Báo cáo khoa học: Molecular dynamics structures of peptide nucleic acidÆDNA hybrid in the wild-type and mutated alleles of Ki-ras proto-oncogene ppt

Molecular dynamics structures of peptide nucleic
acidÆDNA hybrid in the wild-type and mutated alleles
of Ki-ras proto-oncogene
Stereochemical rationale for the low affinity of PNA in the presence
of an A C mismatch
Thenmalarchelvi Rathinavelan and Narayanarao Yathindra
Department of Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai, India
Institute of Bioinformatics and Applied Biotechnology, ITPB, Bangalore, India
Peptide nucleic acids (PNAs) stand out from the rest
of the nucleic acid mimetic, in that they consist of an
uncharged N-(2-aminoethyl) glycine (Fig. 1) backbone
scaffold [1,2]. These enable them to defy protease and
nuclease digestion, and therefore serve as promising
contenders as antigene and antisense agents [3–10].
PNA mediated transcription inhibition occurs either
by strand invasion or by conventional triplex forma-
tion [1,2]. In the former, PNA displaces one of the
strands of the DNA duplex by forming Watson and
Crick (WC) base pairs leading to a PNAÆDNA duplex
(duplex invasion) or by forming WC and Hoogsteen
Keywords
enthalpy-entropy contribution; fluctuating
A .C mismatch hydrogen bond; mismatch
containing PNAÆDNA hybrid; point mutation
Correspondence
N. Yathindra, Institute of Bioinformatics and
Applied Biotechnology, G-05, Tech Park
Mall, ITPB, Bangalore-560 066, India
Fax: +91 80 2841 2761
Tel: +91 80 2841 0029
E-mail:

(Received 13 April 2005, revised 3 June
2005, accepted 14 June 2005)
doi:10.1111/j.1742-4658.2005.04817.x
The low affinity of peptide nucleic acid (PNA) to hybridize with DNA in
the presence of a mismatch endows PNA with a high degree of discriminat-
ory capacity that has been exploited in therapeutics for the selective inhibi-
tion of the expression of point-mutated genes. To obtain a structural basis
for this intriguing property, molecular dynamics simulations are carried
out on PNAÆDNA duplexes formed at the Ki-ras proto-oncogene, compri-
sing the point-mutated (GAT), and the corresponding wild-type (GGT)
codon 12. The designed PNA forms an A. C mismatch with the wild-type
sequence and a perfect A T pair with the point mutated sequence. Results
show that large movements in the pyrimidine base of the A C mismatch
cause loss of stacking, especially with its penultimate base, concomitant
with a variable mismatch hydrogen bond, including its occasional absence.
These, in turn, bring about dynamic water interactions in the vicinity of
the mismatch. Enthalpy loss and the disproportionate entropy gain associ-
ated with these are implicated as the factors contributing to the increase in
free energy and diminished stability of PNAÆDNA duplex with the A C
mismatch. Absence of these in the isosequential DNA duplex, notwith-
standing the A C mismatch, is attributed to the differences in topology of
PNAÆDNA vis-a
`
-vis DNA duplexes. It is speculated that similar effects
might be responsible for the reduced stability observed in PNAÆDNA
duplexes containing other base pair mismatches, and also in mismatch con-
taining PNAÆRNA duplexes.
Abbreviations
DD
wt

, DNA duplex with A .C mismatch; DD
mut
, DNA duplex with A .T pair; LNA, locked nucleic acid; MD, molecular dynamics; PD
wt
,
PNAÆDNA duplex with A. . .C mismatch; PD
mut
, PNAÆDNA duplex with Watson and Crick A .T pairing; PNA, peptide nucleic acid; RMSD,
root mean square deviation; T
m
, melting temperature; WC, Watson and Crick.
FEBS Journal 272 (2005) 4055–4070 ª 2005 FEBS 4055
base pairs leading to a PNAÆDNAÆPNA triplex (triplex
invasion). Duplex strand invasion mechanism has the
advantage of targeting any sequence in a DNA duplex,
without the stringent prerequisite of a polypurine tract
as in the conventional triplex-mediated transcription
repression.
PNAÆDNA duplexes are more stable than their iso-
sequential DNA duplexes at moderate salt levels, as a
consequence of reduced electrostatic repulsion caused
by the conspicuous absence of phosphates in the PNA
strand [11,12]. Another distinctive characteristic of
PNAÆDNA complex formation has been the high
degree of discrimination for sequence selectivity with
the complementary strand of DNA [11,13] given the
significantly less stable nature of the PNAÆDNA
duplex in the presence of even a single mismatch. This
is found to be true for a variety of mismatches [14–16],
a property that is in sharp contrast to mismatch con-

taining DNA duplexes. This unique feature is utilized
to detect point mutation [17–24], selective amplifica-
tion ⁄ suppression of DNA target using PCR clamping
[25–27], and selective suppression of replication [28]
and gene expression [29,29a] by suitable choice of base
sequence in PNA. A case in point is its utility in selec-
tive inhibition of gene expression in the mutational
hotspots of ras proto-oncogenes. Normal ras proto-
oncogenes express p21, an important signal transduc-
tion protein, and a single mutation at one of the few
critical positions of ras proto-oncogenes results in a
single amino acid substitution in p21 [30] causing
malignancy [31]. One such point mutation, occurring
in codon 12 of the Ki-ras proto-oncogene, replaces
GGT with GAT [32] (capped region in Scheme 1) in
one of the alleles of pancreatic cells. This leads to pan-
creatic cancer [32], as Asp (GAT) replaces Gly (GGT)
in p21. A selective inhibition of the mutated Ki-ras
proto-oncogene can be effected by designing a PNA so
as to form a mismatch (PD
wt
) with the wild-type allele
(unmutated proto-oncogene), and a perfect WC base
pair (PD
mut
) with the mutated allele (mutated proto-
oncogene). The logic is that the former, in view of the
mismatch, is rendered a less stable PNAÆDNA complex
promoting normal expression, while the latter
(mutated) forms a stable PNAÆDNA duplex (without

