; the larger the number of
essential motions, the greater the complexity.
Eigenvectors of the same dimension obtained from two trajectories
can be compared by means of the absolute and relative similarity
indexes given in eqs 1 and 2,51,54,55 which measure the similarity between
the essential deformation pattern of the two systems.
AB )
( )
n
A
i
B 2
j
(1)
where the self-similarity indexes, AAT, are calculated by comparing

eigenvectors obtained with the first and second parts of the same
trajectory.
Entropy Calculation. Intramolecular entropies were determined
using Schlitter56 and Andreocci-Karplus57 methods (see eqs 3 and 4)
and considering only atoms common to DNA and RNA (see above).
The time dependence of the entropy estimates was corrected using the
standard exponential extrapolation method.51,58
S 0.5k
S)k
Ri
Ri
( )
e2
ln 1 + R
-1
(3)
2
i
- ln(1 - e-Ri)
(4)
where Ri ) pi/kT; denotes the eigenvalues obtained by diagonalization of the mass-weighted covariance matrix, and the sum extends
to all nontrivial vibrations.
Stiffness Analysis. The positional fluctuations of atoms along the
trajectory were used to derive force constants to describe the elastic
deformability of the hybrid and their constituent strands.51,59-63 The
stiffness associated with the essential movements was determined from
the eigenvalues obtained by diagonalization of the Cartesian covariance
matrix41 (see eq 5). Alternatively, the stiffness matrix K (with entries
Kij) associated with deformability along helical parameters was
determined (see eq 6) by inverting the covariance matrix C (with entries
Cij ) (Xi - Xi0)(Xj - Xj0)). Diagonal elements in K represent
contributions to deformation arising from individual helical variables,
while off-diagonal components account for coupling terms. Note than
once the force matrix is known, the deformation energy of a given
configuration can be calculated by eq 7, where the subindex 0 stands
for the equilibrium value.
Ki ) kT/i
(5)
where i is the eigenvalue (in angstroms2) associated with the essential

movement i determined by diagonalization of the covariance matrix;
T is the temperature (in kelvin); k is the Boltzmann constant, and Ki
is a force constant associated with the essential motion.
j)1 i)1
where Ai is the unit eigenvector i of molecule A; n is the minimum

number of essential motions that account for a given variance in the
trajectory, and the dot denotes a scalar product.
K ) kTC-1
Edef )
Kii
(2)
(45) Darden, T.; York, D.; Pedersen, L. J. Chem. Phys. 1993, 98, 10089.
(46) Ryckaert, J. P.; Ciccotti, G.; Berendsen, H. J. C. J. Comput. Phys. 1977,
23, 327.
(47) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein,
M. L. J. Chem. Phys. 1983, 79, 926.
(48) Case, D. A.; Pearlman, D. A.; Caldwell, J. W.; Cheatham, T. E., III; Ross,
W. S.; Simmerling, C. L.; Darden, T. L.; Marz, K. M.; Stanton, R. V.;
Cheng, A. L.; Vincent, J. J.; Crowley, M.; Tsui, V.; Radmer, R. J.; Duan,
Y.; Pitera, J.; Massova, I.; Seibel, G. L.; Singh, U. C.; Weiner, P. K.;
Kollman, P. A. AMBER6; University of California: San Francisco, CA,
1999
(49) Lu, X. J.; Shakked, Z.; Olson, W. K. J. Mol. Biol. 2000, 300, 819.
(50) Gelpi, J. L.; Kalko, S. G.; Barril, X.; Cirera, J.; de La Cruz, X.; Luque, F.
J.; Orozco, M. Proteins 2000, 45, 428.
(51) Orozco, M.; Perez, A.; Noy, A.; Luque, F. J. Chem. Soc. ReV. 2003, 32,
350.
(52) Wlodek, S. T.; Clark, T. W.; Scott, L. R.; McCammon, J. A. J. Am. Chem.
Soc. 1997, 119, 9513.
(53) Sherer, E. C.; Harris, S. A.; Soliva, R.; Orozco, M.; Laughton, C. A. J.
Am. Chem. Soc. 1999, 121, 5981.
(54) Cubero, E.; Abrescia, N. G. A.; Subirana, J. A.; Luque, F. J.; Orozco, M.
J. Am. Chem. Soc. 2003, 125, 14603.
4912 J. AM. CHEM. SOC.
VOL. 127, NO. 13, 2005
Kij
2 (X - X ) + 2 (X - X )(X - X )
2
AB
AB ) 2 T
(AA + TBB)
(6)
io
io
jo
(7)
i*j
Helical force constants were determined at the local (using local

helical parameters as determined by X3DNA) and global (using the
parameters defined by Lankas et al.59,60) levels. The local analysis was
performed by using all of the snapshots collected from the MD
trajectory.
Once the stiffness constants for global or local parameters are
determined, the average deformation energy of the duplex can be
(55) Rueda, M.; Kalko, S. G.; Luque, F. J.; Orozco, M. J. Am. Chem. Soc. 2003,
125, 8007.
(56) Schlitter, J. Chem. Phys. Lett. 1993, 215, 617.
(57) Andricioaei, I.; Karplus, M. J. Chem. Phys. 2001, 115, 6289.
(58) Harris, S. A.; Gavathiotis, E.; Searle, M. S.; Orozco, M.; Laughton, C. A.
J. Am. Chem. Soc. 2001, 123, 12658.
(59) Lankas, F.; Sponer, J.; Hobza, P.; Langowski, J. J. Mol. Biol. 2000, 299,
695.
(60) Lankas, F.; Sponer, J.; Langowski, J.; Cheatham, T. E. Biophys. J. 2003,
85, 2872.
(61) Olson, W. K.; Gorin, A. A.; Lu, X. J.; Hock, L. M.; Zhurkin, V. B. Proc.
Natl. Acad. Sci. U.S.A. 1998, 95, 11163.
(62) Matsumoto, A.; Olson, W. K. Biophys. J. 2000, 83, 22.
(63) For comparison purposes with present Figure 9, note that Figure 4 of ref
46 has a typo error, and values in the y-axis need to be multiplied by a
factor of 10.
Figure 1. Root-mean-square deviations (RMSD for backbone atoms in

angstroms) for the two hybrid trajectories; (top) starting 1FIX, and (bottom)
from InsightII A-form, with respect to A (red), B (blue), and NMR (black;
generated from 1EFS by (i) change of the sequence to the target one and
(ii) restricted optimization of nucleobases to remove bad contacts) structures.
Values displayed here correspond to the central 10-mer segment of the
duplex.
computed considering a common limit of deformation for each helical

coordinate. This limit should (i) make all helical deformation equally
important in the definition of the distortion energy, and (ii) allow the
comparison between DNA2, RNA2, and DNARNA stiffness. Thus, we
defined the consensus perturbation for each helical parameter as twice
the largest standard deviation found for this parameter in the three
trajectories. Conformations are then randomly generated by moving
randomly each individual helical parameter within these limits.
Results and Discussion
Convergence of the Trajectories. There is a clear displacement in the two trajectories (in less than 1 ns) from the A-form
to an A/B conformation, closer to the A- than to the B-form,
but clearly different from the canonical A-helix (see Figure 1).
Both simulations sampled the same region of the configurational
space, which is close to the conformation found in NMR
experiments (1EFS64 was used here for reference; see Figure
1). Analysis of the trajectories clearly demonstrated that, as
previous MD simulations suggested,30,36,41 the fast repuckering
of the 2-deoxyriboses from pure North to South/South-East
conformations is responsible for the structural transitions
detected in the simulations.
Taking data from our longest trajectory (very similar results
can be obtained considering the 5 ns one starting from pure
A-form), we can quantify deviations in the converged structure
with respect to reference conformations. The average RMSD
(values taken for the central 10-mer backbone (including C1)
in the last 10 ns of the trajectory) with respect to the starting
conformation is 2.9 ( 0.5 , a value which is slightly larger
than that found when RMSD is computed from a classical NMR
(64) Hantz, E.; Larue, V.; Ladam, P.; Le Moyec, L.; Gouyette, C.; Huynh Dinh,
T. Int. J. Biol. Macromol. 2001, 28, 273.
ARTICLES
Figure 2. Distributions of selected local helical parameters in DNA2, RNA2,

and DNARNA trajectories. Rotational values are in degrees, and the
translational ones are in angstroms. DNA2 (blue), RNA2 (red), and hybrid
(green).
(1EFS 64) conformation (2.2 ( 0.3 ). The RMSD with respect

