Spectroscopic and Molecular Dynamics Evidence for a Sequential
Mechanism for the A-to-B Transition in DNA
Kelly M. Knee,* Surjit B. Dixit,yColin Echeverrı ´a Aitken,* Sergei Ponomarev,yD. L. Beveridge,y
and Ishita Mukerji*
*Molecular Biology and Biochemistry Department, andyChemistry Department and Molecular Biophysics Program, Wesleyan
University, Middletown, Connecticut 06459
and d(CGCAAATTTCGC), using circular dichroism spectroscopy, ultraviolet resonance Raman (UVRR) spectroscopy, and
molecular dynamics (MD) simulation. Circular dichroism spectra confirm that these molecules adopt the A form under conditions
of reduced water activity. UVRR results, obtained under similar conditions, suggest that the transition involves a series of inter-
mediate forms between A and B. Cooperative and distinct transitions were observed for the bases and the sugars. Independent
MD simulations on d(CGCGAATTCGCG)2show a spontaneous change from the A to B form in aqueous solution and describe a
kinetic model that agrees well with UVRR results. Based on these observations, we predict that the mechanism of the transition
involves a series of A/B hybrid forms and is sequential in nature, similar to previous crystallographic studies of derivatized
duplexes. A simulation in which waters were restrained in the major groove of B DNA shows a rapid, spontaneous change from B
to A at reduced water activity. These results indicate that a quasiergodic sampling of the solvent distribution may be a problem in
going from B to A at reduced water activity in the course of an MD simulation.
The B form of DNA is well known to be predominant in
biological systems. However, conformational changes from
B in the direction of A DNA are implicated in protein-DNA
and drug-DNA interactions, and there is leading evidence for
a functional role for A as well as B forms in genome structure
and function (1). The A-to-B transition in DNA has also
served as a prototype case for testing out and validating
empirical energy functions and force fields used in molecular
dynamics simulations on nucleic acids (2–4). A number of
recent experimental (5–8) and theoretical (4,9–12) studies
havereportedonthissystem.Moststudiestodate have focused
on the preferential stability of A and B DNA as a function of
water activity and salt concentrations, whereas the mecha-
nism of the B-to-A transition has received less attention.
Unique new information on this problem can now be ob-
tained from ultraviolet resonance Raman (UVRR) spectros-
copy, which monitors the local structures of individual
nucleotide bases in a sequence. Molecular dynamics (MD)
simulations can provide a detailed computational model
for the conformational change, which must of course be ex-
perimentally validated. We report herein a combined exper-
imental/theoretical study of the B-to-A transition in selected
DNA oligonucleotides based on UVRR spectroscopy and
MD computer simulation. The particular objective of this
study is to elucidate base-pair sequence and structural effects
on the transition mechanism. UVRR spectroscopy has been
applied to explicitly examine different base types through the
intensity and frequency of their vibrational modes as a
function of water activity.
Experiments were carried out on three dodecamer se-
quences, d(CGCGAATTCGCG), d(CGCGAATTGCGC),
and d(CGCAAATTTCGC). Corresponding MD simulations
including water and counterions performed on d(CGCGA-
ATTCGCG) with an A-form initial structure and the spon-
taneous transition of the A- to B-form dodecamer during the
initial phase of the MD simulation provides a theoretical
model of the transition mechanism (Fig. 1). The essential
results of the experimental and theoretical studies are in close
accord, and are found to support the idea of a sequence-de-
pendent, sequential mechanism for the transition rather than
a concerted, all-or-none mechanism. Detailed analyses of
DNA structures and solvation obtained from the MD simu-
lations provide further information leading to an improved
understanding of the transition mechanism.Inaddition, some
new insights into sampling issues in obtaining conforma-
tional transitions in MD on DNA for mixed solvent systems
have been obtained.
The structural difference between B and A forms of DNA
resides essentially in the sugar puckers (f) and the helix-base
parameters x-displacement (XDP), inclination (INC), and
slide (SLD), according to the definition of these parameters
(13). In B DNA the nucleotide basepairs are perpendicular to
the helix axis (INC ¼ 0) and centered (XDP, SLD ¼ 0), and
Submitted July 17, 2007, and accepted for publication January 10, 2008.
Kelly M. Knee and Surjit B. Dixit contributed equally to this work.
Address reprint requests to D. L. Beveridge, Chemistry Department and
Molecular Biophysics Program, or Ishita Mukerji, Molecular Biology and
Biochemistry Department, Wesleyan University, Middletown, CT 06459.
Editor: Jonathan B. Chaires.
? 2008 by the Biophysical Society
Biophysical JournalVolume 95 July 2008257–272257
the sugar pucker is C29-endo. In the A form, the basepairs are
tilted (INC ¼ 20?) such that they are displaced from the helix
axis by ?4.0 A˚, and the sugar pucker is C39-endo.
DNA is unique in the extent to which its structure depends
on solvation. The early fiber diffraction experiments that
provided experimental data crucial to the discovery of the
double helix also revealed that DNA structure was sensitive
to the relative humidity of the sample fibers (14). The B form
is stable at relative humidities of 95% and the A form is
preferentially stabilized below 74%. Subsequent fiber dif-
fraction by Arnott and co-workers (15) elucidated the mo-
lecular structures of the canonical B and A forms of DNA.
The preferential stability of B- and A-form DNA in solution
was first reported by Ivanov and co-workers (16) based on
CD spectroscopy, and was found to depend on water activity
reported the B-to-A transition to be cooperative (17).
There has been considerable research into the forces in-
volved in the preferential stability of right-handed DNA
helices and the A-to-B transition. Solvent accessibility (18),
base-stacking interactions (19), the economics of phosphate
hydration (20), hydrophobic pressure, enthalpic stabilization
of B form by the minor-groove spine of hydration (21,22),
and electrostatic effects associated with the explicit organi-
double helix (23–26) have all been invoked as plausible and
possible explanations. Based on analysis of MD simulation
trajectories, Jayaram et al. (27) proposed that the molecular
origins of the conformational preferences of A and B DNA in
water and 85% EtOH lie primarily in the differential free-
energy contributions from interphosphate repulsion, coun-
terion condensation, and solvation.