mismatch) causing inhibition of gene expression. Using
this strategy, a differential proliferation effect of the
wild-type (with A C mismatch), and mutated (with
A T pair) alleles of Ki-ras proto-oncogene, has been
reported recently [29,29a]. Needless to say, a structure-
based rationale is obligatory to comprehend the causa-
tive factors for the destabilization of PNA ÆDNA in the
presence of mismatch compared to DNA duplex. Inci-
dentally, no structural information either from NMR,
X-ray crystal structure or modelling is available for
PNAÆDNA duplex with a mismatch. It is in this con-
text, molecular dynamics (MD) simulations have been
carried out on PNAÆDNA and DNA duplexes, formed
out of a sequence present in the Ki-ras proto-onco-
gene, in the presence and absence of an A C mis-
match. Results reveal that enthalpic loss and the
concomitant, but disproportionate entropic gain due to
interrupted stacking, fluctuating nature of the hydro-
gen bond and water organization in the vicinity of the
mismatch might be the contributing factors for the
increase in free energy and diminished stability of
PNAÆDNA vis-a
`
-vis DNA duplex.
Fig. 1. Schematic representation of a section of peptide nucleic
acid (PNA) chain along with notations for the backbone and side
chain torsion angles: a(C6¢-N1¢-C2¢-C3¢), b(N1¢-C2¢-C3¢-N4¢), c(C2¢-C3¢-
N4¢-C5¢), d(C3¢-N4¢-C5¢-C6¢), e(N4¢-C5¢-C6¢-N1¢), n(C5¢-C6¢-N1¢-C2¢),
v1(C8¢-C7¢-N4¢-C3¢), v2(N9 ⁄ N1-C8¢-C7¢-N4¢)andv3(C4 ⁄ C2-N9 ⁄
N1-C8¢-C7¢). Planar peptide unit is enclosed in a rectangle. Peptide

hydrogen atom alone is shown for clarity.
Effect of A .C mismatch in PNAÆDNA and DNA duplexes T. Rathinavelan and N. Yathindra
4056 FEBS Journal 272 (2005) 4055–4070 ª 2005 FEBS
Results
For convenience of discussion, and to be consistent
with the strategy of designing of PNA for gene suppres-
sion through PNAÆDNA duplex formation (see above),
the 15mer PNAÆDNA duplexes formed with an A C
mismatch (wild-type allele) and with WC A T pairing
(mutated allele) are referred to as PD
wt
and PD
mut
,
respectively (Scheme 1). Likewise, the corresponding
isosequential DNA duplexes are referred to as DD
wt
(with an A C mismatch) and DD
mut
(with A T
pair), respectively. Because base stacking and base pair-
ing interactions are the major sources of stabilization
of nucleic acid duplexes, their comparison, especially in
the vicinity of the mismatch in PD
wt
compared with
DD
wt
duplex may give clues towards deciphering the
origin of the destabilization and hence, diminution of

the melting temperature (T
m
) in the former.
Base stacking in the vicinity of A C mismatch
in PNAÆDNA and DNA duplexes
Intra strand base stacking at the AC(6–7) (Fig. 2A) &
CC(7–8) steps (Fig. 2B) of the DNA strand, and
GA(23–24) (Fig. 2C) and AT(24–25) (Fig. 2D) steps of
the PNA strand, flanking the A24 C7 mismatch in
PD
wt
(Scheme 1), and the corresponding AT(6–7) &
TC(7–8) steps of the DNA strand (Fig. 2E,F), and
GA(23–24) & AT(24–25) (Fig. 2G,H) steps of the
PNA strand in PD
mut
(Scheme 1) are monitored.
Base stacking at the CC(7–8) step of PD
wt
(Fig. 2B),
and the TC(7–8) step of PD
mut
(Fig. 2F) of the DNA
strand show significant differences. This is due to con-
siderable movement of cytosine (C7) of the A24 C7
mismatch of PD
wt
, leading to large fluctuations in its
interaction with the adjacent pyrimidine base (C8).
This results in hardly any stacking between them.

Only occasionally, C5-H group of cytosine (C8) over-
laps with C7 and, O2 of C7 overlaps with C8. On the
other hand, sustained stacking persists by way of par-
tial overlap of T7 and C8 (Fig. 2F) at the correspond-
ing TC(7–8) step of PD
mut
. A totally unstacked
situation is seldom seen here indicating that occur-
rence of an A24 C7 mismatch brings about signifi-
cant reduction in adjacent base stacking in PD
wt
compared to PD
mut
.
On the other hand, stacking at the AC(6–7) step in
the DNA strand of PD
wt
is retained during the entire
simulation in spite of the large movement of C7
(Fig. 2A). This occurs due to the coordinated move-
ments of C7 and A6 which ensure stacking through-
out. Similarly, stacking persists at the corresponding
AT(6–7) step in PD
mut
(Fig. 2E) through interaction of
A6 with either the six-member ring of T7 or through
the methyl group and ⁄ or O4 of T7. Thus, base stack-
ing prevails at the AC(6–7) step (Fig. 2A) of PD
wt
,

