Báo cáo khoa học: Solution structure of 2¢,5¢ d(G4C4) Relevance to topological restrictions and nature’s choice of phosphodiester links docx

Solution structure of 2¢,5¢ d(G
4
C
4
)
Relevance to topological restrictions and nature’s choice of phosphodiester links
Bernard J. Premraj
1
, Swaminathan Raja
1
, Neel S. Bhavesh
2
, Ke Shi
3
, Ramakrishna V. Hosur
2
,
Muttaiya Sundaralingam
3
and Narayanarao Yathindra
1
1
Department of Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai, India;
2
Department of Chemical
Sciences, TIFR, Colaba, Mumbai, India;
3
Department of Chemistry, The Ohio State University, Columbus, OH, USA
The N MR structure o f 2¢,5¢ d(GGGGCCCC) was deter-
mined to gain insights into the structural differences between
2¢,5¢-and3¢,5¢-linked DNA duplexes that may be relevant

in elucidating nature’s choice of sugar-phosphate links to
encode genetic information. The oligomer assumes a duplex
with extended nucl eotide repeats formed out of mostly
N-type sugar puckers. With the e xception of the 5 ¢-terminal
guanine that assumes the syn glycosyl conformation, all
other bases prefer the anti glycosyl conformation. Base pairs
in the duplex exhibit slide ()1.96 A
˚
) and intermediate values
for X-displacement ()3.23 A
˚
), as in ADNA, while their
inclination to the helical axis is not prominent. Major and
minor grooves display features intermediate to A and
BDNA. The duplex structure of iso d(GGGGCCCC) may
therefore be best characterized as a hybrid of A and BDNA.
Importantly, the results confirm that even 3 ¢ deo xy 2¢,5¢
DNA supports duplex formation only i n the presence of
distinct slide (‡ )1.6 A
˚
) a nd X-displacement (‡ )2.5 A
˚
)for
base pairs, and hence does not favor an ideal BDNA
topology characterized by their near-zero values. Such
restrictions on base pair movements in 2¢,5¢ DNA, w hich are
clearly absent in 3¢,5¢ DNA, are expected to impose con-
straints on its ability for deformability of the kind ob served
in DNA during its co mpaction and interaction with proteins.
It is therefore c onceivable t hat selection pressure relating to

the optimization of t opological features might have been a
factor in the rejection of 2¢,5¢ links in preferenc e to 3¢,5¢ link s.
Keywords: structure of 2¢,5¢ DNA; evolution of 3¢,5¢ vs. 2¢,5¢
links in nucleic acids; AB hybrid structure ; restrained base
pair movements; topological restrictions in 2¢,5¢ DNA.
Nature’s selection of 3 ¢,5¢ linkages ( instead of 2¢,5¢ linkages)
in nucleic acids, to encode genetic information, is intriguing.
Thefactthat2¢,5 ¢ links are formed i n a bundance and serve
as a template in nonenzymatic reactions suggest that they
might have been the ancestors of the biotic 3¢,5¢ links, which
could h ave e volved from a pool of 3¢,5¢ and 2 ¢,5¢ links [1].
Nucleic a cids with 2¢,5¢ links satisfy one of the critical
features required for the fidelity of replication, namely that
theyassociatetoformWatsonandCrickbase-paired
duplex structures [2–5], although with weaker affinity than
3¢,5¢-linked DNA strands. However, detailed knowledge
about stereochemistry, polymorphism and topological
properties of 2¢,5¢ DNA duplexes, which may provide
insights into the factors that determine nature’s choice of
sugar-phosphate links from a stereochemical perspective, is
sparse [6–9]. In fact, there are only two reports of NMR
structure determination – one on a 2 ¢,5¢ DNA fragment [10]
and one on a 2¢,5¢ RNA fragment [ 11] – both of w hich
suggest an A-type duplex structure with s ome stereochem-
ical details that differ from genomic DNA and RNA
duplexes. In this context, it is relevant to recognize the
results from recent modeling studies on 2¢,5¢ nucleic ac ids,
which suggest that 2¢,5¢ DNA cannot form a 10-fold
BDNA-like duplex (like 3 ¢,5¢ DNA) without the mandatory
slide (‡ )1.6 A