Recently, Vargason et al. (8,28) reported a crystallo-
graphic map of the conversion of B to A DNA based on a set
of 13 crystal structures of d(GGCGCC), with various struc-
cytosine bases. This study provided a model for the B-to-A
conformational transition in terms of a set of distinct con-
formational intermediates, each of which is an equilibrium
state with respect to the free energy of the particular crystal.
The duplex in some instances was indicated to be partly A
form and partly B form, depending on local sequence com-
position, and some of the intermediate structures bear little
resemblance to either the starting or ending states. If the
mechanism of the transition can be successfully modeled
based on these intermediate structures, the conversion of
SLD precedes the change in base-pair inclination in going
from B to A, and changes in XDP occur continuously. In
these results, sugar puckers show a reasonably sharp transi-
tion point from B-like C29-endo to A-like C39-endo structures
(with some intermediate structures exhibiting a mixture of
C29- and C39-endo-type sugars). In the intermediate structures,
an extension and unwinding of the helix occurs along with
the change in sugar pucker. It remains to be established
whether these results from crystallography carry over from
equilibrium states to dynamics and are applicable to the so-
lution as well as to the crystalline state. Experimental data on
the dynamics of the B-to-A transition are relatively sparse. A
A-to-B transition based on stopped-flow, electric-field-jump
experiments. They report time constants for the transition in
the range of 10 ms, and present evidence for a significant
activation-energy barrier in going from B- to A-form DNA.
A number of CD and related studies on DNA have been
reported which characterized the sequence dependence of the
preferential stability of A and B forms of DNA (20). In gen-
eral, ApA and ApT steps are relatively rigid and resist tran-
sition from B to A, and the B/A transition is viable only for
derived from a 5-ns-long MD simulation starting from the canonical B-form structure. (c) An ensemble of structures derived from a 5-ns-long MD simulation
starting from the canonical A-form DNA structure. (d) Canonical A-form structure. The nucleotides are color coded as follows: red, adenine; blue, thymine;
green, guanine; and yellow, cytosine.
Structural representation of the d(CGCGAATTCGCG)2DNA sequence. (a) Canonical B-form DNA structure. (b) An ensemble of structures
258 Knee et al.
Biophysical Journal 95(1) 257–272
sequences with a sufficient number of GC basepairs (30,31).
Similarly, conversion to the A form using Co31(NH3)6re-
quires GpG steps and has not been demonstrated with se-
quences containing a majority of AT basepairs (25,32). As
noted above, the B form is favored under conditions of high
water activity in solution or high relative humidity in fibers.
When the water activity is lowered, or the relative humidity of
a DNA fiber is ,;75%, B-form sequences convert sponta-
neously to A form. Several groups (33,34) have examined the
experimental data on the preferential stability as a function of
sequence and B/A philicity has been expressed based on di-
meric and trimeric base-pair steps. It is important to note that
the solution structures of the A- and B-DNA endpoints of the
transition are expected to contain some structural differences
relative to the ideal canonical A and B forms of DNA inferred
from fiber diffraction (35).
In addition to CD, infrared (36–38), and Raman (5,39)
spectroscopic methods have been applied to the study of
B- and A-form DNA. Raman spectroscopic markers for the
B and A forms of DNA are well established (5,39). Certain
structural parameters of DNA can be obtained from these
methods, particularly the configuration of the sugar rings, the
angles of phosphates, and overall base geometries. A disad-
vantage of these methods, including CD spectroscopy, is that
the information on sequence dependence cannot generally be
resolved. NMR studies involving 2D nuclear Overhauser
effect spectroscopy do not resolve the DNA helical param-
eters, but the recent addition of reduced dipolar coupling has
measurably improved these determinations. However, elu-
cidation of a reliable molecular structure from NMR data
UVRR has introduced some new dimensions to the study
of nucleic acid structure (40–42). The resonance enhance-
ment intrinsic to UVRR spectroscopy makes it possible to
selectively investigate the behavior of individual types of
nucleotide bases and, in the case of propitious sequences,
individual bases. As a consequence of this resonance selec-
tive enhancement and detection, stacking interactions and
H-bonding of relevant functional groups can be attributed to
distinct residue types. Thus, UVRR spectroscopy can be a
source of more detailed information regarding local structure
of DNA as a function of sequence and conformation.
In previous studies, the UVRR method has proven useful
in evaluating the structure and stacking of DNA molecules,
cation coordination, and H-bonding (43,44). From mea-
surements of the premelting transition in DNA A-tracts, the
relative strength of cross-strand 3-centered H-bonds could be
determined (45). More recently, UVRR spectroscopy has
been applied to the study of protein-DNA interactions, and
subtle changes in DNA structure associated with protein
binding were readily detected (46–48).
MD computer simulation including explicit consideration
ofsolventhas beenapplied extensivelyto model thedynamic
structure of DNA oligonucleotides (Fig. 1) (49–54). MD
simulations are found to be generally in close accord with
NMR-derived solution structures (7,55–59). MD simulations
have provided new insights into the solution structures of
these molecules, which include essentially straight A-tracts
(60), highly flexible purine-pyrimidine steps that can serve
as a locus for axis bending (56), and support for the ‘‘non-
A-tract’’ model of DNA curvature (49). MD studies of se-
quence effects on B-form DNA structures has been advanced
by recent simulations on all 136 unique tetranucleotide base-
acid force fields (63).
The interconversion from B to A DNA has been investi-
gated via molecular dynamics simulations as described in
(2) were the first to report MD studies of A- and B-form
conformational stability in explicit solvent. Subsequently,
Sprous et al. (4) reported MD simulations on d(CGCGAA-
TTCGCG)2in A and B forms. The motivation for these
studies was directed more toward assessing the performance
of force fields, with the A-to-B transition as a prototype case.
Both Cheatham et al. (2) and Sprous et al. (4) independently
observed that in MD based on the AMBER parm94 force
field, B-form DNA was stable in water and the A form was
stable in a mixed ethanol/water solvent with reduced water
activity. The MD model A-form DNA in aqueous solution
was observed to convert spontaneously into the B form.