and the AT(6–7) step of PD
mut
(Fig. 2E). Likewise, the
extent of intra strand base stacking at the GA(23–24)
and AT(24–25) steps of the PNA strand in both PD
wt
(Fig. 2C,D) and PD
mut
(Fig. 2G,H) is essentially sim-
ilar. Thus, A24 C7 mismatch leads to an almost
complete loss of stacking only at the CC(7–8) step
(PD
wt
), while the stacking is maintained in the other
steps that flank the mismatch.
Scheme 1. Sequences encompassing codon 12 (capped) of the Ki-ras proto-oncogene of wild type (wt) and mutated (mut) alleles. Bold-italic
regions in both wild-type and mutated sequences represent the PNAÆDNA duplex. Mismatch (wild-type) and the corresponding ideal WC
base pairs (mutated) are underlined. The C- and N-termini of the PNA are considered as equivalent to 3¢ and 5¢ ends of a nucleic acid chain,
respectively.
T. Rathinavelan and N. Yathindra Effect of A C mismatch in PNAÆDNA and DNA duplexes
FEBS Journal 272 (2005) 4055–4070 ª 2005 FEBS 4057
It is clear from Fig. 3A–D that presence of an A C
mismatch in DD
wt
, seemingly does not influence adja-
cent base stacking at the mismatch site. Although
stacking at the CC(7–8) step is found to be only margi-
nal in DD
wt
during the first 220 ps, quite similar to

that seen in the PD
wt
, it is enhanced significantly
beyond 220 ps, so much so that almost a complete
overlap of adjacent pyrimidines is observed (Fig. 3B).
Stacking interactions at the neighbouring AC(6–7),
GA(23–24) and AT(24–25) steps of the A24 C7 mis-
match site are also maintained (Fig. 3A,C,D). It is
noteworthy that although stacking at the AC(6–7) step
fluctuates, a complete loss of stacking is seldom found
(Fig. 4A). Stacking persists either through the overlap
A
B
C
D
E
F
G
H
Fig. 3. Stereo diagram of adjacent bases at
various steps flanking the A24 .C7 mis-
match in DD
wt
: (A) AC(6–7); (B) CC(7–8); (C)
GA(23–24) and (D) AT(24–25), and their
equivalent steps in DD
mut
: (E) AT(6–7); (F)
TC(7–8); (G) GA(23–24) and (H) AT(24–25).
Notice that stacking prevails in all the steps,

both in DD
wt
and DD
mut
. C7 and A24
involved in A .C mismatch in DD
wt
and the
equivalent T7 and A24 in DD
mut
are col-
oured red. Trajectories corresponding to
every 20 ps are shown.
E
F
G
H
A
B
C
D
Fig. 2. Stereo diagram of adjacent bases at
various steps flanking the A24 .C7 mis-
match in PD
wt
: (A) AC(6–7); (B) CC(7–8);
(C) GA(23–24) and (D) AT(24–25), and their
equivalent steps in PD
mut
: (E) AT(6–7); (F)

TC(7–8); (G) GA(23–24) and (H) AT(24–25).
Note the interruption of the stack at the
CC(7–8) step (B) in PD
wt
, while base stack-
ing prevails at all the steps in PD
mut
(E–H).
Large movements of C7 at CC(7–8) step (B)
are apparent. C7 and A24 bases involved in
A .C mismatch in PD
wt
and, the equivalent
T7 and A24 bases in PD
mut
are coloured
red. Trajectories corresponding to every
20 ps are shown.
Effect of A .C mismatch in PNAÆDNA and DNA duplexes T. Rathinavelan and N. Yathindra
4058 FEBS Journal 272 (2005) 4055–4070 ª 2005 FEBS
of the amino group of C7 with A6 (Fig. 4B) or
through the overlap of the six-member ring of C7 with
A6 (Fig. 4C). Thus, unlike in PD
wt
(Fig. 2B), uninter-
rupted stacking prevails at all the steps of DD
wt
.
Interestingly, the extent of stacking at the TC(7–8)
step of DD

mut
with A T pair (Fig. 3F), is similar to
that at the CC(7–8) step of DD
wt
(Fig. 3B). Further-
more, it is evident that although the exact mode of
stacking interactions at the AT(6–7) (Fig. 3E), GA(23–
24) (Fig. 3G) and AT(24–25) (Fig. 3H) steps in DD
mut
appear to be different from the equivalent steps in
DD
wt
(Fig. 3A,C,D), the degree or extent of stacking is
comparable. This suggests that the stacking interaction
persists in the adjacent steps of both DD
wt
and DD
mut
.
On the other hand, as noted above, stacking is inter-
rupted in PNAÆDNA duplex with an A C mismatch.
Variation of A C mismatch hydrogen bond
in PNAÆDNA and DNA duplexes
Fluctuations in the position of C7 of the A24 C7
mismatch in PD
wt
discussed above are also found to
influence the nature of A24 C7 mismatch hydrogen
bond. It is found that hydrogen bond fluctuates
between N6(A24) N3(C7) and ⁄ or N6(A24). O2(C7)