˚
) and X-displacement (‡ )2.5 A
˚
)[9].Witha
view to probe further i nto the structural properties o f 2¢,5¢
DNA, we report here a high-resolution NMR study of the
2¢,5¢ DNA fragment that pos sesses a g uanine tract f ollowed
by a cytosine t ract, to d iscern also possible sequence e ffects.
The results show that iso d(GGGGCCCC) [d(G
4
C
4
)]
assumes a duplex that conforms to neither a canonical
BDNA nor an A DNA family, but a duplex characterized
by featu res of both A and BDNA. Possible implications
of this on the topological restrictions of 2¢,5¢ DNA, and
its rejection by nature, are discussed.
Materials and methods
DNA synthesis and NMR sample preparation
The 2¢,5¢-linked 3 ¢ deoxy (GGGGCCCC) (iso DNA), was
synthesized at 1 lmol scale on an in-house Applied
Biosystem 391 automatic DNA synthesizer using solid-state
phosphoramidite chemistry [12]. The universal support
(purchased from BioGene) was used as the solid support for
the synthesis. The standard concentration of phospho-
ramidite was d iluted with a n equal volume of acetonitrile.
The products were cleaved off the column w ith 5 mL of
37% ammonium hydroxide containing 5% LiCl. The
Correspondence to N. Yathindra, Department of Crystallography and

Biophysics, U niversity of M adras, Guindy C ampus, Chennai-600 025,
India. Fax: + 9 1 4 4 2230 0122,
2
Tel.: + 91 44 2235 1367,
E-mail:
Abbreviations:d(G
4
C
4
), d(GGGGCCCC); LALS, linked atom least
squares; RDC, residual dipolar couplings.
(Received 4 March 2004, revised 30 A pril 2004, ac cepted 21 May 2 004)
Eur. J. Biochem. 271, 2956–2966 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04225.x
solution was incubated in a 55 °C water bath for 16 h and
then lyophilized. The pellet from lyophilization was dis-
solved in 5% NaHCO
3
and purified by FPLC. The collected
peak elution was lyophilized and the sample stored at
)20 °C. NMR samples (0.6 m
M
)ofthe2¢,5¢ DNA fragment
were prepared in 20 m
M
potassium phosphate buffer
containing 0.5 m
M
EDTA and 100 m
M
KCl. For experi-

ments in D
2
O, the s amples were lyophilized and redissolved
in D
2
O. UV melting studies show that the T
m
of iso d(G
4
C
4
)
is  32 °C under identical buffer conditions.
NMR data acquisition
NMR experiments were carried out on a 600 MHz Varian
Unity-plus spectrometer. 1D spectra in H
2
O were r ecorded
using the jump-and-return pulse sequence for H
2
O sup-
pression at different t emperatures in t he range o f 2–45 °C
[13]. 2D NOESY spectra in H
2
O were recorded at 2 °Cwith
mixing times of 80 ms and 300 ms. Phase-sensitive N OESY
spectra in D
2
O [14] w ere recorded with mixing times of 7 0,
120, 150, 200, 250 and 300 ms; and TOCSY spectra [15]

were recorded with mixing times of 30 and 90 ms at 2 °C.
The DQF-COSY spectrum [16,17] and 2D J-resolved
spectra [18] were recorded in D
2
Ofor
1
H–
1
H coupling
constant estimation. For the various experiments, the time
domain data c onsisted of 2048 c omplex points i n t2 and
300–400 complex points in t 1 dimension. The relaxation
time delay was between 1 and 3 s for the different 2D
experiments.
Experimental restraints
Data processing and analysis were carried out using
VNMR
and
FELIX
packages [ 19] on a Silicon graphics work station.
Based on the relative intensities and build-up, the cross
peaks in the NOESY s pectra (obtained in D
2
O at various
mixing times), are classified as strong, medium-strong,
medium, and weak, and the interproton distances are
restrained, respectively, to the ranges 2–3 A
˚
, 2.5–3.5 A
˚