to the B-form is larger (5.1 ( 0.7 with respect to Arnotts
values and 3.5 ( 0.6 with respect to the MD-averaged
conformation of B-DNA) than that with respect to A-form (3.3
( 0.4 from Arnotts40 values and 2.8 ( 0.5 from the MDaveraged conformation of the A-RNA). In summary, MD
simulations drive the structure of the hybrid from fiber or crystal
conformations to NMR-like conformation, which is not a pure
A-form, but that, in general, is clearly closer to this conformation
than to the B-form.
General Structural Characteristics. Helical analysis shows
that the average twist angle is 31, which is close to that found
for the RNA duplex (30) and smaller than that for the DNA
one (33) (see Figure 2). With regard to slide, the hybrid is
also closer to RNA than to DNA. For the rest of the local helical
parameters, the hybrid is either intermediate between DNA and
RNA or does not change significantly between the three kinds
of duplexes (see Figure 2). The intermediate A/B character of
the hybrid becomes more evident when looking to more general
descriptors, such as the shortest C1-C1 intrastrand distance41
or the global (see Methods) helical parameters (see Figure 3).
Interestingly, compared with the DNA duplex, both RNA and
hybrid duplexes seem to be less sequence-dependent, as reflected
in a more normal distribution of some helical parameters, such
as twist (see Figure 2).
There are several differences in the distribution of the
backbone dihedral angles (available upon request) with respect
to pure DNA and RNA duplexes. In general, the differences
are due to a strong asymmetry between the two strands in the
hybrid, which seems to retain some kind of predilection for the
J. AM. CHEM. SOC.
VOL. 127, NO. 13, 2005 4913
Noy et al.
ARTICLES
Figure 3. Distributions of selected global helical parameters in DNA2,

RNA2, and DNARNA trajectories. Rotational values are in degrees, and
the translational ones are in angstroms. See color code in Figure 2.
distributions found in corresponding pure duplexes (see Figure

4 for selected examples). Such a predilection is clearly reflected
in the sugar puckering since all riboses are found in the North
conformation (as expected for RNA), while 2-deoxyriboses
mainly populate South and South-East conformations (only
8-9% is found in North conformations) (see Figure 5). The
change between S T E T N puckerings of 2-deoxyriboses is
very fast (subnanosecond time scale), which indicates that
present results cannot be ascribed to limited sampling in our
MD simulations (see Figure 5). We must also notice that the
amount of East conformers in the DNA strand of the hybrid is
not different than that detected in normal DNA duplexes, which
supports suggestions by James and co-workers26,27 that the
anomalous sugar spectra found for the DNARNA hybrids are
not due to a displacement of the 2-deoxyriboses to the East
conformation, but to a fast interchange between North and South
puckerings.
The strand asymmetry in sugar puckering generates a unique
groove distribution in the hybrid. The major groove is clearly
wider than that of pure RNA and only 2 narrower than
that of pure DNA (see Figure 6). The minor groove of the hybrid
is 2 narrower than that of pure RNA and clearly wider than
that of pure DNA. Very interestingly, the minor groove of the
hybrid seems to be less sensitive to sequence effects (particularly
to A-tracks) than does that of the DNA duplex (see Figure 6),
leading to a more normal distribution in the hybrid. This
difference is probably due to the geometrical restrictions
imposed by the fixed conformation of the riboses in the RNA
strand.
Molecular Recognition Properties. The unique groove
geometry of the hybrid generates a special recognition pattern
VOL. 127, NO. 13, 2005
Figure 4. Distributions of selected backbone dihedrals in DNA2, RNA2,

and DNARNA trajectories. Taking values for individual strands (the two
DNA strand in DNA2 (blue), the two RNA strands in RNA2 (red), the DNA
strand in the hybrid (magenta), and the RNA strand in the hybrid (orange).
Values are in degrees.
compared to DNA and RNA duplexes of the same sequence.

This is noted in the cMIP distribution (Figure 7), which has
characteristics of both DNA and RNA duplexes and an
interesting asymmetry between strands in the major groove since
the most favored region for interaction with small cations is
not centered in the middle of the groove but displaced toward
the RNA strand (see Figure 7). The hybrid is very well hydrated
by an average number of 27.0 water molecules/nucleotide pair
compared with values of 25.3 and 28.2 for DNA and RNA,
respectively. In the minor groove, the amount of highly
structured water is slightly larger (see Figure 7) for DNA than
for RNA, the hybrid being in an intermediate situation. Due to
the presence of the 2-OH group, the backbone is better hydrated
in RNA (12.8 waters/single strand) and in the RNA strand of
the hybrid (12.4 waters) than in the DNA duplex (10.4 waters/
single strand) or the DNA strand (10.6 waters) of the hybrid.
The maximum water residence times around polar groups range
from 100 ps to 1 ns, and no systematic differences are detected
between DNA, RNA duplexes, and the hybrid.
Entropy Calculations. Schlitter and Andreocci-Karplus
methods were used to estimate the intramolecular entropy of
ARTICLES
Figure 5. (Top panel) Distribution of phase angles (in degrees) in DNA2, RNA2, and DNARNA trajectories. Values are obtained by taking the two strands
separately (see color codes in Figure 4). (Bottom panels) Evolution of the phase angle along the 11 ns hybrid trajectory for selected nucleotides.
Table 1. Intramolecular Entropies (in kcal/molK) Computed using
Schlitters (roman font) and Andreocci-Karpluss Methods (italics)
for DNA, RNA, and HYBRID Double Strand and Extrapolated to
Infinite Simulation Time (see Methods)a
Figure 6. Distribution of minor and major groove widths in DNA2, RNA2,

and DNARNA trajectories. Distances (in angstroms) are computed as the
shortest P-P distance along the groove minus 5.8 (van der Waals radii
of phosphates).
the duplexes. Both methods suggest that DNA is the most

disordered structure, followed by the hybrid and finally by the
RNA (Table 1). The difference in total entropy between RNA
and DNA is 0.24 kcal/molK,41 and 0.16 kcal/molK between
RNA and the hybrid. Therefore, while structural parameters
point out that the hybrid is closer to pure RNA, the hybrid is
closer to pure DNA in terms of structural disorder. As found in
a previous analysis of homoduplexes,41 such an entropic
difference arises from the backbone (entropy estimates obtained
considering only the nucleobases are nearly identical for the
three duplexes; see Table 1). Interestingly, when entropies are
computed considering only the first three essential movements,
DNA is the most ordered helix, while RNA and DNARNA
show the same level of structural disorder. When the calculation
includes the first 10 frequencies, the hybrid appears as the most
disordered structure and RNA as the most ordered one. Clearly,
entropy is not distributed uniformly in the three duplexes (see
Figure 8), a fact that was already recognized in a previous study
of DNA and RNA flexibility.41
The two strands of the hybrid retain the intrinsic flexibility
in their respective homoduplexes. Thus, the DNA strand of the
hybrid has an entropy 0.12 kcal/molK larger than that of the
RNA strand (see Table 2). Quite impressively, the entropy
estimates for the DNA and RNA strands of the hybrid nearly
DNA
(all atoms)
RNA
(all atoms)
HYBRID
(all atoms)
DNA
(nucleobases)
RNA
(nucleobases)
HYBRID
(nucleobases)
DNA
(backbone)
RNA
(backbone)
HYBRID
(backbone)
S (t ) )
S (3)
S (10)
2.14(0.05)
1.93(0.05)
1.90(0.03)
1.71(0.03)
2.06(0.03)
1.85(0.03)
0.91(0.01)
0.84(0.01)
0.91(0.02)
0.83(0.02)
0.91(0.01)
0.84(0.02)
1.43(0.07)
1.34(0.10)
1.18(0.03)
1.09(0.03)
1.34(0.04)
1.24(0.04)
0.0328
0.0327
0.0342
0.0340
0.0341
0.0339
0.0286
0.0285
0.0300
0.0299
0.0299
0.0298
0.0320
0.0319
0.0333
0.0331
0.0332
0.0331
0.0988
0.0983
0.0976
0.0971
0.0997
0.0993
0.0849
0.0845
0.0860
0.0856
0.0858
0.0853
0.0962
0.0958
0.0944
0.0940
0.0973
0.0968
a Partial entropies were computed considering only nucleobases or