However, both studies reported that B-form DNA did not
spontaneously convert to the A form in the low-water-
activity mixed solvent at 300 K. This was initially addressed
as a force-field limitation and a subsequent local alteration of
the force field was designed to make this transition more
favorable (65). However, the performance of the resulting
MD model DNA deteriorated with respect to other structural
features. In these studies, the expectation of a B-to-A tran-
sition at low water activity was based on generic patterns
of environmental effects on DNA conformational stability
rather than specific behavior of a DNA sequence and the
question of whether in fact this sequence was observed ex-
perimentally to form A DNA at low water activity was raised
by Pichler et al. based on infrared studies of hydrated films
(38). The exact solvent conditions that favored A- or B-form
DNA for this sequence were not well delineated. Elucidation
of these issues, as well as the mechanism underlying them,
motivated the study described here.
Recently, Pastor studied the B-to-A transition in a TATA
box sequence using MD based on the CHARMM nucleic
acids force field (12) and found evidence in support of a
sequential ‘‘slide first, roll later’’ mechanism that is opposed
by DNA electrostatics and favored by increasing condensa-
tion of sodium ions. The latter phenomenon was noted earlier
calculations of Jayaram et al. (27), with cross correlations
validation of the results. Banavali and Roux (64) recently
reported a free energy profile for the B-to-A transition of
d(CTCGAG) in water with minimal salt based on the
Mechanism for DNA A-to-B Transition259
Biophysical Journal 95(1) 257–272
CHARMM force field. The free energy difference between
canonical A and B forms was found to be at least 2.8 kcal/
mol, and the simulation with root mean-square deviation
constraints sampled a continuum of hybrid structures with no
of this calculation raise a number of additional issues, since
some base-flipping events and other effects were noted that
are presumably artifacts. Also the MD structures of A and
B DNA deviate significantly from the idealized canonical
forms, which are not appropriate endpoints for a determina-
tion of the free energy difference in solution.
In this study, the mechanism of the A-to-B transition is
revisited to address the relative importance of base step,
solvation, and electrostatics in the transition. The combined
methodologies of UVRR spectroscopy and MD simulation
provide new insights into the transition, since the UVRR
results identify regions of change and stability, and the MD
simulations provide a structural basis for interpretation of
Raman data. These results particularly point to a sequential
mechanism for the transition, in which reorganization of
water plays a central role.
Experiments were carried out on three dodecamer sequences, d(59-CGCG-
AATTCGCG-39), d(59-CGCGAATTGCGC-39), and d(59-CGCAAATTT-
CGC-39), as a function of water activity. The 59 to 39 sequences are shown;
however, all experiments were performed on duplexes with the appropriate
complementary strands. Oligonucleotides were synthesized in 1-mmol
quantities (Integrated DNA Technologies, Coralville, IA). The deblocked
and desalted oligomers were purified by polyacrylamide gel electrophoresis.
Purity was checked by analytical gel electrophoresis. For all spectroscopic
experiments, samples were dialyzed against a buffer containing 0.1 M NaCl
and 0.67 mM NaPO4, pH 7.0. For the trifluoroethanol (TFE) experiments,
only buffer was used, as minimal salt concentrations were chosento promote
in a water bath for 5 min, followed by slow cooling to room temperature.
Annealed samples were subsequently diluted 10-fold with either TFE or
buffer,to producetwo 1310?4-M samples(strand),oneinbuffer (0%TFE)
and one in 85% TFE.
CD experiments were performed using a Jasco Spectropolarimeter J-810
with a six-cell Peltier temperature controller. Spectral scans were measured
from 300 to 190 nm, with a scan speed of 20 nm/min and an 8-s response
time. Three scans were averaged at each molar hydration point.
A Q-switched, Nd:YLF pumped Ti:sapphire laser system (Quantronix, East
Setauket, New York) was used to generate the excitation wavelengths, by
frequency-tripling or quadrupling the output of the Ti:sapphire using barium
borate crystals, as previously described(43,45).Samples werecontained in a
3 3 3-mm quartz cuvette and continuously stirred for the duration of the
experiment. All spectra were collected at 15-min intervals in separate cycles;
if any degradation in the sample was observed, that scan was discarded. The
0% and85%TFEspectraarisefrom2hof averageddata,andall otherpoints
result from 1 h of averaged data. Spectra were acquired with a slit width of
170 mm and calibrated against ethanol, acetone, and pentane. Relative
spectral shifts are accurate to 0.25 cm?1and absolute frequencies are accu-
rate to 1 cm?1. Samples were examined at room temperature and were
normalized to the TFE band occurring at 1459 cm?1. Curve fitting of the
spectra was accomplished with a mixed Lorentzian and Gaussian function,
using constant peak frequencies. All data manipulation and analyses were
performed using GramsAI (ThermoGalactic, Salem, NH).
Percent relative molar hydration (MH) was calculated as the mole frac-
tion of water at each TFE concentration using the equation XH2O¼
ðnH2O=ðnH2O1nTFEÞÞ3100; where n refers to the number of moles of water
or TFE. Concentrations of DNA and salt were neglected, as they remained
constant. Transition midpoints were treated independently and analyzed
using a Boltzmann function with Origin v. 6.0. (MicroCal, Northampton,
MA): y ¼ ððA1? A2Þ=11eððx?xoÞ=dxÞÞ1A2:
Molecular dynamics simulations
Four simulations on the duplex d(CGCGAATTGCGC) are considered here.
Simulationson both the B andA formsof this sequencereportedearlier were
simulationof the canonicalA-formstructureinan ;85%(v/v)ethanol/water
mixture,i.e., ;30%relativeMH,andinwater,andthethird andfourthareof
the canonical B-form structure in water and at 30% MH. Considerable new
analysis of solvation was carried out for this project. Details of the calcula-
tions are as follows: The AMBER utility NUCGEN was employed to create
the canonical A- and B-DNA starting structures based on fiber diffraction
data (35). For simulations in the ethanol/water mixture, the united-atom
OPLS-ethanol model (66), which is computationally effective and closely
approximates the correct density, heat capacity, and heat of vaporization
at a concentration of 30% MH relative to ethanol. The simulation protocol is
similar to those reported earlier (4,9). A ‘‘biphasic’’ initial configuration, as
described by Cheatham et al. (9), was constructed by adding TIP3P water
molecules in the first 6 A˚from the DNA surface followed by the requisite
number of ethanol molecules to make up the 30% MH ethanol/water solvent
mixture. For the simulations in water, the TIP3P (68) water model was
employed for the solvent such that the solvent extends up to 13 A˚from the
surface of the solute in all the directions.