,
3–4.5 A
˚
, and 3.5–5.5 A
˚
. The narrow bounds are mostly
used for strong intranucleotide cross peaks, for which
distance ranges are small and known. As the distance ranges
for the observable NOEs are not so large, the NOE distance
bounds used are c onsidered to be realistic. The interproton
distances involving the exchangeable protons in the H
2
O
NOESY spectra are restrained to the ranges 2–4 A
˚
and 3.5–
5.5 A
˚
, corresponding to the s trong and w eak cross peaks,
respectively. At this level o f NOE inte nsity quantification,
spin diffusion is not expected to influence the distance
restraints to a significant extent. Even so, the larger the
number of distance restraints, the better it is for internal
consistency, and the structures derived would be more
reliable. A total of 162 NOE restraints were collected, of
which 115 were intranucleotide and 47 internucleotide
NOEs.
Base (H8/H6) s ugar proton NOEs, especially to the H1¢,
also enable deriving constraints on the glycosyl torsion
angles. The H8/H6–H1¢ distance is very short ( 2.3–2.5 A

˚
)
for a syn conformation and r elatively much l onger ( 3.5–
4.0 A
˚
)forananti conformation. Thus, the H8/H6–H1¢
NOE will be very strong, even at short mixing times (such as
60–70 ms) if the glycosyl torsion angle is in the syn domain,
whereas, u nder the sa me conditions, the peak will be nearly
absent for an anti conformation. We observe that G1 has a
syn conformation, while all others are in the anti domain
(spectra presented in Results).
The 2D J-resolved spectra provides precise values of the
J(H1¢–H2¢) coupling constants (Table 1). The observed
coupling constants are very small, indicating that the
sugar geometry b elongs largely to the N domain (in the N
domain this coupling constant is near 0–2 Hz, whereas it
varies between 9 and 10 Hz in the S domain). A common
practice is to consider the sugar geometry as an equilib-
rium mixture of N and S types, and the coupling
constants as weighted averages. However, there are also
reports in the literature [18] where the sugar ring is
believed t o be rigid, and is primarily of a single type, at
least in the interior of the duplex. In the present case, we
observe that the terminal residues, for example, C8 and
G2, where one would have expected greater dynamism,
exhibit very small values ( 1.5Hz) for J(H1¢–H2¢). If one
considers an equilibrium model, for a 10% contribution of
the S domain, the contribution to t he coupling c onstant
would be around 1 Hz. Moreover, it is c lear from the

steepness of the curve displaying the dependence of
coupling constants on pseudorotation phase angle P
(Fig. 1 ), that the P range i n the N domain is not going
to be very different regardless of whether the S contribu-
tion is explicitly considered. Thus, from t he small values
of the coupling constants for the terminal residues, it is
evident t hat the sugar geometries are dominantly in the N
domain only. This will be also true for the internal
residues. Now, in the N domain, especially in the P range
30–80°, the dependence of H1¢–H2¢ coupling on P is very
steep and this significantly narrows the range of permis-
sible P-values for a given coupling constant value [18,20].
Taking these factors into consideration, sugar puckers
were restrained to the P ranges indicated in Table 1 a nd
these were then converted to respective dihedral angle
ranges in the sugar rings.
The hydrogen bond restraints were given as two distances
per hydrogen bond (a total of 36 restraints) for the central
hexamer (see below). Based on the observation that the
peak count for t he duplex is the same as expected from a
single strand in the various spectral data (indicating that the
duplex is highly symmetric and the two strands are
Table 1. J(H1¢–H2¢) coupling and the c orresponding ranges of phase ang le of pse udorotation (P°).
G1 G2 G3 G4 C5 C6 C7 C8
J(H1¢–H2¢) Hz 2.9 1.7 3.5 2.9 3.8 1.8 5.1 1.5
Range of phase angle
of pseudorotation (P°)
49–58 35–47 55–64 49–58 57–66 37–48 68–77 33–45
Ó FEBS 2004 2¢,5¢ DNA with hybrid features of A and BDNA
1