backbone atoms. In all cases, values were determined considering all
frequencies, as well as only the first 3 and 10 ones.
match the average values obtained for the same strands in

homopolymers of the same sequence (see Table 2). Finally, the
entropy contributions associated with the first 10 essential
movements of the DNA and RNA strands of the hybrid follow
very closely those observed for the same strands in homopolymers (see Figure 8). In summary, the two strands of the hybrid
maintain their intrinsic entropy in pure duplexes, which is not
much altered by the counterpart, and the global entropy
properties of the hybrid should then be understood as a
combination of the local entropies of the two strands.
Essential Dynamics. As previously found in other studies,51,55
global twistings and bendings are the movements that explain
most variances in the three duplexes. The first modes in all
nucleic acids are associated with very small stiffness constants
J. AM. CHEM. SOC.
VOL. 127, NO. 13, 2005 4915
Noy et al.
ARTICLES
Figure 7. (Top) Classical Molecular Interaction Potential (in purple energy contour -3 kcal/mol), showing the best region for interaction of the different
duplexes with a classical Na+ probe. (Bottom) Solvation map (in purple density contour 2.5 g/cm3) density for the three duplexes.
(below 10 cal/mol2; see Figure 9), reflecting the extreme

plasticity of nucleic acids along their preferred deformation
pathways.63 As suggested by entropy plots (Figure 8), the DNA
appears to be very stiff for the first deformation modes, but the
situation changes for higher essential movements. The hybrid
shows stiffness constants similar to those of the RNA for the
first five components, while for lower modes, they approach
those of the DNA homoduplex (see Figure 9).
Similarity measurements for the 10 first essential modes
(those explaining more than 70% variance of all trajectories)
reveal that the nature of these movements is quite common to
the three duplexes, especially in movements involving nucleobases (see Table 3). The essential deformation modes of the
hybrid are slightly more similar to those of the RNA than to
those of the DNA (see Table 3). However, the hybrid is able to
VOL. 127, NO. 13, 2005
capture characteristics of the essential dynamics of DNA which

were not present in the RNA duplex and vice versa. Thus,
similarity indexes between the hybrid and the two homoduplexes
are larger than that obtained between pure DNA and RNA
duplexes, the difference being especially clear when a large
number (500) of eigenvectors are used in the comparison (see
Table 3).
The essential dynamics of the RNA and DNA strands of the
hybrid is significantly different, creating a unique asymmetry
in the essential deformation modes of the hybrid. Thus, the
relative similarities, , between the DNA and RNA strands of
the hybrid are 76% for 10 modes and 88% for 250 modes
(see Table 3), while when the dynamics of the two complementary strands of pure DNA and RNA duplexes is compared,
similarity indexes very close to 1.0 are obtained. The essential
ARTICLES
Figure 9. Force constants (in cal/mol2) assigned to the most essential

movements of the three duplexes. Color code as in Figure 2.
Table 3. Relative () Similarity Indexes between the Essential
Movements of DNA, RNA, and HYBRID at the Duplex (top) and
Single Strand (bottom) Levelsa
all atoms
Figure 8. Entropies (in kcal/molK) assigned to different deformation

modes of the three duplexes. (Top) Values for the two strands. (Bottom)
Values for individual strands. Color code as in Figure 4.
Table 2. Same as Table 1, but Considering Each Strand
Independentlya
DNA
(all atoms)
RNA
(all atoms)
HYBRID DNA
(all atoms)
HYBRID RNA
(all atoms)
DNA
(nucleobases)
RNA
(nucleobases)
HYBRID DNA
(nucleobases)
HYBRID RNA
(nucleobases)
DNA
(backbone)
RNA
(backbone)
HYBRID DNA
(backbone)
HYBRID RNA
(backbone)
a
S (t ) )
S (3)
S (10)
1.11(0.05)
1.03(0.07)
0.99(0.02)
0.91(0.04)
1.12(0.02)
1.04(0.03)
1.00(0.02)
0.92(0.03)
0.49(0.01)
0.45(0.01)
0.48(0.01)
0.44(0.01)
0.49(0.01)
0.45(0.01)
0.48(0.01)
0.44(0.01)
0.75(0.09)
0.71(0.14)
0.62(0.04)
0.59(0.08)
0.75(0.03)
0.70(0.04)
0.63(0.03)
0.60(0.06)
0.0310
0.0308
0.0321
0.0320
0.0324
0.0323
0.0318
0.0317
0.0268
0.0266
0.0280
0.0279
0.0281
0.0280
0.0277
0.0276
0.0303
0.0301
0.0313
0.0311
0.0317
0.0316
0.0310
0.0308
0.0928
0.0923
0.0909
0.0905
0.0944
0.0940
0.0912
0.0908
0.0786
0.0782
0.0793
0.0789
0.0796
0.0792
0.0787
0.0783
0.0906
0.0901
0.0877
0.0873
0.0922
0.0918
0.0882
0.0877
For pure duplexes, values are the averages of the two strands.
movements of the RNA strand in the hybrid and in a pure RNA

duplex are very similar (90% identity for 10 modes), while the
maintenance of dynamics of the DNA strand is slightly worse
(identity 77% for 10 deformation modes (95% for 250
modes)). In summary, the two strands of the hybrid have a
surprising tendency to maintain the essential dynamics of the
DNA and RNA strands in pure homoduplexes. Such a predilection is especially strong for the RNA strand, whose deformability
pattern in the hybrid is almost identical to that found in pure
RNA duplexes.
nucleobases
backbone
HYBRID/RNA
HYBRID/DNA
DNA/RNA
Duplex
0.788/0.911
0.859/0.988
0.722/0.921
0.782/0.990
0.682/0.850
0.701/0.970
0.805/0.935
0.728/0.919
0.692/0.870
HYB_RNA/RNA
HYB_RNA/DNA
HYB_DNA/RNA
HYB_DNA/DNA
HYB_RNA/HYB_DNA
Single Strand
0.904/0.919
0.875/0.979
0.766/0.852
0.846/0.984
0.771/0.866
0.847/0.986
0.772/0.952
0.904/0.982
0.763/0.878
0.842/0.993
0.866/ 0.961
0.739/0.851
0.744/0.883
0.775/0.961
0.769/0.880
a Values in roman correspond to the calculations using the first 10 modes,

while values in italics are obtained considering the first 500 (duplex) or
250 (single strand) modes.
Table 4. Diagonal Elastic Force Constants for Deformations along

a Reduced Set of Global Helical Parameters (angular force
constants in cal/moldeg2 and displacement force constants in
kcal/mol2) Computed for the Central 10-mer Portion of DNA,
RNA, and HYBRID Duplexesa,b
DNA
RNA
HYBRID
global tilt
global roll
global twist
global stretch
4.29(0.05)
2.98(0.05)
4.56(0.06)
5.92(0.07)
5.74(0.11)
4.76(0.12)
5.58(0.07)
12.89(0.21)
9.52(0.23)
1.51(0.039)
0.80(0.009)
0.79(0.018)
a Standard deviation values have been calculated by averages using

different groups of snapshots taken every 5 ps. b The complete stiffness
matrixes are available upon request.
Helical Stiffness. As noted in previous papers,41,65 it is not