(Fig. 5). As C7 approaches A24, it engages in
N6(A24) N3(C7) hydrogen bonding, and when C7
moves away from A24 along the major groove, the
other possible hydrogen bonding schemes emerge
(Fig. 5A–C). Extreme movement of C7 away from
A24 can even result in the absence of both the hydro-
gen bonds (Fig. 5D). These are apparent in Fig. 5E.
MD simulations extended up to 4 ns further substanti-
ates the variable nature of the hydrogen bond (Fig. 5).
These clearly indicate the absence of a stable hydrogen
bond for the A24 C7 mismatch in PD
wt
.
In sharp contrast, a stable N1(A24) N4(C7) hydro-
gen bond (Fig. 6B) prevails in DD
wt
, although the ini-
tial N6(A24) N3(C7) hydrogen bond (Fig. 6A) lasts
for a short duration (200 ps) (Fig. 6C–F). The transi-
tion to the favoured hydrogen bond occurs as a result
of movement of A24 rather than C7 (of the DNA
strand) as found in PD
wt
and persists till the end of
4 ns dynamics. Further, A C mismatch hydrogen
bond in DD
wt
is different from that found in PD
wt
(Fig. 5). An earlier MD simulation (just over 100 ps)

based on NMR data on DNA duplex, pointing to the
Ki-ras proto-oncogene having an A23 C8 mismatch
(Scheme 1) instead of A24 C7 as in the present study,
has indicated the possibility of all the three schemes
for A23 C8 mismatch hydrogen bonding [33], but
without preference for any one of them. However, it is
found here that A24 C7 favours N1(A24) N4(C7)
hydrogen bond.
In any case, the present analysis clearly points to the
greater changeability and destabilization of the A C
mismatch hydrogen bond in PNAÆDNA than in DNA
duplex. As expected, these bring forth significant varia-
bleness in the water interactions surrounding the mis-
match.
Water interaction in the vicinity of A C
mismatch
Figure 7A–L depicts the nature of water interaction in
the neighbourhood of A24 C7 mismatch in PD
wt
.
Water interaction along the minor groove side of
A24 C7 mismatch is conserved to the extent that
either N1(A24) or O2(C7) or both, are involved in
interaction with water. This is true irrespective of
Fig. 4. Stacking interactions seen at AC(6–7) step of DD
wt
. Note
the prevalence of stacking interaction (B and C) almost throughout
dynamics (see also text) despite the fluctuations. Complete loss of
stacking interaction is seldom seen (A).

T. Rathinavelan and N. Yathindra Effect of A C mismatch in PNAÆDNA and DNA duplexes
FEBS Journal 272 (2005) 4055–4070 ª 2005 FEBS 4059
the presence or absence of N6(A24) N3(C7) and⁄ or
N6(A24) O2(C7) hydrogen bond. On the other hand,
water interaction along the major groove is influenced
by the nature of the mismatch hydrogen bond. When
N3(C7) is not involved in hydrogen bond with
N6(A24), it engages itself in a variety of interactions
with water along the major groove side as shown
in Fig. 7A,E,F,I. In the absence of both
N6(A24) N3(C7) and N6(A24) O2(C7) hydrogen
bonds due to the displacement of the C7 towards the
major groove, N3(C7) and O2(C7), both are engaged
in interaction with water (Fig. 7D). These are demon-
strative of significant fluctuations in the water structure
in the vicinity of A24 C7 mismatch in PD
wt
. In con-
trast, such variation is not observed in DD
wt
due to
the strong preference for N1(A24) N4(C7) hydrogen
bond (Fig. 7M–T). As a result, N6(A24) and N4(C7)
are involved in a variety of water interaction on the
major groove side (Fig. 7N–T). Similarly, N3(A24),
N3(C7) and O2(C7) are also engaged in water interac-
tion most of the time (Fig. 7N–T). Thus, it is apparent
that water interaction associated with the atoms parti-
cipating in A24 C7 mismatch does not show fluctu-
ation as in the case of PD

wt
.
Fig. 5. Interaction between the A24 (blue)
and C
7
mismatch bases in PD
wt
(A–D) and
variation of N6(A24). . .N3(C7) and
N6(A24) .O2(C7) hydrogen bond distances
(F & H), and angles (G & I) over 4 ns dyna-
mics. Large movement of C7 and the asso-
ciated variable hydrogen bonding pattern for
A24. . .C7 mismatch are clear from the
superposition (E).
Effect of A .C mismatch in PNAÆDNA and DNA duplexes T. Rathinavelan and N. Yathindra
4060 FEBS Journal 272 (2005) 4055–4070 ª 2005 FEBS
Conformation of the PNA strand in PNAÆDNA
duplexes
Like in DNA, backbone conformation of the PNA
scaffold is governed by six backbone torsion angles,
a(C6¢-N1¢-C2¢-C3¢), b(N1¢-C2¢-C3¢-N4¢), c(C2¢-C3¢-
N4¢-C5¢), d(C3¢-N4¢-C5¢-C6 ¢), e(N4¢-C5¢-C6¢-N1¢) and
f(C5¢-C6¢-N1¢-C2¢). These are found to be confined to
the trans ⁄ gauche
–
, gauche
+
, gauche
+