Electroneutrality was established by adding 22 sodium cations (69) to the
system. The positions of the ions were randomized such that they were at
least 5 A˚from the DNA and 5 A˚away from each other. All MD simulations
were performed in TPN ensemble using AMBER version 7 or 8 and the
parm94 version of the Cornell et al. force field (70), and employing the
particle-mesh Ewald procedure (71,72) for the treatment of long-range in-
teractions. The MD protocol of minimization, heating, equilibration, and
production dynamics was carried out in the following steps. Minimization
involved 100 steps of steepest descent followed by 250 steps of conjugate
gradient method. Heating from 0 to 300 K was done over 10 ps, followed by
equilibration at 300 K for an additional 40 ps with SHAKE constraints (73).
During the equilibration phase, a flat well restraint on the C19-C29-C39-C49
torsion was introduced to maintain the angle between 30? and 40? in the
A-form structure, set at 25 kcal/mol/deg2for the first 30 ps and then reduced
to 5 kcal/mol/deg2. The production simulations were pursued without any
restraints for simulation times of 10 ns and longer using 2-fs time steps.
The analysis of the composition of a molecular fluid requires an inter-
pretation of the statistical distribution functions in structural and energetic
terms. A theoretical approach for this problem was mapped out several years
ago for pure liquids by Ben-Naim (74) based on the generalized molecular
distribution functions and the closely related quasicomponent distribution
function involves developing the distribution of particles with certain well-
defined values of a compositional characteristic on the statistical state of the
system (75). The basis for a general compositional analysis of the statistical
state of molecular fluids must be a unique definition of the local solution
260 Knee et al.
Biophysical Journal 95(1) 257–272
environment of each identifiable substructure-atom, function group, or
subunit of the solute. The proximity criterion accomplishes this by uniquely
identifying each solvent molecule with a well-defined solute entity in each
configuration. The proximity analysis partitions the solvent according to the
proximal solute atoms and calculates distribution properties for each parti-
tion. The solute atoms can be partitioned independently or into functional
groups defined by their chemical identity. All the proximity calculations
were performed using the MMC program (76). The solvent-accessible sur-
face area calculations on the snapshots in the MD trajectory have been
performed using the program SurfRacer 3.0 (77). The atomic radius set re-
ported by Alden and Kim (18) was employed. A spherical probe radius of
1.4 A˚corresponding to the regular radius of a water molecule was employed
in all the solvent-accessible surface area calculations.
CD spectra for all three duplexes considered in this study
preferential stability of the B and A forms at high and low
water activities and were suitable for our study. The CD
function of increasing relative percentages of TFE in Fig. 2 a.
At relatively low concentrations of TFE (.77% MH), the
spectrum exhibits a maximum at 283 nm and a minimum at
252 nm of comparable magnitude. These conservative fea-
tures above 220 nm are consistent with those previously ob-
served for B-form DNA (78). As the TFE concentration is
increased, the maximum at 283 nm decreases in intensity and
shifts to 270 nm (Fig. 2, inset). In previous work, spectro-
a peak at 270 nm, the shape of which was strongly sequence-
dependent (16,31). To ensure that the transition was not
associated with the palindromic nature of the d(CGCGA-
ATTCGCG) sequence, similar CD studies were performed
with other DNA dodecamers, which are of similar base
composition but different sequence. For all duplexes exam-
ined, a decrease in ellipticity at 250 nm coupled with a shift in
maximum to 270 nm is observed with increasing concentra-
tions of TFE (Fig. S2 in Supplementary Material, Data S1).
The magnitude of the ellipticity at 270 nm measured at 41%
MH lies in the 10–20 De (M?1cm?1) range. This is in good
accord with previous measurements of the B-to-A conversion
in TFE, in which peaks of similar ellipticity were observed at
270 nm and were identified as being diagnostic of the A form
(16,31). For all duplexes examined, the spectrum obtained at
41% MH exhibits an increase in ellipticity at 270 nm, a
decrease at 250 nm and an increase at 210 nm relative to the
B-form spectrum. These spectral features have been previ-
ously assigned to A-form DNA (33,78) and indicate that the
d(CGCGAATTCGCG)2duplex has adopted the A form with
increasing concentrations of TFE.
The CD intensity at 252 nm for d(CGCGAATTCGCG)2is
plotted as a function of molar hydration in Fig. 2 b. The
change in intensity occurs over a relatively narrow range of
hydration, consistent with previous studies. The results in-
dicate that the global change in conformation from B- to
A-form DNA occurs at 65% molar hydration. The d(CGC-
GAATTGCGC)2duplex, which is exactly the same base
composition as the d(59-CGCGAATTCGCG-39) duplex, has
a transition midpoint of 68.3%, whereas the d(59-CGCAA-
ATTTCGC-39) duplex exhibits a transition midpoint at
63.2% molar hydration (Fig. S2 in Data S1). As expected,
the increase in number of AT basepairs in going to
d(59-CGCAAATTTCGC-39) results in a transition midpoint
at a significantly lower level of hydration, that is, the more
AT-rich sequence converts to the B form at lower water ac-
tivity, consistent with conventional wisdom on the relative
propensities of the basepairs to form A- and B-form DNA. In
summary, the CD studies indicate that A/B interconversion
does occur in each of the sequences studied. The average
transition midpoint is consistent with the general idea of
the preferential stability of B form at high water activity and
UVRR spectra: ribosyl conformation
Three different excitation wavelengths were used to gain
a molecular picture of the DNA oligonucleotides changing
from A to B form in solution (Fig. 3). To selectively moni-
tor the deoxyribose-phosphate backbone, an excitation
wavelength of 210 nm was used (43,45). At this excitation
wavelength, the peak intensities of ribose modes for dG res-
idues are preferentially enhanced (41,42). The vibrational
frequencies of ribose modes are sensitive to conformation,
with the C29-endo configuration associated with a peak at 685
665 cm?1(5,39). This feature is often used as an indicator of
DNA conformation, but the results presented below indicate
that ribose conformation does not necessarily reflect an
overall A- or B-form structure. In this work, we find that this
mode shifts to lower frequency as the relative hydration is
decreased (Fig. 3). A transition midpoint of 68.3 6 2.3% is
obtained from a plot of the frequency as a function of relative
hydration (Fig. 4); this value is within the range of that de-
termined by CD spectroscopy (see above) (Table 1).
endo configurations, the Raman bands were fit assuming that
band. This fitting indicates that even at 41.5% MH, the
population of dG ribose modes in the C39-endo configuration
is 94% and not 100% (Fig. 5 a). This reflects a situation
wherein the dynamic structure of the duplex in solution in-
volves both B-form and A-form ribose structures in the
Boltzmann ensemble. Because UVRR spectroscopy super-
imposes all dG ribose modes, it is not possible to distinguish
which individual ribose moieties have adopted the C39-endo
configuration at41.5% MH and whichoneshave remained in
the C29-endo configuration. The sugar conformation of other
Mechanism for DNA A-to-B Transition261
Biophysical Journal 95(1) 257–272
bases cannot be examined because of spectral interference
from TFE bands.