clear whether DNA is more flexible than RNA in terms of global
helical deformations. For the global twist, RNA is more rigid
than DNA, but for global stretch and tilt, the reverse situation
is found (see Table 4). Thus, DNA is more or less flexible than
RNA depending on the type of global deformation. The hybrid
has an intermediate behavior, though it is slightly closer to RNA
than to DNA (see Table 4). Thus, the global twist of the hybrid
is twice more difficult than for DNA and only 23% easier than
for RNA. For the global stretch, the force constants in the hybrid
and in RNA are identical and nearly one-half of that found for
DNA. Finally, the hybrid is 20% more flexible than both DNA
and RNA in terms of global roll deformation and more rigid
(50 and 20% relative to RNA and DNA, respectively) for
changes in the global tilt (see Table 4). Considering a common
isotropic distortion (i.e., a common distortion for all the duplexes
defined to weight the same all the possible helical deformation;41,63 see Figure 10), there are no clear differences between
the global flexibility of DNA, RNA, and DNARNA duplexes
(Figure 10).
(65) Perez, A.; Noy, A.; Lankas, F.; Luque, F. J.; Orozco, M. Nucleic Acids
Res. 2004, 32, 6144.
J. AM. CHEM. SOC.
VOL. 127, NO. 13, 2005 4917
Noy et al.
ARTICLES
Table 5. Diagonal Elastic Force Constants for Deformations along Local Helical Parameters of DNA, RNA, and HYBRIDa,b
DNA
RNA
HYBRID
a
tiltc
rollc
twistc
shiftd
slided
rised
31.16(0.31)
26.48(0.22)
27.04(0.17)
18.50(0.15)
15.06(0.08)
12.82(0.12)
14.92(0.12)
51.72(0.21)
33.12(0.17)
1.22(0.01)
1.37(0.01)
1.58(0.01)
1.90(0.02)
3.19(0.02)
2.42(0.01)
7.20(0.01)
6.18(0.06)
7.01(0.05)
Standard deviations are determined as noted in Table 4. b The complete stiffness matrixes are available upon request. c In cal/moldeg2
Figure 10. Elastic energy associated with helical isotropic distortions for
the three nucleic acids. (Top) Perturbations in local helical parameters.
(Bottom) Perturbations in global helical parameters. Perturbations along
each helical variable are chosen as twice the largest standard deviation for
this helical parameter in DNA2, RNA2, and DNARNA duplexes.
For most local helical parameters, RNA is stiffer than DNA,

and in general, random local deformation is easier for DNA
than for RNA41,65 (see Table 5 and Figure 10). Once again, the
behavior of the hybrid is intermediate between that of DNA
and RNA (see Table 5), but the local pattern of deformability
of the hybrid is unique, and not just a simple scaled average of
that of DNA and RNA homoduplexes (Table 5). For example,
deformation in rise and tilt is equally difficult for RNA2 and
the hybrid, but it is much easier to unwind or bend (twist and
roll stiffness) the hybrid than a pure RNA duplex. The isotropic
deformation energy of the hybrid is slightly closer to that of
DNA than to that of the RNA duplex (see Figure 10), but this
situation changes if the hybrid is deformed more along a helical
coordinate than along the others (anisotropic perturbation). Once
again,41,65 the flexibility in nucleic acids emerges as a very
complex concept especially for a molecule such as DNARNA
with a very asymmetric pattern of deformability.
RNase H Susceptibility. RNase H has binding constants in
the micromolar range for several oligonucleotides with A-like
conformations, including RNA duplexes and DNARNA hybrids, but does not bind DNA duplexes66-71 or other B-form
nucleic acids.70 The crystal structure of a RNase H suggests
that the enzyme does not recognize a pure canonical A-form
and that some distortion in the helix occurs.72-75 It is also known
that the enzyme does not show any marked sequence specific(66) Altmann, K. H.; Fabbrot, D.; Dean, N. M.; Geiger, T.; Monia, B. P.; Muller,
M.; Nicklin, P. Biochem. Soc. Trans. 1996, 24, 630.
(67) Agrawal, S.; Iyer, R. P. Pharmacol. Ther. 1997, 76, 151.
(68) Crooke, S. T.; Bennett, C. F. Annu. ReV. Pharmacol. Toxicol. 1996, 36,
107.
(69) Han, G. W.; Kopka, M. L.; Cascio, D.; Grzeskowiak, K.; Dickerson, R. E.
J. Mol. Biol. 1997, 269, 811.
(70) Lima, W. F.; Crooke, S. T. Biochemistry 1997, 36, 390.
(71) Stein, H.; Hausen, P. Science 1969, 166, 393.
(72) Ding, J.; Hughes, S. H.; Arnold, E. Biopolymers 1997, 44, 125.
(73) Ding, J.; Das, K.; Hsiou, Y.; Sarafianos, S. G.; Clark, A. D.; Jacobo-Molina,
A.; Tantillo, C.; Hughes, S. H.; Arnold, E. J. Mol. Biol. 1998, 284, 1095.
VOL. 127, NO. 13, 2005
In kcal/mol2
ity14,21,76,77 and is inactive against single-stranded oligonucleotides.69,70 Finally, in DNARNA duplexes, only the RNA strand
of the hybrid is degraded.69,70
All experimental data demonstrate that, despite the general
similarity between RNA2 and the hybrid, no appreciable amount
of RNA duplex is degraded by the enzyme.66-70,76 The reasons
for this extreme specificity have been obscure for decades since
it is not easy to find structural determinants which are different
for DNARNA and RNA2. Thus, all crystal structures of the
DNARNA hybrid are nearly identical to those found for pure
RNA duplexes (see Introduction). This fact could explain why
both RNA duplexes and DNARNA hybrids are recognized by
the enzyme, but does not justify why RNA duplexes are
inhibitors and DNARNA hybrids are substrates.
Crystallization conditions might bias the conformation of the
DNARNA hybrid, and both NMR and MD simulations suggest
that the hybrid adopts in physiological conditions an intermediate
A/B-form. In fact, analysis of NMR or MD structures allows
the determination of a few structural differences between DNA
RNA and RNA2, such as the narrower minor groove in the
hybrid compared with RNA duplex. On the basis of this finding,
several authors have suggested that a minor groove with a width
of 8-9 is a necessary requisite for degradation by RNase
H.16,21-24 The cMIP profiles in Figure 7 confirm that the average
recognition pattern of the minor groove of the hybrid is different
than that of the RNA duplex, providing an apparent support to
the hypothesis that a narrow minor groove is the structural
determinant for RNase H specificity. However, a more detailed
analysis of the structures shows that there is large overlap in
the distribution of widths of DNARNA and RNA duplexes,
and 20% of the time, the RNA duplex displays a minor groove
with an ideal width of 8-9 (see Figure 6), which would
imply some susceptibility of RNA duplexes to degradation by
RNase H. Furthermore, cMIP calculations on hybrid-chimeras,
which are recognized and degraded by RNase H,32,33 show very
anomalous minor grooves, leading to cMIP distributions far from
that expected for a normal DNARNA hybrid (compare Figures
7 and 11) and, in some cases, identical to that of a normal RNA
duplex (Figures 7 and 11). In summary, equilibrium geometry
can easily explain why B-type helical structures do not bind
the enzyme, but it cannot explain the discriminative ability of
RNase H between RNA duplex and DNARNA hybrid.
Discarding sequence effects and the equilibrium geometry
as the unique determinants for the specificity of RNase H, we
can consider that the reduced stability of the DNARNA hybrid
compared to that of the RNA homoduplex78-81 can be a key
(74) Jacobo-Molina, A.; Ding, J.; Nanni, R. G.; Clark, A. D.; Lu, X.; Tantillo,
C.; Williams, R. L.; Kamer, G.; Ferris, A. L.; Clark, P. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 6320.
(75) Sarafianos, S. G.; Das, K.; Tantillo, C.; Clark, A. D.; Ding, J.; Whitcomb,
J. M.; Boyer, P. L.; Hughes, S. H.; Arnold, E. EMBO J. 2001, 20, 1449.
(76) Oda, Y.; Iwai, S.; Ohtsuka, E.; Ishikawa, M.; Ikehara, M.; Nakamura, H.
Nucleic Acids Res. 1993, 21, 4690.
(77) Roberts, W. R.; Crothers, D. M. Science 1992, 258, 1463.
(78) Riley, M.; Maling, B. J. Mol. Biol. 1966, 20, 359.
ARTICLES
Figure 11. Classical Molecular Interaction Potential (energy contours -3 kcal/mol) for two hybrid structures known to be the substrate of RNase H (1DRN,
1DHH) and a reference RNA2 duplex which is not degraded by the enzyme.
determinant of the different susceptibility of DNARNA and