(Eur. J. Biochem. 271) 2957
equivalent), NCS restraints were imposed to ob tain sym-
metry between the two strands forming the duplex.
Structure calculation
Structure calculation of the iso d(GGGGCCCC) was
carried out using
X
-
PLOR
3.8.5 [21]. The topology and
parameter files w ere appropriately modified t o handle 2¢,5¢
linkages to obtain optimum geometry at the 2 ¢,5¢ phospho-
diester linkage. Ideal A- and B-type duplex models for iso
DNA (possessing helical parameters identical to those of the
canonical ADNA and B DNA), obtained p reviously [9]
using the linked atom least squares (LALS) refinement
approach [22], were used as the starting models for structure
calculation. This is justified considering that the NMR
spectra in water clearly estab lish Watson and Crick b ase
pair formation between antiparallel strands. The model iso
ADNA duplex is characterized by the s ame value of slide,
X-displacement and the helical parameters, as ADNA. On
the other hand, the iso BDNA model, while possessing the
same h elical parameters as BDNA, is distinguished by a
nonzero slide ( ‡ )1.7 A
˚
) and X-displacemen t (‡ )2.5 A
˚
), in
sharp c ontrast to the ideal BDNA that is characterized b y

zero values for them. Nonzero slide and X-displacement are
found to b e mandatory to generate a 10-fold 2¢,5¢ duplex,
even with 3¢ deoxy sugars [9]. Thus, the iso BDNA and iso
ADNA models are very different from each other, and
choosing these two as initial models removes any starting
model bias i n the results of c alculation. Such a strategy a lso
saves computational efforts compared to starting the
calculation from a completely extend ed structure. In the
latter case, m uch effort i s expended for the formation of
the base pair itself .
Syn conformation was imposed for the 5¢ end guanine
(see below). The initial model was subjected to restrained
energy minimization using the conjugate gradient algorithm
and was guided by the experimental NOE distance restraints
as well as dihedral restraints. A conform ational search w as
performed on the octamer duplex using the Ôsimulated
annealingÕ protocol [23], f ollowed by s tructure refinement
using the Ôgentle refineÕ protocol of
X
-
PLOR
3.8.5. A distance-
dependent dielectric constant was used throughout the
structure calculation to mimic the presence of high dielectric
solvent, typically for simulating water (when explicit water is
not used). The starting s tructure was heated t o 1000 K , and
sets of 100 structures tha t are s ignifican tly different from
one another were extracted during high-temperature
dynamics. Each of the structures was subjected to 18 ps of
high-temperature dynamics followed by s low cooling to

100 K , at steps of 50 K. During each cooling step the
structures were subjected to 500 fs of molecular dynamics.
Finally, the structures were energy minimized using the
conjugate gradient algorithm. This was followed by a
refinement using the Ôgentle refineÕ protocol, where each of
the structures was subjected to 20 ps of m olecular dynamics
at 300 K. Average coordinates over the last 10 ps of
molecular dynamics simulation were computed and then
refined by conjugate gradient minimization. The NOE
distance restraints, hydrogen bond restraints (given as two
distances per hydrogen bond), and dihedral restraints on the
sugar conformation were applied throughout the entire
calculation with force constants of 50 kcalÆmol
)1
ÆA
˚
)2
,
100 k calÆmol
)1
ÆA
˚
)2
and 300 kcalÆmol
)1
ÆA
˚
)2
, respectively.
NCS restraints w ith a force constant of 300 kcalÆmol