UVRR spectra: base interactions
An advantage of the UVRR technique is the ability to se-
lectively enhance contributions from the different bases
through judicious choice of excitation wavelength. Thus, dG
residues are enhanced through an excitation wavelength of
240 nm and dA residues contribute strongly to the spectrum
when excited at a wavelength of 260 nm. Contributions from
dT bases are also observed using an excitation wavelength of
260 nm. Under these excitation conditions the modes that are
observed primarily arise from ring-stretching vibrations.
ATTCGCG)2DNA at increasing percentage of
molar hydration. Inset shows an expansion of
the region between 230 and 300 nm. DNA
concentration was 100 mm (strand). (b) Inten-
sity at 252 nm plotted as a function of percent
(a) CD spectra of d(CGCGA-
quence d(CGCGAATTCGCG), excited
at 210, 240, and 260 nm. Far left panel
shows an expanded view of the region
containing modes assigned to the dG C29-
endo and C39-endo sugar puckers. Spectra
are shown at 41%, 70%, and 100% molar
hydration. Vibrational modes referenced
in the main text are indicated on the
100% molar hydration spectra. Peaks
appearing at 1460 and 1283 cm?1are
assigned to residual TFE modes present
in the spectra after subtraction. Each spec-
trum results from 1 h of acquisition.
UVRR spectra of the se-
262 Knee et al.
Biophysical Journal 95(1) 257–272
Because of the effect of Raman hypochromism, which is
directly related to absorption hypochromism, the intensity of
the base modes can be related to base-stacking interactions.
For all of the modes examined, the intensity of the modes
increases upon adopting the B form (Fig. 3). The increase in
intensity is attributed to a reduction in base-stacking inter-
actions because of the greater distance or rise in base steps
(3.4 A˚forBvs. 2.8A˚forA) and themore pronouncedhelical
twist between base steps (36? for B vs. 33? for A) in B-form
DNA. The increased intensity upon adoption of the B form
has also been observed in other Raman studies of DNA
The intensity changes of the 1362 and 1485 cm?1modes
obtained with an excitation wavelength of 240 nm are shown
as a function of molar hydration in Fig. 4. Based on a com-
parison of the relative Raman cross sections (41,42,79), these
modes arise primarily from dG stretching motions rather than
dA residues because of the 240-nm excitation wavelength.
The mode occurring at 1362 cm?1arises from dG C2¼N3-
C4-N9stretching motions, with contributions from N7ring-
stretching vibrations and the 1485-cm?1mode arises from
C2-H and C8-H bending modes, coupled with C8-C9stretch-
ing motions. Since these motions lie primarily in the plane of
the ring, the enhancement mechanism is associated with the
absorption of the base itself and changes in intensity can be
related to changes in hypochromicity associated with stack-
ing interactions. The transition midpoints obtained from the
intensity changes as a function of molar hydration yield a
value of 85 6 1.0% (Table 2). Interestingly, this transition
midpoint differs significantly from that obtained for the
change in ribose conformation and suggests that the base-
stacking geometry of the dG residues changes at a higher
molar hydration compared to the ribose.
Using an excitation wavelength of 260 nm, the intensities
of modes at 1332 and 1575 cm?1can also be monitored as a
function of hydration (Fig. 4). These modes, which arise
primarily from dA residues at this excitation wavelength,
result from imidazole and pyrimidine ring-stretching vibra-
tions, respectively. The intensity change as a function of
molar hydration yields transition midpoints of 69% and 75%,
respectively. These midpoints are suggestive of a mean
transition midpoint for the AT basepair of 72% MH, which
differs significantly from that obtained for the ring-stretching
modes arising from dG residues (Fig. 4). Thus, these results
indicate that dA residues do not change their base-stacking
geometries under thesame conditions as dGresidues and that
the dA residues require significantly lower levels of molar
quency, (b) AT mode intensity, (c) GC mode intensity, and (d) dT carbonyl
mode frequency as a function of molar hydration, as observed by UVRR for
d(CGCGAATTCGCG)2. The left axis scale corresponds to the squares in
each plot, and the right axis scale to the stars. Transition midpoints and DDG
values for each component are reported in Table 2. Original data are shown
in Fig. 3.
Plots of changes in (a) sugar pucker vibrational mode fre-
d(CGCGAATTCGCG)2basepair-axis parameters and
pseudorotation phase angles
Relaxation times of some global
Average for G and C bases
Average for A and T bases
Parameters were calculated from the first-order exponential decay fit of the
x displacement (XDP), inclination (INC), and phase angle of sugar pucker
in strands 1 (PHA1) and 2 (PHA2).