RNA2 to the action of RNase H. This possibility is indirectly
supported by the fact that modified oligonucleotides designed
to make more stable DNARNA hybrids5,67,68,82 fail to produce
RNase H-susceptible hybrids. However, a few modified oligonucleotides have been generated displaying simultaneously good
stability, specificity, and also RNase H susceptibility.30,83-86
Thus, an intrinsic instability in the duplex does not appear to
be a requisite for RNase H susceptibility.
In summary, to our understanding, and without rejecting a
possible role for structure and for specific interactions (for
example, those involving 2-OH groups), flexibility emerges as
the major differential trend that can use RNase H to discriminate
between both duplexes.32,33,87 Our MD simulations show that
the pattern of deformability of the hybrid is quite different than
that of a pure RNA duplex. Not only is the hybrid, in general,
more flexible than the RNA duplex but also are there several
specific local deformations which are much easier for the hybrid
than for the RNA duplex, which provides more possibilities for
deformability in the helix during the catalysis. Interestingly, our
simulations strongly suggest that the RNADNA hybrid has a
strong asymmetry in terms of flexibility between both strands.
It is suggested that this unique asymmetry might be used by
the enzyme to distinguish between the DNA and RNA strands,
something that for a duplex showing a more symmetric pattern
of flexibility will be very difficult.
(79) Sugimoto, N.; Nakano, S.; Katoh, M.; Matsumura, A.; Nakamuta, H.;
Ohmichi, T.; Yoneyama, M.; Sasaki, M. Biochemistry 1995, 34, 11211.
(80) Nakano, S.; Kanzaki, T.; Sugimoto, N. J. Am. Chem. Soc. 2004, 126, 1088.
(81) Freier, S. M.; Altmann, K. H. Nucleic Acids Res. 1997, 25, 4429.
(82) McKay, R. A.; Miraglia, L. J.; Cummins, L. L.; Owens, S. R.; Sasmor, H.;
Dean, N. M. J. Biol. Chem. 1999, 274, 1715.
(83) Moulds, C.; Lewis, J. G.; Froehler, B. C.; Grant, D.; Huang, T.; Milligan,
J. F.; Matteucci, M. D.; Wagner, R. W. Biochemistry 1995, 34, 5044.
(84) Barnes, T. W.; Turner, D. H. J. Am. Chem. Soc. 2001, 123, 4107.
(85) Barnes, T. W.; Turner, D. H. Biochemistry 2001, 40, 12738.
(86) Tonelli, M.; Ulyanov, N. B.; Billeci, T. M.; Karwowski, B.; Guga, P.; Stec,
W. J.; James, T. L. Biophys. J. 2003, 85, 2525.
(87) Nakamura, H.; Oda, Y.; Iwai, S.; Inoue, H.; Ohtsuka, E.; Kanaya, S.;
Kimura, S.; Katsuda, C.; Katayanagi, K.; Morikawa, K.; Miyashiro, H.;
Ikehara, M. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 11535.
Our results support a complex mechanism of action for RNase

H. In a first step, the enzyme should bind any duplex showing
a general conformation not far from the A-form and should reject
B-type structures. In a second step, the enzyme should distort
the duplex in a very asymmetric way, leaving the rigid RNA
near the cleavage site while the DNA strand is pointed to the
exterior. It is expected that this type of deformation will be too
energetically costly for the rigid RNA duplex. Overall, we
suggest the differential flexibility of RNA duplex and the hybrid,
and especially, the asymmetry in the flexibility pattern of the
latter constitutes the basis for the selective mechanism of action
of RNase H. We propose that future designs of oligonucleotides
for antisense purposes should explicitly consider these flexibility
issues to guarantee the enzymatic susceptibility of the resulting
hybrid.
Conclusions
Extended state-of-the-art MD simulations are able to reproduce with accuracy the structural properties of DNARNA
hybrids, even when the starting conformation is far from the
equilibrium conformation in solution.
The equilibrium geometry of the hybrid is closer to the
A-form than to the B-form. All riboses show North puckerings,
but 2-deoxyriboses are mostly in the South and South-East
regions, which lead to the definition of grooves intermediate to
those typical of the A- and B-forms.
The flexibility of the DNARNA hybrid is unique and not a
simple average of that of pure DNA and RNA hybrids. Quite
surprisingly, each strand in the hybrid maintains well its essential
dynamics in pure duplexes, which generates a strong asymmetry
in the pattern of deformability of the helix.
Analysis of the different putative mechanisms that will allow
RNase H to distinguish between different duplexes strongly
suggests that while equilibrium structure can be enough for the
enzyme to discard DNA2, only the differential flexibility pattern
can justify the ability of RNase H to discriminate between DNA
RNA and RNA2.
J. AM. CHEM. SOC.
VOL. 127, NO. 13, 2005 4919
Noy et al.
ARTICLES
Acknowledgment. We thank Dr. Chattopadhyaya for helpful

discussions, and the Centre de Supercomputacio de Catalunya
for computer resources. We also thank the financial support of
the Spanish Ministry of Education and Science (SAF2002-
VOL. 127, NO. 13, 2005
04282; BIO2003-06848), Fundacio La Caixa, and Fundacion

BBVA. A.N. is a fellow of the Spanish Ministry of Education
and Science, and A.P. is a DURSI fellow.
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33303338 Nucleic Acids Research, 2007, Vol. 35, No. 10

doi:10.1093/nar/gkl1135
Published online 25 April 2007
Theoretical study of large conformational transitions

in DNA: the B$A conformational change in water
and ethanol/water
Agnes Noy1, Alberto Perez1, Charles A. Laughton2 and Modesto Orozco1,3,4,*
1
Molecular Modeling and Bioinformatics Unit, Institut de Recerca Biome`dica & Instituto Nacional de
Bioinformatica, Parc Cientfic de Barcelona, Josep Samitier 1-5, Barcelona 08028, Spain, 2School of Pharmacy
and Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, UK, 3Departament de
Bioqumica i Biologia Molecular. Facultat de Biologa. Universitat de Barcelona. Avgda Diagonal 645, Barcelona
08028, Spain and 4Computational Biology Program, Barcelona Supercomputer Centre, Jordi Girona 31,
Edifici Torre Girona, Barcelona 08028, Spain
ABSTRACT
We explore here the possibility of determining
theoretically the free energy change associated
with large conformational transitions in DNA, like
the solvent-induced B$A conformational change.
We find that a combination of targeted molecular
dynamics (tMD) and the weighted histogram
analysis method (WHAM) can be used to trace this
transition in both water and ethanol/water mixture.
The pathway of the transition in the A!B direction
mirrors the B!A pathway, and is dominated by two
processes that occur somewhat independently:
local changes in sugar puckering and global
rearrangements (particularly twist and roll) in the
structure. The B!A transition is found to be a
quasi-harmonic process, which follows closely the
first spontaneous deformation mode of B-DNA,
showing that a physiologically-relevant deformation
is in coded in the flexibility pattern of DNA.
INTRODUCTION
DNA is a exible and polymorphic structure, whose
conformation changes as a response to the presence
of ligands, variations in the temperature or modications
in the solvent (13). Such exibility is implicitly coded in
the structure of the DNA (48) and is crucial to its
biological functionality, since, for example, binding
of DNA-regulatory proteins can require dramatic changes
in the structure of nucleic acid. Understanding DNAs
exibility and conformational transition is thus a necessary step in transforming structural data into biologically
important information.
In general, the determination of DNA structure is no
longer the challenge for experimental techniques had
it used to be, both NMR and X-ray techniques are now
able to provide accurate structures for most sequences
(more than 2500 (experimentally-solved) DNA structures

are deposited in the Protein Data Bank in June 2006).
Unfortunately, these experimental techniques are less
eective at discovering exibility or tracing conformational transitions, and so for this we currently make major
use of simulation techniques like molecular dynamics
(MD) to study these transitions at the atomic level.
However, many conformational transitions in DNA occur
on the micro or millisecond time scale, while timescales
currently accessible by atomistic MD are in the 550 ns
range. To overcome this problem, it is common to use
biasing techniques to move simulations along a reaction
coordinate at an articially high rate, but many of the
transitions we wish to study involve large conformational
changes, where many internal degrees of freedom move
in a coordinated way. In this situation, it is dicult to
apply standard biasing techniques based on the regular
change of internal degrees of freedom along the reaction
coordinate.
In summary, the analysis of large conformational
changes is still a major challenge for both experimental
and theoretical techniques. A clear example is the B$A
transition of DNA duplexes (912). It has been known
since 1953 that physiological DNA is mostly B-form,
while physiological RNA is always in the A-conformation
(1,2,912). However, the binding of some proteins to
DNA can induce local B!A changes, which are needed
to form some protein-DNA complexes crucial for the
control of gene functionality (1315). The B!A transition can also be brought about by other stress conditions,
like crystal lattice restrictions (16 and references therein),
or the addition of a large proportion of ethanol, which is
believed to displace water molecules hydrating the helix,
leading to a pseudo-anhydrous environment where the
most compact A-form is more stable (1,12,17,18).
The B$A conformational transition has been also the
subject of numerous theoretical studies, aimed at understanding the mechanism of the transition and the
atomic reasons for the A/B preference in water and
*To whom Correspondence should be addressed: Tel: 34 934037155; Fax: 34 934037157; Email: modesto@mmb.pcb.ub.es
2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acids Research, 2007, Vol. 35, No. 10 3331