, gauche
+
, near
cis and trans range of conformations, respectively, in
both PD
mut
and PD
wt
(Fig. S1A–F of Supplementary
material). The side chain torsion angles, v1(C8¢-C7¢-
N4¢-C3¢), v2(N9 ⁄ N1-C8¢-C7¢-N4¢) and v3(C4 ⁄ C2-
N9 ⁄ N1-C8¢-C7¢) favour the cis, trans ⁄ gauche
–
and
gauche
+
conformations, respectively (Fig. S1G–I of
Supplementary material). It is noteworthy that both
backbone, as well as side chain, conformations of the
PNA strand observed in the present study generally
fall in the same range of conformational angles seen in
the crystal structures of PNAÆDNA duplex [34] and
(PNA)
2
ÆDNA triplex [35]. These are also broadly
similar to the results obtained from earlier MD simu-
lations on PNAÆDNA complexes [36,37]. Some differ-
ences seen from the NMR structure may be due to
under-determination of the backbone structure by
NMR as acknowledged by the authors [38]. Inciden-

tally, a designed PNA analogue with b ¼ gauche
+
region, similar to that observed in the current investi-
gation, readily forms complex with both DNA and
RNA [39–41].
A
B
C
D
EF
Fig. 6. Hydrogen bonding schemes (A and
B) observed for the A24 .C7 mismatch in
DD
wt
. Variation of hydrogen bond distances
(C and E) and angles (D and F) over 4 ns
dynamics. Note the strong preference for
N1(A24) .N4(C7) hydrogen bond beyond
220 ps (C and D).
T. Rathinavelan and N. Yathindra Effect of A C mismatch in PNAÆDNA and DNA duplexes
FEBS Journal 272 (2005) 4055–4070 ª 2005 FEBS 4061
Base backbone hydrogen bonds and a(N1¢-C2¢)
and e(C5¢-C6¢) correlation in the PNA strand
Interestingly, a near-neighbour bond correlation between
the torsion angles a(N1¢-C2¢)ande(C5¢-C6¢) associated
with the peptide unit is recognized. It is observed that
whenever a undergoes a conformation change from the
most preferred trans ⁄ gauche
–
to the gauche

+
conforma-
tion, a concomitant change occurs in e from a cis to a
trans conformation (Fig. 8) as found earlier [42]. These
ensure stacking as well as the WC hydrogen bond
(Fig. 9). Other transitions lead to a totally unstacked
situation (data not shown). Further, the (gauche
+
, trans)
conformational state for (a,e) promotes an intramolecu-
lar O6¢ H-N2 (G) hydrogen bond between guanine
and the carbonyl of the peptide (Fig. 10I). However,
this is not possible for the (trans ⁄ gauche
–
, cis) conforma-
tion as O6¢ orients towards the solvent with the amide
(N1¢) hydrogen pointing inside the helix. On the other
hand, this facilitates in the formation of hydrogen
bond with N3 of purines and O2 of pyrimidines either
directly (Fig. 10A,C,E,G) or through water molecules
Fig. 7. Interaction of water (orange) with
A24. . .C7 mismatch in PD
wt
(A–L) and DD
wt
(M–T) during the dynamics. Variation in
hydration pattern in PD
wt
(A–L) depending
on the A24 .C7 mismatch hydrogen bond-

ing is readily apparent.
Effect of A .C mismatch in PNAÆDNA and DNA duplexes T. Rathinavelan and N. Yathindra
4062 FEBS Journal 272 (2005) 4055–4070 ª 2005 FEBS
(Fig. 10B,D,F,H). Interestingly, water mediated N3 N1¢
[34] and O2 N1¢ [34,35] interactions are found in the
minor groove side of the PNAÆDNA duplex [34] and
(PNA)
2
ÆDNA tripl ex [35] crysta l structures.
PNAÆDNA duplex structure
Average structure of the central 11mer of PD
wt
over
2.5 ns dynamics is shown in Fig. 11A. Root mean
square deviation (RMSD) of the entire trajectory
(2.5 ns) with respect to the average structure lies in the
range  0.7–2.5 A
˚
for both PD
wt
and PD
mut
.
Average value of helical twist corresponding to the
central 11mer is  27° in both PD
wt
and PD
mut
leading
to a 13-fold duplex. This is similar to that observed in

an NMR study of a PNAÆDNA hybrid [38]. Average
value of rise, slide, X-displacement and propeller twist
correspond to values around 3.3 A
˚
, )1.2 A
˚
, )3.7 A
˚
and )10.7°, respectively, for PD
wt
, and 3.3 A
˚
, )1.3 A
˚
,
)4.0 A
˚
and )10.6°, respectively, for PD
mut
. Average
widths of minor and major grooves are around 9.5 A
˚
and 25 A
˚
in both PD
wt
and PD
mut
.
Sugar puckers in DNA strands favour the C2¢ endo

conformation in both PD
wt
and PD
mut
. Interestingly,
C7 involved in A C mismatch seems to favour the
C4¢ exo sugar pucker, although C2¢ endo is seen dur-
ing the dynamics (data not shown).
In general, stacking interaction is nearly similar in
both PD
mut
and PD
wt
(data not shown) except at steps
on either side of the mismatched A C hydrogen
bond.
DNA duplex structure
Average structure of the central 11mer of DD
wt
over
2 ns dynamics is shown in Fig. 11B. RMSD of the
entire trajectory (2 ns) corresponding to DD
wt
and
DD
mut
varies from 1.2–2.1 A
˚
and 1.0–2.8 A
˚