Mechanism for DNA A-to-B Transition 263
Biophysical Journal 95(1) 257–272
hydration to convert to the A form. For both base types, the
transition midpoints observed are at higher water activity
relative to that observed for the ribose conformation. These
findings are in good qualitative agreement with the structure
of the d(CCCCGGGG)2duplex sequence determined by CD
and NMR spectroscopic techniques (6), in which the base-
stacking properties were found to be A-like and the sugar
pucker was observed to be B-like. The NMR-determined
structure was interpreted to be an intermediate in the A-to-B
UVRR spectra of the A and B forms of DNA also provide
some insight into the relative hydration of the grooves
through measurement of H-bonding strength. The relative
strength of H-bonding interactions is inferred from the car-
bonyl stretching frequency of relevant functional groups. A
decrease in frequency results from a reduction in the force
strength of H-bonding (45,80). The thymine C4¼O group
points into the major groove and is readily monitored using
an excitation wavelength of 210 nm (41,45). Under condi-
tions of increasing concentrations of TFE, the frequency of
the C4¼O shifts from 1662 to 1653 cm?1(Fig. 4). The
A-form than in B-form DNA. Crystallographic structures
have revealed a more ordered water structure in the major
groove of A-form DNA, and the decrease in frequency of
the C4¼O is attributed to stronger H-bonding with locally
ordered water molecules. Examination of the MD simula-
tions and canonical structures also indicates that H-bonding
geometries of the A and B forms are not substantively
In summary, the UVRR results make it possible to monitor
the ribose conformations, as well as the base stacking of dG,
dA, and dT. An examination of the series of equilibrium
states of the system as the relative hydration is increased
reveals that the sugar moieties change to C29-endo followed
by A- to B-type changes in base-stacking interactions. There
is evidence of stronger H-bonding to solvent in the major
groove of A-form DNA than in the B-form. The UVRR re-
the B-to-A transition, as opposed to an all-or-none, simple
Since earlier reported MD involved relatively short trajec-
tories, MD simulations on B and A forms of d(CGCGAA-
TTCGCG) were extended to 60 ns and 20 ns, respectively.
The overall behavior of the extended simulations was es-
sentially similar to that reported (Fig. 1) (4), and provides a
more rigorous basisfordetailed analysis ofthesolvation.The
MD simulationmostrelevanttothemechanism oftheB-to-A
transition is a simulation on the d(CGCGAATTCGCG) du-
plex in aqueous solution, beginning with the sequence in the
canonical A form. A comparison of the canonical A- and
B-form structures with the ensemble of structures generated
by the MD simulations is given in Fig. 1. During the initial
phase of the MD, there is a rapid interconversion of the se-
quence from A- to B-form, and a detailed structural analysis
provides the MD-predicted mechanism. In going from A to
B, the sugar puckers and the helical parameter INC transition
to B-form values within 500–750 ps. The basepair dis-
placement XDP convertsfrom A toBvalues inthe 750–1250
ps time range. The A-to-B interconversion is complete by
1500 ps of MD, and the resulting B-form structure is quite
similar to that found in the simulation of the sequence in
aqueous solution that began with the canonical B form. This
finding agrees with experiment, i.e., the B form is the pre-
ferred conformation in aqueous solution.
To be more quantitative, we have monitored the relaxation
observed during the simulation starting with the A-form
endo ribose conformations determined from the
fitting of UVRR bands at 665 and 685 cm?1(Fig.
3). (b) Normalized frequency of sugar puckers for
the d(CGCGAATTCGCG)2 sequence calculated
over the course of the 60-ns B-DNA simulation in
water and the 20-ns A-DNA simulation in 85%
(a) Population of C39-endo and C29-
DDG values for the d(CGCGAATTCGCG) sequence
UVRR-observed transition midpoints and
Tm (% MH)
68.35 6 2.3
72.2 6 0.7
69.2 6 1.4
75.3 6 2.5
84.6 6 1.1
85.4 6 1.5
87.5 6 6.0
264Knee et al.
Biophysical Journal 95(1) 257–272
structure in water. The relaxation times are calculated on the
basis of exponential fit of the decay in the autocorrelation
function of these parameters (Table 2). Neglecting the outlier
observed in sugar pucker relaxation time of the G10 nucle-
otide, we observe that, on average, the sugar transition from
C39-endo to C29-endo conformations relax faster than XDP and
INC. With respect to sequence effects, the AATT tract con-
These observations are consistent with those obtained from
UVRR spectroscopy, described above, which indicate that
the mechanism of the A-to-B transition is sequential, with
sugars converting from A form to B form, followed later by
the changes in base stacking. The structure passes through
an A/B hybrid form, with the AT tract converting at lower
relative hydration than theGC tracts. The MD results point to
populations of mixed ribose conformation for A DNA in
ethanol and B DNA in water but also indicate that small
fractions of other ribose conformations, not resolved in the
UVRR spectra, may be present. Interestingly, MD simula-
tions predict that even A DNA in ethanol/water contains
;20% of the C29-endo configuration and that the ribose
conformation is more heterogeneous in this conformation
than in B DNA (Fig. 5 b). In summary, the MD and UVRR
results agree well on the issue of a sequential mechanism,
i.e., one in which changes happen in sequence as opposed to
‘‘all or none.’’ Since water activity is the agent of change, we
now look to the simulation results to obtain an understanding
of the manner by which solvation effects the transition.
Our analysis of solvation examines the general nature of
the A and B forms of d(CGCGAATTCGCG) based on cal-
culation ofsolvent accessibility, and isfollowedbya detailed
analysis of the MD results using the proximity method.
Calculations of the solvent-accessible surface area of the four
nucleotides in the A- and B-form structures, computed from
the MD trajectories, are shown in Fig. 6. The solvent ac-
cessibility analysis isbroken downin terms of theconstituent
atoms in the sugar, phosphate, and major and minor groove
regions. Although the grooves together contribute ;20% of
the net solvent-accessible surface area, the related change on
going from the A- to the B-form structures is large, and
comparable to that found for the phosphate and sugar groups
(Fig. 6 c). There is a net reduction in the solvent-exposed
surface area for both the AT and GC basepairs in the A-form
structures, compared to the B-form, consistent with the idea
of reduced hydration in A DNA. Moreover, the change in
solvent-accessible surface area for the two forms is distinctly
different for the AT and GC basepairs. Specifically, the sol-
vent accessibility of AT basepairs in the major groove in the
the solvent-accessible surface areas of the G, A, C,
and T nucleotides in the MD simulation of the
central 10 basepairs of the CGCGAATTCGCG
sequence in A-form DNA (a) and B-form DNA
(b). (c) Difference in the average solvent-accessible
surface area (in A˚2) between the A and B confor-
mations for the four nucleotides and the AT and GC
(a and b) Percentage composition of
Mechanism for DNA A-to-B Transition265
Biophysical Journal 95(1) 257–272
A-form structure is greatly reduced. This finding is indicative
of a higher level of water rearrangement in the immediate
proximity of the AT basepairs, compared to the GC base-
pairs, during the B-to-A transition. This finding is in good
qualitative agreement with experimental results, which indi-
cates that AT-rich regions convert from the A to B form at
a lower level of relative hydration. Thus, the differences in
accessible surface area for GC and AT basepairs can be
correlated with their relative differences in energy in the
transition, and these differences are reflected in the experi-
In the B-form structures, phosphate groups are the most
solvent-accessible components of the nucleotides, contrib-
uting 49.5% of the total, followed by sugars at 30.5%. The
corresponding contribution of the phosphates and sugars in
the A-form structure is 47.25 and 34.25%, respectively. For
all the nucleotides, the solvent-accessible surface area of the
sugar and minor groove regions is larger in the A form, but is
consistently lower for the phosphate group and the major
groove. The hydrophobic sugar moieties are more solvent-
groups experience decreased solvent exposure since the in-
terphosphate distance is only sufficient to accommodate one
water molecule. The increased accessibility of the surface to
the solvent is a result of the shallow nature of the minor
groove region in the A-form structure, whereas the con-
comitant decrease in major groove accessibility is a result of
the deeper, narrower groove dimension (81).