other solvents. The latest generations of both CHARMM
and AMBER force-elds (the ones most used in simulations of nucleic acids) recognize the A-form as the only
stable conformation of duplex RNA, and the B-form as
the most important conformation of DNA duplex in
aqueous solution (1828). In fact, MD trajectories of
DNA duplexes started in the A-form convert very rapidly
to the B-conformation (23), showing that MD simulations
are able to drive the DNA from an incorrect conformation
to a correct one. Unfortunately, MD simulations are
unable to detect reversible transitions due to the limited
length of current trajectories, which makes very unlikely
to sample unstable regions of the conformational space.
This lack of reversibility in the transition precludes
the determination of the free energy associated with
the conformational change and the determination of the
atomic mechanism of the transition. For this reason,
these simulations need to be biased to force them to
sample reversibly the B$A transition.
In this article, we re-visit the B$A transition for a
duplex DNA dodecamer using the techniques of essential
dynamics, unbiased molecular dynamics simulations and a
combination of targeted MD (tMD (2931)) and the
weighted histogram analysis method (WHAM (32))
in both water and a mixture of 85:15 ethanol/water.
Using these techniques, we were able to trace the B$A
transition in a smooth reversible way providing estimates
of the associated free energy and of the molecular
mechanism of this conformational change.
METHODS
Model selection
We felt that in order to obtain reliable conclusions on
B/A preference, a DNA duplex containing at least one
helix turn should be used as the model system (i.e. at least
1012 base pairs); smaller duplexes might not provide
a good model of interactions along the major and
minor grooves and would maximize the eect of
artifactual end-eects. Unfortunately, the increase in the
size of the duplex leads to a parallel increase in the
computational diculty of the simulation. Thus, as a
compromise, we decided to use Dickersons dodecamer
(dGCGCAATTGCGC)2 a B-type 12-mer oligo (33)
for which several experimental structures are available
(see http://ndbserver.rutgers.edu) and for which dozens
of simulations have been published (4,3437).
Systems setup for aqueous simulations
Input coordinates for trajectories started in the B-form
were generated by taking Dickersons crystal geometries
(33). The experimental structure was neutralized by
adding Na in the best regions according to classical
molecular interaction potential (CMIP) calculations (38)
and immersed in a rectangular box of water (aroubnd
4454 TIP3P molecules). As described elsewhere (38), MIP
neutralization protocol allows the denition of a reasonable ionic atmosphere around DNA based on Poisson
Boltzman potentials, reducing then the time needed for
counterion equilibration. The solvated systems were then
optimized, thermalized and pre-equilibrated using our

standard protocol (3941). The nal conformations were
then re-equilibrated (constant pressure and temperature:
1 atm, 298 K) for 1 ns more to ensure the stability of the
trajectories. Simulations starting in the A-conformation
were generated by rst building a standard A-type DNA
duplex of the desired sequence, which was then neutralized
and hydrated as below. Optimization, thermalization and
pre-equilibration was carried out following the same
procedure than for B-DNA but adding an harmonic
restraint (K 160 kcal/mol.rad2) that restrained all the
d angles to 808. The same restraints were used in
equilibration (4 ns) and in production runs; when they
are removed the structure changes quickly to the B-form.
Ten dierent structures obtained during the equilibration of the A-form (with d angles restrained to 808) were
used to perform 10 parallel un-restrained 3 ns MD
simulations. Within this short simulation time the A!B
transition was completed in all replicas, which allowed us
to obtain reasonable statistics on the microscopic mechanism of the unbiased transition.
Systems setup for ethanol/water simulations
Equilibrated boxes containing 85% ethanol: 15% water
were generated from Monte Carlo simulations (300
million congurations) at constant pressure (1 atm) and
temperature (300 K). These boxes were then used
to solvate neutralized B and A-DNAs (starting conformations of B and A-DNAs were those obtained at the end
the respective MD equilibrations in water (see above)).
The solvated systems were thermalized (T 298 K) and
pre-equilibrated using 2 ns of MD where only the
solvent was free to move. Finally, all the systems were
equilibrated for 4 ns in both A and B-conformations.
The d angles were restrained to 808 in A-simulations
and 1208 in B-simulations. Fifteen structures obtained
during the last 1 ns of the restrained B-type simulations
were used to start 3 ns unbiased MD simulations
intended to capture spontaneous B!A transitions in
(85:15) ethanol/water. Mirror calculations with unbiased
trajectories started in the A-form were used to conrm
that the A-form is a stable minimum in the ethanol/water
mixture considered here.
Targeted MD simulations
These simulations drive a transition by introducing
a harmonic penalty that forces the sampled structure to
be at a given RMSd from a reference(s) structure(s).
By changing smoothly the target RMSd, we can then
approach or separate the molecule from reference
geometries. Several formalisms can be introduced to
introduce the harmonic restraints (25), but in our hands
smoother and more reliable A$B transitions were
obtained by using eq. (1) (see results). As described
below, a selected group, rather than all the atoms (25)
were used to compute the RMSd.
Erest Krest RMSdX,Xfinal RMSd2

where RMSd() is the desired RMSd to nal structure
at point of the transition path.
After several preliminary tests, simulations were
performing using windows of 0.25 A with Krest 0.1 kcal/
mol A2 atom for all windows except the terminal ones,
where we used spacing 0.1 A and Krest 8 (water) or
2 (ethanol/water) kcal/mol A2 atom. Unless otherwise
noted, each window was simulated for 1.2 ns, the rst
0.2 being considered as equilibration. To make transitions
as smooth as possible, the end point of each window was
used in general as starting point for the next. Using these
conditions smooth transitions and good overlap between
windows was obtained.
The biased samplings obtained were used to derive
potentials of mean force (PMF) for the transition using
the WHAM method (32). Free energies were recovered
by integrating the PMF using suitable boundaries
(see below).
Essential dynamics
Unbiased B-DNA and A-RNA 10 ns trajectories
were used to derive the essential dynamics of relaxed
B and A-forms (4,7,8,42,43). The rst eigenvector
(that describing the most important deformation mode)
of the relaxed B-DNA (or A-RNA) trajectory was
compared with the B!A transition vector (see eq. (2))
to obtain an estimate of the overlap between normal
B-DNA uctuations and B!A transitions.
relax
trans

2

jrelax j jtrans j
where relax stands for the rst eigenvector describing the
essential dynamics in A or B-forms and trans represents
the transition vector A!B or B!A.
As described in Results, the rst eigenvector of
the essential dynamics of B-DNA correlates well with
the B!A transition vector. We then explored the
harmonic energy needed to animate this eigenvector
(from the B-form) to reach conformations close to the
A-form. For this purpose, we add vibrational energy to
the mode obtaining the associated displacement through
eq. (3) from which perturbed geometries were determined.
Pursuing this approach, we performed calculations of the
dimension-less Mahalanobis distance (dM; see eq. (4))
between B and A forms. The Mahalanobis distance
is a unit-less metric, directly related to the deformation
energy (see eq. (5)) which denes the minimum pathway
in essential space between two conformations. Thus,
using dM calculations, we can trace the B!A transition
by activating many dierent essential modes of the
B-equilibrium trajectories computing then the energy
needed to reach structures closer (to a certain threshold)
to the A-form.