, respect-
ively, with respect to their average structure. Even
Fig. 8. Correlation between the backbone torsion angles, a(N1¢-C2¢)
and e(C5¢-C6¢)inPD
wt
(red) and PD
mut
(black). Notice the prefer-
ence for (a,e) . (trans ⁄ gauche
–
,nearcis) compared to (a,e) .
(gauche
+
, trans) conformation.
Fig. 9. Stereo plots illustrating the stacking interaction at the GC step when (a,e) . (trans ⁄ gauche
–
, near cis) (A and B) and (a,e) . (gauche
+
,
trans) (C and D) conformations. O6¢(G). . .N2(G) hydrogen bond (C and D) is shown in dotted line (see also Fig. 10 and text). Hydrogens at
C2¢,C3¢,C5¢ and C8¢ are not shown for clarity.
T. Rathinavelan and N. Yathindra Effect of A C mismatch in PNAÆDNA and DNA duplexes
FEBS Journal 272 (2005) 4055–4070 ª 2005 FEBS 4063
though RMSD is rather large for DD
mut
during the
first 500 ps of the dynamics, it stabilizes later.
RMSD of DD
mut
and DD

wt
falls between 1.0 and
2.0 A
˚
, beyond 500 ps representing the equilibrium
state.
The overall conformation of the helix is of B type.
Average value of the major groove width is 16.8 A
˚
for
DD
wt
and 18.0 A
˚
for DD
mut
, while the average widths
of the minor groove are 11.4 A
˚
and 11.3 A
˚
for DD
wt
and DD
mut
, respectively.
Fig. 10. Dependence of backbone .base
hydrogen bond interactions in PNA on a and
e correlation. Note that hydrogen bond
between N1¢ (backbone) and base (O2 ⁄ N3)

may be direct (A,C,E,G) or through water
(B,D,F,H) when (a,e) . (trans ⁄ gauche
–
, near
cis) and N1¢. . .N1¢ repeat is compact (5.5 A
˚
).
Direct hydrogen bond between O6¢ and N2
(I) is seen for (a,e) . (gauche
+
, trans) when
N1¢. . .N1¢ repeat is extended (6.5 A
˚
). Hydro-
gens at C2¢,C3¢ and C5¢ are not shown for
clarity.
Effect of A .C mismatch in PNAÆDNA and DNA duplexes T. Rathinavelan and N. Yathindra
4064 FEBS Journal 272 (2005) 4055–4070 ª 2005 FEBS
Overall averages of X-displacement, rise, twist, pro-
peller twist and slide correspond to )1.1 A
˚
, 3.2 A
˚
,
32.4°, )10.5° and )0.2 A
˚
, respectively, for DD
wt
and
)0.8 A

˚
, 3.2 A
˚
, 33.5°, )10.8° and )0.2 A
˚
, respectively,
for DD
mut
.
Conformation angles, a(P-O5¢), b(O5¢-C5¢), c(C5¢-
C4¢), e(C3¢-O3¢), f(O3¢-P) and v(C1¢-N1 ⁄ N9) assume
the preferred gauche
–
, trans, gauche
+
, trans, gauche
–
and anti conformations, respectively, in both DD
wt
and DD
mut
. As a consequence of N1(A
24
) N4(C
7
)
hydrogen bond, e and f torsions at A
6
C
7

step favour
a BII conformation (e ¼ gauche
–
& f ¼ trans) [43,44].
The overall preference for sugar puckers conforms to
the C2¢ endo domain.
Discussion
Base pair mismatch in duplex DNA contributes an ele-
ment of instability resulting in lowering of the melting
temperature and increase in free energy. Surprisingly,
the presence of a mismatch in PNAÆDNA duplexes
[11,14–16] has a pronounced effect in reducing the T
m
,
with a free energy penalty of about 15 kJÆmol
)1
per
base pair [16]. This characteristic property is taken
advantage of in the selective inhibition of gene expres-
sion of a point-mutated gene by appropriate design of
a PNA [29,29a]. Factors that contribute to the sharp
decrease in free energy could arise from (a) reduced
binding enthalpy caused by the loss of hydrogen bond
and weak stacking and also (b) entropic and enthalpic
contribution resulting from the nature of interaction of
water in the vicinity of mismatch [16]. However, lack
of structural information on mismatch containing
PNAÆDNA duplex has prevented possible correlation
with thermodynamic data. It is with a view to fill in
this gap that MD simulations are performed for

PNAÆDNA duplexes with and without the base pair
mismatch corresponding to the Ki-ras proto-oncogene
sequence (Scheme 1). Such in silico studies have proved
to be invaluable in understanding the dynamic nature
of the structure and interactions of nucleic acids, and
especially so, in discerning the comparative influence
of base pair matches vis-a
`
-vis normal ones. Choice of
the aforementioned sequence is because of the proven
efficacy of the designed PNA to down-regulate the
gene expression of the mutated allelle, while sustaining
the transcription of the wild-type allele [29,29a]. The
present investigation is the first report on the structure
and dynamics of a PNAÆDNA duplex with a mis-
match. The results of the analysis are also compared
with the MD simulations on isosequential DNA
duplexes in the presence (DD
wt
) and absence (DD
mut
)
of an A C mismatch.
Our results point to considerable fluctuations in
cytosine base (C7) of the A C mismatch in PD
wt
leading to transition between the two possible hydro-
gen bonds, N6(A24) N3(C7) and N6(A24) O2(C7)
(Fig. 5). This, together with the occasional tendencies
for complete loss of hydrogen bonding interactions, is