)1
ÆA
˚
)2
were imposed t o obtain symmetry between the t wo strand s
of the duplex.
Results
1D and 2D proton spectra
The 1D
1
H NMR spectrum (Fig. 2A) of the octamer iso
d(GGGGCCCC) displays three peaks corresponding to
the imino protons at 13.25 (G4), 12.80 (G2) and 12.70
(G3) p.p.m., expected from Watson and C rick base pairs i n
an antiparallel duplex. Sequence-specific assignments for the
exchangeable and nonexchangeable protons were made
from the NOESY and TOCSY spectra following the
procedures developed f or 3¢,5¢ duplexes [24]. T he observa-
tion of NOE changes from G imino to C amino protons of
nonterminal base pairs in the NOESY water spectra
(Fig. 2 B) further substantiates the formation of Watson
and Crick base pairing between G and C. The uninterrupted
self and sequential connectivity from H8/H6 to H1¢
(Fig.3A),aswellasH8/H6toH2¢ (Fig. 3B) in the N OESY
spectra suggest a right-handed helical structure. These
sequential connectivities are consistent throughout the
various regions of the spectra. From the temperature
dependence of the G imino resonances in 1D spectra in
H
2

O (data not shown), the melting temperature of the
duplex was seen to be  30 °C.
Tables 2 a nd 3
3
show the chemical shift values for a ll the
assigned sugar and base protons. The stereospecific assign-
ments involving the 3¢ and 3¢¢ protons were based on the
2¢)3¢ and 2 ¢)3¢¢ NOE intensities in the 70 m s NOESY
spectrum. As the H2¢–H3¢ proton separation is shorter t han
the H2¢–H3¢¢ separation, irrespective o f the sugar confor-
mation, the H2¢–H3¢ NOE intensity should be stronger at
shorter mixing times. The relative intensities of the cross-
peaks of the interproton base to sugar NOEs in the NOESY
spectrum (Fig. 3C), at mixing times varying from 70 to
300 m s, indicate that the 5¢-terminal guanine exists in the
Fig. 1. Plots showing the dependence of the 3 -bond coupling constants
(J) on the phase a ngle of ps eudorotation (P).
2958 B. J. Premraj et al. (Eur. J. Biochem. 271) Ó FEBS 2004
syn conformation, while other bases favor t he anti confor-
mation. This is a recurring feature found in 2¢,5¢-linked
dimers [25–27] and oligomers [10,11]. In the crystal struc-
tures o f 2 ¢,5¢-linked dinucleoside monophosphates, the syn
conformation is stabilized by an intramolecular hydrogen
Fig. 2. NMR spectra and the NOESY spectrum. (A) 1D H
2
O
exchangeable NMR spectra of iso d(GGGGCC CC) in 100 m
M
KCl,
pH 7.0, and at 2 °C, showing the imino and amino proton signals. (B)

Selected region of the NOESY spectrum (mixing time 300 m s) in H
2
O
solution showing N OE correlation sfromGiminotoCaminoprotons.
CNH
2(i)
and CNH
2(e)
refer to the internal (H-bon ded) and external
(free) amino p roton s of the cytosine base.
Fig. 3. (H8/H6)–H1¢ cross-peak region of a 300 ms 2D NOESY
spectrum o f iso d(GGGGCCCC) in D
2
Osolutionat2°C, showing the
uninterrupted sequential connectivities from (A) (H8/H6) to H1¢ protons
(B) (H8/H6) to H2¢ proto ns (C). Stacked plot of the H8/H6 to H1¢
region showing a high intensity for the H8–H1¢ cross-peak of G1,
suggesting syn glycosyl conformation for the terminal G1 residue .
Ó FEBS 2004 2¢,5¢ DNA with hybrid features of A and BDNA
1
(Eur. J. Biochem. 271) 2959
bond between the purine N3 and O5¢H of the sugar residue,
besides sugar O4¢–base (syn) base interaction [9,25–27].
The (H1¢–H2¢) coupling constants derived from 2D
J-resolved spectra clearly indicate that all of the 3¢ deoxy
sugars belong to the N type, except for C7 which has a
slightly higher coupling constant (5.1 Hz).
Structural features of 2¢,5¢ d(GGGGCCCC)
The 3D structure of 2¢,5¢ d(GGGGCCCC) was obtained by
simulated annealing molecular dynam ics using