2E 1=2
3
x
kb T
where E is the vibrational energy, kb is Boltzmans
constant, T is absolute temperature, is the eigenvalue
(in distance2 units).
dM
2
n
X
4
i1
xi
1=2
i
!2 31=2
5
where xi is the displacement along individual eigenvectors

and i stands for the corresponding eigenvalue
(in distance2 units). The sum extends of n-important
essential movements, in this article we consider the rst
10th ones, which accounts for more than 85% of the DNA
variance.
E
kB T 2
d
2 M
Technical details
All simulations were carried in the isothermalisobaric
ensemble (P 1 atm, T 300 K). Monte Carlo simulations (for preparing the hybrid solvent box) were
performed using the BOSS3.4 computer program (44)
allowing internal rotations in the solvent molecules,
but no other internal changes. A non-bonded cuto of
12 A was used in conjunction with periodic boundary
conditions to reduce the number of non-bonded interactions in these Monte Carlo simulations. Molecular
dynamics (MD) calculations were carried out using the
AMBER8 computer programs (45) and long-range
electrostatic eects were taken into account by means of
the Particle Mesh Ewald method (46). PARM99 was used
to represent DNA interactions (47,48), while TIP3P (49)
and all-atoms OPLS (50) parameters were used for
the solvents. The use of SHAKE (51) to maintain all the
bonds at equilibrium distance allowed us to use 2 fs
as the integration step size.
Analysis of trajectories was carried out using PTRAJ
module included with the AMBER8 release as well as
software developed in house. All simulations were
performed using the Mare Nostrum supercomputer at
the Barcelona Supercomputer Center.
RESULTS AND DISCUSSION
Unbiased A!B trajectories
As reported by others (1924), MD simulations of DNA
using the PARM99 force-eld produces a spontaneous
A!B conformational change in water in short simulation
times, which makes possible the study of the atomic
mechanism of such an unforced transition. Ten dierent
trajectories starting from well-equilibrated A-form
conformations move to the B-conformation within the
3 ns simulation times. Extension of ve of these trajectories to 10 ns did not shown any back-transition to the
A-form (data not shown, but available upon request).
The analysis of the RMSd plots from canonical A and
B-forms show that the transition starts very quickly,
and after around 500 ps, the lines of RMSd(versus A) and
RMSd(versus B) cross for the rst time (see Figure 1).
After this period, the structures uctuate around

Table 1. Dot products between the unit vectors representing the rst
essential movement of dierent trajectories. A value of 1 (or 1)
indicates a perfect match between essential deformation movements.
Note that the rst essential movement obtained in the unbiased A!B
simulation or in the tMD B!A one should correspond to the B!A
transition vector. The non-optimum overlap between them (around
90%) gives an indication of the uncertainties in the denition of this
transition vector
DNA
RNA
B!A (unbiased)
Figure 1. TOP: Average variation with time of the RMSd (from A

and B canonical structures) of 10 unbiased trajectories starting from
A-form in water. Standard deviations are shown. MIDDLE: average
variations of d and number of sugars in North conformation for the
same trajectories. BOTTOM: Variations in the average minor groove
width (mGw) and Zp.
DNA
RNA
B!A (Unbiased)
B!A (tMD)
1.00
0.81
1.00
0.94
0.81
1.00
0.87
0.90
0.86
Very interestingly, the transition vector correlates very

well (see Table 1) with the rst deformation mode of both
relaxed B and A-forms (taken from ref. (7)). This indicates
that the deformation pattern needed to perform the
biologically relevant B$A transition is implicitly coded
in the polymeric structure of these nucleic acids and that in
a rough approximation, the A-form can be interpreted as
a vibrational-activated state of B-DNA. This hypothesis
is supported by analyzing the structures obtained
by moving the B-DNA along the rst essential mode
(see Figure 2). Thus, a simple deformation along the rst
mode yields structures with RMSd(A) RMSd(B) for
vibrational energies around 4 kcal/mol, and for deformation energy around 12 kcal/mol to structures that are
at only 2 A from the A-form (see Figure 2). The activation
of additional deformation modes (up to 10) using
the Mahalanobis metric allowed us to slightly reduce the
RMSd from target A-form (to 1.7 A), but at the
expense of a very large vibrational energy (19 kcal/mol).
Clearly, B!A transitions follows closely the rst
mode, and local rearrangement from a distorted to the
real A-conformation requires some local, probably
non-harmonic deformations.
Targeted MD transitions in water
a conformation with B-like properties (see Figure 1;

individual plots for trajectories are shown at http://
mmb.pcb.ub.es/A-B), but still not far from the A-form
in terms of RMSd. For all 10 trajectories, the transition in
RMSd only happens after major changes in sugar
puckering, supporting the idea that sugar re-puckering is
the driving force for A!B transition (24). Once delta
change, helical parameters like roll and twist are adjusted
to the standard B values. Thus, in just 100 ps half of the
sugars which were originally in the N-conformation
(d below 958 have moved into the E and S regions
(d greater than 958) and after 500 ps only four sugars are
(in average) in the N-region. Slow re-puckering of these
residual North sugars occur along the 13 ns simulation
period. Analysis of individual sugars shows that in
general, sugars in purine nucleotides change faster than
those in pyrimidines. Within purines G shows faster
transitions than A and within pyrimidines C is that with
the slower rate of N!S transitions.
Unbiased simulations provide an intuitive picture of

the A!B transition in water, but they are not able to
provide any estimate of the thermodynamics, or kinetics
of the process. Essential dynamics allows us to obtain
only a rough qualitative picture of the B!A transition,
making necessary the use of more accurate non-harmonic
techniques like the tMD/WHAM one.
Our previous work (24) and the unbiased simulations
discussed above suggested that sugar puckering could be
a good variable to discriminate between B and A-form.
Accordingly, we undertook a study of the B!A transition
by means of restrained MD simulations, slowly changing
the d angles of all sugars from 140 to 708. As shown
in Figure 3, and in data from equilibrium simulations in
Figure 4, the B!A transition happens for d values around
908. Note that such a transition is clear not only in the
RMSd from A and B-conformations, but also in the
width of the minor groove (mGw) and in Olsons (52)
Zp parameter (see Figure 3). Thus, a structure can be
assigned to the A-family if RMSd(A)5RMSd(B),
Figure 2. Representation of the energy (black), and RMSd with respect

to A (in red) and B (in blue), when a relaxed DNA structure is
perturbed along its rst essential mode. RMSds were computed with
reference to the MD-averaged A and B-conformations. Blue line is not
smooth because perturbation along rst essential mode is made using
MD-averaged B-conformation.
Zp41.0; d5958 and mGw414 A. When only the

RMSd criteria is fullled, the structure should be labeled
as distorted B-form. These threshold need to be consider
to validate when a tMD simulation is really driving the
structure to the target conformation and not to a distorted
geometry, which might have small RMSd to target
conformation but very poor internal geometry.
While the use of restraints on delta permitted the
required transition, and was extremely useful to dene
clear threshold values to classify structures as A or B, it
does not allow us to easily recover the free energy
associated to the conformational change. Thus,
we decided to follow the A$B transition by dening the
RMSd function considering only heavy atoms in the sugar
ring (Reference set 1, see Figure 5). Additional calculations were performed considering an extended set of
restraint atoms which include all ring atoms plus
O5 (Reference set 2) and an alternative set of restraint
atoms formed by the phosphate group and O4 (Reference
set 3). Using any of these three RMSd denitions and
the restraint function shown in eq. (1), we were able
to reproduce correct B!A transitions (see Figure 6 and
Figure S1 in Supplementary material), where both starting
and end structures ts the canonical B and A-forms.
It is worth noting that such smooth transitions cannot
be so easily obtained with other choices of the
RMSd function. Caution is then necessary against
a un-supervised pure-force use of the targeted MD
technique to follow complex conformational transitions.
It is worth to note that the transition pathway closely
follows the direction of the rst essential mode of relaxed
DNAs and RNAs (see Table 1), conrming the results
obtained for unbiased A!B transitions. This nding
demonstrates that the ability to change between the B and
A-conformers is implicitly coded in the structure of nucleic
acid duplexes. Interestingly, irrespective of the set of
atoms used to dene the RMSd with respect to target
structures the transition is similar: for RMSd() 2.0 A,
the RMSd(A) and RMSd(B) lines cross, but until very
late in the reaction coordinate (RMSd() 1.0 A) full
transition is not achieved (see Figure 6 and Supplementary
Figure S1).
The PMF associated to the B!A transitions in water
are well converged and seems robust to the selection of the
Figure 3. TOP: Prole of RMSds (from A in red and B in blue)

obtained from MD trajectories where the target d (dt) was varied from
140 to 708 (see text). Proles corresponding the average d are shown in
the MIDDLE panel and those for the average mGw (black) and
Zp (pink) are shown BOTTOM.
Figure 4. Distribution of TOP: d angle, BOTTOM-left: mGw

and BOTTOM-right: Zp for equilibrium trajectories of B-DNA blue
and A-RNA (red). Trajectories were taken from ref.(7).
set of restrained atoms and to changes in the forward

or reverse direction of the transition (see Figure 7).
As predicted, the A!B PMF takes the form of a downhill
landscape, where the A-form is not dened as a minimum.
Integration of the PMF proles provides a direct
estimate of the free energy associated with conformational
transitions. Inspection of Figure 6 (see text) allowed us to
Figure 5. Representation of dierent sets of atoms used to restrain the

system in tMD simulations of the B$A transition in water and 85:15
ethanol/water mixture.
Figure 7. Potential of mean force (PMF) obtained from tMDWHAM

calculations of the B!A transition in water. Proles were obtained
using dierent sets of atoms to dene the target RMSd in each window
(see insert and text) and using both forward and reverse directions.
Table 2. Free energy (in kcal/mol) dierence for B!A conformational

transition in water and 85:15 ethanol/water obtained by integration of
the PMF curves. For denition of the states, see text
Figure 6. Variation of TOP: RMSds (from A in red and B in blue),