expected to contribute towards disfavoured enthalpy
(single hydrogen bond or complete loss of hydrogen
bond) leading to destabilizing conditions. It should
also be noted that the fluctuating nature of A C mis-
match hydrogen bond concomitantly brings forth the
dynamical nature of water interaction, particularly on
the major groove side (Fig. 7A–L). This should give
rise to stabilizing (increase in) entropy contributions.
Additional factor that could contribute towards lower-
ing enthalpy arises from unstacking of C7 with the
adjacent C8 in the DNA strand of PD
wt
(Fig. 2). It is
probable that unequal compensation between enthalpy
loss and entropy gain might lead to unfavourable free
energy for PNAÆDNA duplex in the presence of an
A C mismatch. This argument gains support from
a thermodynamic study [16], which shows that a
Fig. 11. Stereo plot of the central 11mer of the average structure
of PD
wt
(A) and DD
wt
(B) with A24 .C7 mismatch (blue). PNA
strand is shown in green (A).
T. Rathinavelan and N. Yathindra Effect of A C mismatch in PNAÆDNA and DNA duplexes
FEBS Journal 272 (2005) 4055–4070 ª 2005 FEBS 4065
PNAÆDNA duplex with an A C mismatch (mediated
between C G and A T pairs) results in unfavourable
change in the value of enthalpy (DH ° ) from )198 to

)85 kJÆmol
)1
while the entropy (TDS°) increases from
)154 to )50 kJÆmol
)1
, in a noncompensating manner,
causing a loss in free energy (DG°) from )44 to
)35 kJÆmol
)1
. This indicates that favourable entropic
contributions are not sufficient to offset the loss in
enthalpy and this results in the less stable nature of
the PNAÆDNA duplex in the presence of an A C mis-
match.
In sharp contrast, an A C mismatch in DNA
duplex (DD
wt
), strongly prefers N1(A24) N4(C7)
hydrogen bond (Fig. 6) and a relatively stable water
environment around the mismatch (Fig. 7M–T), con-
comitant with an uninterrupted base stacking at the
steps flanking (Fig. 3) the A C mismatch. These are
expected to contribute towards the relatively higher
stability of DD
wt
through (favourable) enthalpy and
(unfavourable) entropy compared to PD
wt
.
Thus, it can be inferred from these results that the

fluctuating nature of hydrogen bond (entropically
favoured but enthalpically disfavoured), substantial
rearrangement of water around the A. C mismatch
(entropically favoured), and interrupted base stacking
(enthalpically disfavoured) perhaps are the factors that
contribute to considerable increase in free energy due
to the presence of A C mismatch in a PNAÆDNA
duplex compared to an isosequential DNA duplex. In
this context, it may be interesting to point out that
preliminary studies have shown that consideration of
an identical PNAÆDNA duplex with an A C mis-
match used in the current investigation has indeed
been shown to exhibit a much weaker effect on the
proliferation of the pancreatic carcinoma cells contain-
ing only wild-type allele [29] (PD
wt
in the current
study), which has been further confirmed very recently
using RT-PCR [29a]. As discussed above, this is con-
ceivably due to the less stable nature of the PNAÆDNA
duplex with an A C mismatch. However, formation
of a PNAÆDNA duplex with a prefect WC pair (PD
mut
in the current study) significantly reduces both mRNA
and protein levels in cells, perhaps due to the higher
stability of duplex [29,29a]. Melting studies also point
to the reduction of T
m
by  13–19° in PNAÆDNA
duplex [14–16] with an A C mismatch.

It may be observed that the A C mismatch hydrogen
bond is different in PD
wt
and DD
wt
. As detailed above,
it fluctuates between N6(A24) N3(C7) and
N6(A24) O2(C7) in PD
wt
. Further simulation (1 ns)
indicates that even at 400 K, the fluctuating nature of
the mismatch hydrogen bond (data not shown) prevails
in PD
wt
. In contrast, a stable N1(A24) N4(C7) hydro-
gen bond is favoured throughout the dynamics in DD
wt
leading to an uninterrupted stacking at the steps flank-
ing the mismatch. The difference in the A C mismatch
hydrogen bond may therefore be attributed to the signi-
ficant differences in the topological features, namely,
minor and major groove widths and X-displacement
( 9.5,  25,  )4 vs.  11,  18,  )1A
˚
) of PNAÆDNA
and DNA duplexes. It is noteworthy that all of these
A C mismatch hydrogen bonds (Figs 5 and 6) noticed
during the dynamics of PNAÆ DNA and DNA duplexes
are found in the crystal structure of ribosomal subunit
[45,46] and tRNA [47,48].

The above deductions are also expected to hold true
for other mismatch containing PNAÆDNA duplexes,
wherein a similar drastic reduction in T
m
accompanied
by increase in free energy is reported [16], although the
nature of the stacking, mismatch hydrogen bond and its
fluctuating character in these cases may be governed to
some extent by the sequences flanking the mismatch.
Interestingly, base pair mismatch is also found to induce
depression of T
m
[14] in PNAÆRNA hybrid enabling
mutation-selective translational effect on wild-type and
mutated Ha-ras mRNAs [49]. Similar mismatch induced
reduction in T
m
has also been reported for PNAÆPNA
duplex [50]. It may be that the causative factors even
here might be those attributed in the context of
PNAÆDNA hybrid in view of similar topology (large
X-displacement and wider major groove width) for
PNAÆRNA [51] and PNAÆPNA [34,52] duplexes. Inter-
estingly, a more recent study reports that presence of a
mismatch in PNAÆlocked nucleic acid (LNA) hybrid
duplex also leads to depression in the T
m
[53], perhaps
resulting from similar topological difference of
LNAÆPNA hybrid compared to DNA duplex.