The details of the MD-calculated water structure around
the A and B forms of DNA have been examined based on
proximity analysis of water molecules in MD trajectories of
A-form DNA in an ethanol/water mixture and of B-form
DNA in water (82). The analysis is presented in Table 3 in
terms of the four structural moieties, i.e., sugar, phosphate,
and major and minor grooves, for each of the central eight
basepairs in the dodecamer DNA. In particular, we report the
volume of the first solvation shell, the corresponding coor-
dination numbers, and binding energies for the stable A- and
B-form structures. Since the ethanol/water mixture contained
528 water molecules, the number of water molecules closest
to that in the B-DNA simulation in aqueous solution was
included in this proximity analysis. The ions are assumed to
be part of the solute in the binding-energy calculations. The
first shell volume of the phosphate and major-groove func-
tional group proximity region is much smaller in the A-form
structure, whereas the sugar moiety has higher accessibility
in the A-form structure. This high accessibility of the sugar
moiety is likely to be an important factor in destabilizing this
structure in aqueous medium, in which the A-form sponta-
neously transitionstoa B-form structure due atleast inpart to
hydrophobic pressure. The coordination numbers indicate
that the number density of water molecules in the proximity
region of the functional groups is much smaller in the
A-DNA structures. On average, 16 water molecules are co-
ordinated in the first two shells of the A-form structure,
compared to an average of 34 water molecules observed in
the B-form structure. The reported first-shell solvation en-
ergy is the interaction energy between the solute functional
groups and the water molecules in their corresponding
proximity region, averaged over the 5-ns MD trajectory. It is
interesting to note that although the average coordination
number is much lower, the average interaction energy of the
water molecules in the major groove of the A-form DNA
structure, especially in the central region, is more favorable
sodium ions also occurs within the major groove of A DNA.
The localization of water is in good accord with Raman
spectroscopic results, which suggests that the water mole-
cules are more tightly bound in the major groove of A-form
DNA than in the B-form structure. The water molecules in
the first shell of the B-DNA structure are bound much more
strongly in the minor groove compared to the major groove,
of hydration. In the B-DNA structure, the proximity analysis
reveals that, on average, the GC basepairs bind to water
molecules more strongly than the AT basepairs, in accord
with experimental volumetric results (83).
The CD studies have established that A/B interconversion
does occur in each of the sequences studied. The average
transition midpoint is consistent with the general idea of
preferential stability of the B form at high water activity and
as a function of sequence. The UVRR results make it pos-
stacking of dG, dA, and dT. Examination of a series of
equilibrium states as a function of increasing hydration shows
that the sugar moieties change to C29-endo, followed by A- to
B-type changes in base-stacking interactions. Intermediate
forms involve A/B hybrid structures, with AT-rich tracts
assuming the B form, whereas the CG tracts are still A-like.
In the experimental case, the reduced water activity in the
solution was achieved through addition of TFE, whereas in
the computations, the cosolvent used was ethanol. Since
ethanol can precipitate DNA, TFE was used in solution ex-
periments. Although hydrogen-bonding properties of the two
solvents are different, the changes observed are mainly at-
tributed to a reduction in water activity. This conclusion is
supported by our analyses, which indicates that the MD and
UVRR results agree well on the issue of a sequential mech-
anism for the A-to-B transition in DNA and the detailed
nature ofitat themolecular level. The UVRR and MD results
thus collectively support the idea of a cooperative but se-
quential mechanism for the B-to-A transition, as opposed to
an all-or-none, simple two-state mechanism.
In comparing experimentally observed and theoretically
calculated results, it is important to note that the experimen-
tal results report the A/B ratios for a series of equilibrium
266Knee et al.
Biophysical Journal 95(1) 257–272
mixtures, whereas the MD simulation models a kinetic
pathway between the calculated solution structures of the A
and B forms. The features of the A-to-B transition indicated
by the UVRR spectroscopy and the MD simulations are
consistent with the diverse experimental results reviewed in
Background, which pertain to the general aspects of con-
formational stability. At a more detailed level, we have
compared the UVRR/MD sequential model of the transition
to the mechanism proposed on the basis of x-ray diffraction
of various derivatized intermediates (see Fig 9 for our MD-
calculated results). A comparison of the equilibrium model
from experiment and the kinetic model from MD in terms of
the order of events agrees well with the idea of a sequential
mechanism. Comparison with the crystal structure model
indicates that the results obtained on the crystalline solid
carry over to the solution state.
A new perspective on the A-to-B transition was indicated
by the observations that H-bonding to the dT C4¼O group is
stronger in the A form and the transition midpoint occurs at a
high molar hydration (87.5% MH) (Fig. 4; Table 2). The
increased H-bonding strength was attributed to greater sol-
vent interaction and MD simulations point to increased
localization of water and ions in the major groove in the A
form. These results and the accessible surface area analyses
support the idea that reorganization of the solvent is a major
determinant in the transition.
We turn now to a methodological issue relevant to MD
simulations on DNA. Many groups have independently
TABLE 3 Proximity analysis of A- and B-form DNA trajectories
Volume of first solvation shell*Coordination number Ækæy
Basepair positionBasepairB DNAA DNA B DNAA DNA
*Volume of primary solvent shell (A˚3) derived by the proximity method.
yNumber of water molecules in the primary proximity region.