MIDDLE: d angle; BOTTOM: Zp (pink) and mGW (black), obtained
in targeted MD trajectories in pure water using Reference set 1.
classify the PMF into three regions: (i) pure A-form

(RMSd() 1.0 A), pure B-form (RMSd() 2.0 A),
and distorted B-forms (2.0 A4RMSd()41.0 A)
Using these values, the deformation of B-DNA to achieve
structures with RMSd(A) RMSd(B) requires around
3 kcal/mol (see Table 2), while a full B!A transition is
associated with a free energy penalty of 11.4 kcal/mol.
After sending the rst version of this manuscript for
publication, a colleague addressed us to the work by
Zhurkins group (53) who reported empirical scales for the
free energy of B!A transition in water. Applying
Zhurkins data in our duplex, we obtain an empirical
estimate of 11 kcal/mol for the B!A transition, a value
that matches perfectly our estimate. It is also worth noting
Solvent
B!B(dist)
B(dist)!A
B!A(all)
A!A(dist)
Water
(EtOH/water)
3.2
8.1
11.4
0.8
0.4
the very good agreement between rigorous tMD/WHAM

estimates and rough values obtained by essential dynamics
in Figure 2, something that conrms that the B!A
transition is a pure up-hill process which can
be simplied as a vibrational activation of the rst
deformation mode of B-DNA. A small amount of
energy is enough to distort the DNA to conformations
that are not far in terms of RMSd from the A-form,
however, a full transition is very unlikely and requires
major changes in the environment, such as the introduction of proteins or co-solvents.
In summary, all our simulations suggest that in water
the A!B transition is a downhill process, and that the
A-form is not a stable conformation of Dickersons
dodecamer in water. This result is in full agreement with
all previous MD simulations irrespective of the force-eld,
sequence or simulation conditions used (1822,24,25),
which argues against force-eld or simulation protocols.
Special agreement is found with data by Banavali and
Roux (25), who using other force-eld a shorter oligo,
and a dierent functional for the restrain energy obtained
quite similar results, demonstrating that the method, when
applied with common sense is robust. However, these
theoretical results have been severely criticized by Jose and
Porschke (54,55), who found that experimentally the
transition time for the A$B change (around 70:30
ethanol/water) is in the range 100101 ms, which should
indicate the existence of a sizeable transition barrier
and the presence of two well-characterized canonical
forms. We will show below that the criticism is not
justied since the shape of the free-energy curve in pure
water or ethanol-rich solutions is completely dierent, and
conclusions obtained in rich ethanol mixtures cannot be
simply extrapolated to water solution (see next sections).

Study of B!A transitions in 85:15 ethanol/water
by unbiased MD simulations
It is experimentally known that in the presence of
large concentration of ethanol, the DNA changes from a
B to an A-type conformation (1,12,17,18,54,55). Up to 15
MD simulations of Dickersons dodecamer starting from
the A-conformation remain in this region, as previously
found by McConnell and Beveridge (21). Extension of one
of this simulation to 10 ns does not show any dramatic
change from the shorter ones (see Supplementary
Figure S2), suggesting that the force-eld recognize the
A-form as a stable conformation of DNA in ethanol/
water solution. However, 10 unbiased 3 ns trajectories
starting in the B-conformation remains within this
conformational family (see Figure 8), conrming again
previous results by other authors (20,21,55). Overall,
unbiased MD simulations suggest that in the presence of
high concentration of ethanol, the free-energy landscape
is dierent to that in pure water, since at least two minima
(close to the B and A-forms) exist, separated by a sizeable
energy barrier.
Targeted MD transitions in ethanol/water (85:15)
Targeted MD simulations (using restraint set 1) are able to
drive a smooth and complete A!B transition
(see Figure 9) in the presence of a high concentration
of ethanol. Analysis of RMSd and internal geometry
changes along the transition pathway demonstrate that
the A!B transition in 85:15 ethanol/water starts with
a fast change (RMSd() 3.3 A) of sugar puckering from
N to S which leads to an immediate change in the Zp
parameter, and later (RMSd() 2.0 A) to the crossing
of the RMSd(A/B) lines and to the reduction of the minor
groove width to canonical B-values. In summary, we see
in ethanol/water a mirror copy of the transition found
in pure water. Combining results in ethanol/water
and pure water, we can obtain a quite complete picture
of the nature of the A$B conformational change.
Accordingly, when the DNA moves from the A to the
B-form, the driving force is a change in the sugar
puckering that is soon followed by a global arrangement
of the structure. On the contrary, for the B!A transition
a global change of shape and reduction of the width
of the minor groove happens rst, and only when the
global structure is close to the A-form do the sugars
adopt a North puckering, dening at that point a true
A-type structure.
As suggested by unbiased simulations, PMF prole for
the A!B transition in ethanol/water shows the existence
of two minima regions separated by wide region of higher
free energy (see Figure 10). Current tMD simulations
cannot be used to determine precisely the free energy
barrier, but a rough estimate of 2 kcal/mol (A!B) and
1 kcal/mol (B!A) can be derived from the PMF curve
in Figure 10. Clearly, the shape of the free-energy prole
associated with the A$B transition is strongly dependent
on the solvent, the barrier-less A!B transition found
in water is changed to a barrier-limited B!A transition
in 85:15 ethanol/water. The apparent discrepancy between
MD simulations (in water) and experimental studies
Figure 8. TOP: Average variation along the time of the RMSds (from
A and B canonical structures, in A) of 10 unbiased trajectories starting
from B-form in 85:15 ethanol/water mixture. Standard deviations are
shown. MIDDLE: average variations in d and number of sugars in
North conformation for the same trajectories. BOTTOM: variations of
the average minor groove width (mGw) and Zp.
(in high ethanol concentration) of the B$A transition

can then be easily understood, and illustrates that
caution is always necessary when MD simulation results
are interpreted in the light of experimental measures
performed under dierent conditions.
Analysis of geometrical variation along the tMD
simulation pathway allows us to dene boundaries for
integration of the PMF curve. Thus, the pure A-form is
obtained just in a narrow valley (4 A RMSd() 3.3 A),
an intermediate distorted A-conformation is obtained in
the region: 3.3 A RMSd() 2.0 A. Finally, a canonical
B-form is dened in the region: 2.0 A RMSd(). Note
that these partitions agree well with the positioning of maxima and minima in the PMF curve (see
Figure 10). Using these denitions, we conclude that
in 85:15 ethanol/water the A-form (considering both
canonical and distorted species) is 0.8 kcal/mol more
stable than the B-one, while the distortion of the canonical
A-form is disfavored by only 0.4 kcal/mol.

ACKNOWLEDGEMENT
This work was supported by the Spanish Ministry of
Education and Science (BIO2006-01602), Fundacion La
Caixa and the Barcelona Supercomputer Center
(Computational Biology Program). AN and AP are
fellows of the Spanish and Catalan Ministries of Science.
Funding to pay the Open Access publication charge was
provided by Institut de Recerca Biome`dica.
Conict of interest statement. None declared.
REFERENCES
Figure 9. Variation of TOP: RMSds (from A in red and B in blue);

MIDDLE: d angle; BOTTOM: Zp (pink) and mGW (black); obtained
in targeted MD trajectories in 85:15 ethanol/water mixture using
Reference set 1.
Figure 10. Potential of mean force (PMF) obtained from

tMDWHAM calculations of the A!B transition in a mixture of
ethanol (85%) and water (15%).
In summary, tMD simulations show that the presence

of large amounts of ethanol produces a dramatic
change in the A/B conformational equilibrium, in good
agreement with experimental data (see Introduction).
The A-form, which is negligibly populated (ratio
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