Incidentally, parallel PNAÆDNA duplex exhibits a
lower thermal stability (T
m
) compared to its antiparal-
lel counterpart [11]. Although CD data on parallel
PNAÆDNA hybrid deviates from that of DNA duplex
[11], comparison of the MD structure of parallel
PNAÆDNA [36] with the B DNA indicates some
amount of similarity especially with respect to lack of
X-displacement. Based on this, one may speculate that
the effect of mismatch on parallel PNAÆDNA duplex
stability might be similar as in DNA duplex.
In summary, the present study offers a possible
stereochemical basis for the experimentally observed
highly diminished stability of the PNAÆDNA duplex in
the presence of A C mismatch vis-a
`
-vis DNA duplex.
Experimental procedures
Two antiparallel (the C- and N- termini of the PNA are
considered as equivalent to 3¢ and 5¢ ends of a nucleic acids
Effect of A .C mismatch in PNAÆDNA and DNA duplexes T. Rathinavelan and N. Yathindra
4066 FEBS Journal 272 (2005) 4055–4070 ª 2005 FEBS
chain, respectively) 15mer PNAÆDNA hybrid duplexes
(PD
wt
and PD
mut
) represented in Scheme 1 are generated
using the twist angle of 28° and rise of 3.2 A

˚
derived from
the NMR structure of PNAÆDNA duplex [38] (PDB ID
1PDT). Steepest descent energy minimization is carried out
for 1000 cycles using the Discover module of insight ii
software program [54] with the amber force field. Initial
orientation of A C mismatch is such that it forms a
hydrogen bond between N6(A24) and N3(C7) [55] and is
constrained during initial minimization (500 cycles) of PD
wt
(Scheme 1). It is noteworthy that N6(A24) N3(C7) hydro-
gen bond is found along with N1(A24) O2(C7) in DNA
and RNA duplexes [33,56,57] comprising A
+
C mismat-
ches. MD simulation is pursued with amber 6 [58] as des-
cribed below.
Partial charges on the PNA are calculated using pc
gamess Ver. 6.2 [59] (Granovsky, A. A., http://classic.
chem.msu.su/gran/gamess/index.html) and RESP [60] mod-
ule of amber 6, because the same is not available in the
amber standard library. Electrostatic potential calculated
using the HF ⁄ 6–31G* basis set (pc gamess) is used in the
calculation of RESP charges (Figs. S2 and S3A–D of the
Supplementary material). It is noteworthy that the partial
charges for the various atoms of bases are very similar to
when they are part of DNA chain (Fig. S3E–H of the Sup-
plementary material).
Periodic box of TIP3P waters and 14 Na
+

counter ions
to neutralize the charge on the DNA strands of the hybrids
are added using LEaP module of amber 6. This results in
4294 and 4379 number of water molecules for PD
mut
and
PD
wt
systems, respectively, and periodic boxes of sizes
 48 A
˚
·  51 A
˚
·  77 A
˚
and  50 A
˚
·  51 A
˚
·  77 A
˚
are obtained.
Both wild-type DNA (DD
wt
) and mutated DNA (DD
mut
)
duplexes are generated to conform to ideal B DNA using
insight ii [54]. Minimization is carried out as described for
PD

wt
and PD
mut
. Using LEaP module of amber 6, 28 Na
+
net neutralizing counter ions, and periodic box of TIP3P
waters are added to each system. A total number of 3985
and 4239 water molecules are added to the DD
mut
and the
DD
wt
, respectively. The resultant periodic box sizes corres-
pond to  50 A
˚
·  47 A
˚
·  77 A
˚
for the DD
mut
and,
 51 A
˚
·  50 A
˚
·  74 A
˚
for the DD
wt

.
MD for PD
wt
,PD
mut
,DD
wt
and DD
mut
is pursued
with amber 6 following the protocols described elsewhere
[61]. Production run is continued up to 2.5 ns each for
PD
mut
and PD
wt
and 2 ns each for DD
wt
and DD
mut
.
Simulation is carried out under isobaric and isothermal
(300 K) conditions. Long-range electrostatic interactions
are calculated using particle mesh Ewald method [62]
with a tolerance of 0.00001 for direct space sum cut-off.
Simulation is performed with shake (tolerance ¼
0.0005 A
˚
) on the hydrogens [63], a 2fs integration time
and a cut-off distance of 9 A

˚
for Lennard–Jones inter-
action.
Average structure and RMSD are calculated using ptraj
Ver. 6.4 ( />html). moil-view Ver. 9.0 program is used for trajectory
analysis [64]. Conformation angles of the PNA strand are
calculated using an in-house program. Conformation angles
of the DNA strands and helical parameters are extracted
from the output of 3dna Ver. 1.5 [65] using in-house
programs.
Acknowledgements
Financial assistance to the Department under DSA
and FIST programs of UGC and DST, respectively,
are acknowledged. Authors thank Prof. L. E. Xodo,
Department of Biomedical Sciences and Technologies,
University of Udine, Italy for useful discussions and
preprint of his work. TR thanks UGC and CSIR for
research fellowships.
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Supplementary material
The following supplementary material is available
online:
Fig. S1. Bar diagram illustrating the normalized fre-
quency of different backbone (A–F) and side chain

(G–I) torsion angles of the PNA strand of PD
wt
(red)
and PD
mut
(black).
Fig. S2. Partial charges for the different atoms of
PNA chain.
Fig. S3. Partial charges for the bases in: (A–D) PNA
and as given in Parm99.dat forcefield for a DNA chain
(E–H).
Effect of A .C mismatch in PNAÆDNA and DNA duplexes T. Rathinavelan and N. Yathindra
4070 FEBS Journal 272 (2005) 4055–4070 ª 2005 FEBS