Mechanism for DNA A-to-B Transition 267
Biophysical Journal 95(1) 257–272
shown that MD simulations in water using the popular
AMBER parm94 force field equilibrate in a B-form-like
structure regardless of whether the simulation starts in an
A- or B-form structure (4,65). In an ethanol/water solvent
mixture of 30% relative MH, a simulation started from the
canonical A-form structure remains A-like (4,65). According
to conventional wisdom, a B-form structure in such a low-
water-activity state should transition to an A-like structure,
but in this case it was originally reported, and confirmed in
this study, that the MD structure remains B-like in this case.
This discrepancy has raised questions about the quality of the
force field, the length of the simulation, and the sequence
dependence of the transition (4,65). To pursue this issue, we
have extended our MD solvent analysis using a numerical
procedure to determine the average solvent positions, taking
into consideration the diffusional interchanges that would
occur as a result of solvent motion. These so-called generic
sites (84) represent the locations of the peaks in the 3D
density distribution of the solvent. Solvation sites computed
from the trajectories of A- and B-DNA structures in the 30%
MH mixture are shown in Fig. 7. The stable A-form structure
in the ethanol/water mixture shows a strong preference to
localize water and ions in the major groove, in accord with
our Raman spectroscopic results and the solute-solvent in-
teraction energy calculated using the proximity analysis.
However, the solvent analysis for the corresponding B-form
structure in the ethanol/water mixture indicates that the sol-
vent distribution in the groove region has not achieved lo-
calization of water and ions in the major groove equivalent to
that observed for the A-form structure.
sis (84) of the MD trajectories of A- and B-form
d(CGCGAATTCGCG)2DNA in the ethanol/water mixture
with the water locations color-coded by fractional occu-
pancy. A gradient coloring scheme is employed for the
generic water positions, with the sites of high occupancy
(.0.9) shaded blue, those of low occupancy (;0.3) shaded
red, and thoseof intermediate occupancyshadedgreen. The
C, G, T, and A nucleotides are colored yellow, green, blue,
and red, respectively. Sodium ions are colored magenta.
The figureshave been generated using the programPyMOL
(DeLano Scientific, Palo Alto, CA).
Representation of generic solvent site analy-
268Knee et al.
Biophysical Journal 95(1) 257–272
To assess the importance of water structure in the major
groove of the DNA in facilitating the transition from B to A
structures, we constructed a simulation ‘‘experiment’’ in the
ethanol/water mixture that began with a small cluster of 30
water molecules restrained to the vicinity of major-groove
surface atoms of the B-form starting structure. The results of
the 10 central nucleotide pairs in the dodecamer sequence
moves rapidly toward more negative values, which is indic-
ative of a transition toward A-like structures. Reversible
transitions of the sugar pseudorotation phase angle from the
C29-endo toward the C39-endo conformations is also observed,
indicating that the selective hydration of the major groove
does lead to an A-like sugar puckering. (Fig. S3 in Data S1).
We also observe that in this simulation of the CGCGA-
ATTCGCG sequence, the sugars attached to C and G bases
are more likely to be in the C39-endo state than are those at-
tached to the central A and T bases.
This result suggests that the problem with converting B to
A at reduced water activity, observed in MD simulations,
may be a matter of sampling rather than, as previously sus-
pected, a force field problem. The idea is that the B-to-A
transition evidently proceeds if the solvent is structured
properly in the major groove, but this is a highly improbable
event on the nanosecond timescale, and thus the transition is
not observed in the MD simulations. On the timescale of the
the dodecamer sequence CGCGAATTCGCG. The
x-displacement of basepairs in B DNA simulated in
ethanol/water mixture is shown in green. The data
in red represent the x-displacement of B DNA in
ethanol/water mixture simulated with a cluster of
water molecules restrained in the major groove. The
blue and pink horizontal lines at 0 and ?4 A˚
correspond to the values of x-displacement in
canonical B- and A-form DNA structures.
x-displacement of basepairs 2–11 in
Mechanism for DNA A-to-B Transition 269
Biophysical Journal 95(1) 257–272
simulation, this is a quasiergodic problem. In time frames
from microseconds to seconds,even improbable events atthe
nanosecond level can occur and nucleate the conformational
change, analogous to what happens in the observation of
particles undergoing Brownian motion. Although this is only
a leading idea, and not unequivocally demonstrated by the
analysis, our results do indicate that quasiergodic problems
deserve serious consideration in assessing future MD studies
of the B-to-A transition at reduced water activity.
The time actually required for the reorganization of the
solvent remains an open question. The measurements of Jose
and Porschke (29) suggest that the timescale of the A-to-B
transition is 10 ms and that a significant transition barrier
exists. The results presented here indicate that the barrier for
the transition lies in the organization of the solvent. Mazur
had concluded that occupation of ions in the major groove
was required for the transition (11); however, the results of
this study suggest that reorganization of water is equally
important in this transition. The ultrafast dynamic measure-
ments of Zewail and co-workers (85) demonstrate that sol-
vent reorganization can occur on a timescale of 20 ps.
However, these measurements were confined to a relatively
major groove may require a longer timescale. Further studies
are needed to appropriately address this issue.
SUMMARY AND CONCLUSIONS
A combination of methodologies has been utilized to exam-
ine the A-to-B transition. The comparison of UVRR results
with information gleaned from MD simulations has proven
illuminating with respect to the nature and mechanism of the
DNA A-to-B transition. Of significance are the correlations
between UVRR, MD, and crystallography results, which all
point to a sequential mechanism for the A-to-B transition
(Fig. 9) (8). In addition, UVRR and MD results also reveal
of A-to-B DNA conformation, as suggested in earlier NMR
studies (6). Finally, these measurements highlight the sig-
nificance of water and ion positions in facilitating the A-to-B
To view all of the supplemental files associated with this
article, visit www.biophysj.org.
This work was supported by National Science Foundation grant MCB
0316625 (I.M.) and an investigator grant from the Patrick and Catherine
Weldon Donaghue Medical Research Foundation (I.M.). D.L.B. acknowl-
edges support from National Institute of General Medical Sciences 7909.
National Institutes of Health Molecular Biophysics training grant GM
008272 and the Howard Hughes Medical Institute supported K.M.K. and
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