X
-
PLOR
3.8.5
[21]. Experimental restraints and structure convergence
parameters are listed in Table 4. The convergent structures
are clustered into families: BFI (Fig. 4A) with 39 structures,
and BFII (Fig. 4B) with 20 structures when the starting
model was ideally iso BDNA; and AFI (Fig. 4C) with 85
structures and AFII (Fig. 4D) with 15 s tructures when the
starting model was iso ADNA. Structures represented
by BFI and AFI families (FI) differ considerably in their
overall topologies from the structures represented by BFII
and AFII families (FII). The root mean square devi-
ation (rmsd) b etween FI and FII is greater t han 3 A
˚
, while
it is less than 1 A
˚
for structures within FI or FII. The
structures were selected using standard criteria on the basis
of proper c ovalent geometry, the least number of distance
and dihedral violations, symmetry and low energy.
The duplex model AFI (Fig. 5A), c losely resembles BFI
(Fig. 5 B). T he rmsd between the average structure of AFI
(Fig. 5 A) and BFI (Fig. 5 B) is 0.8 A
˚
. Thus, in spite of the
large rmsd (> 4 A
˚

) in the starting structures, the final
structures fall into similar families, indicating that the
structures are not biased by the c hoice of the initial m odel.
This also indicates that the experimental restraints are
sufficient and consistent to define good convergent struc-
tures. In view of this, it is b elieved th at there is no need for
any further refinements using residual dipolar couplings
(RDCs), as often performed in longer DNA stretches
[28–30]. Likewise, we also did not f eel the need f or any
relaxation matrix refinement, w hich takes into account spin
diffusion e xplicitly, which may be r equired if t he NOE data
set is very small. At the same time, relaxation matrix
refinement puts a greater demand on the accuracy of NOE
quantification.
In the final structures, the terminal GC pairs are not well
defined owing to insufficient NOEs. Hence, structural
features manifested in the cen tral hexame r of iso d(G
4
C
4
),
corresponding to the GGGCCC du plex in the f amily AFI,
which has the highest population of converged structures
and also has very good convergence, are considered for
detailed discussion.
Calculated values of X-displacement and slide for the
base pairs in AFI are given in Table 5 . Average values of
X-displacement and slide of GC base p airs at the GG step
(Fig. 5 A) are )3.25 A
˚

and )1.62 A
˚
(Table 5), r espectively.
On the other hand, slide for the GC pair at the GC step that
links the G stretch with the C stretch is rather high
()3.3 2 A
˚
).
The nature of the base stacking interaction in the iso
d(GGGGCCCC) duplex, as seen in AFI, is shown in
Fig. 6A. Stacking at the G
2
G
3
and G
3
G
4
steps involves
overlap of t he six-membered ring of one gu anine with the
imidazole r ing o f the adjacent guanine, while there i s only
Table 3 . Chemical s hifts (p .p.m ) f or iso d(GGGGCCCC)
2
exchange-
able proto ns.
Base H
1
H
22
/H

42
(e) H
21
/H
41
(i)
G1 – – –
G2 12.76 – –
G3 12.71 – –
G4 13.25 – –
C5 – 6.94 8.7
C6 – 6.93 8.58
C7 – 7.12 8.51
C8 – – –
Table 2. Chemical shifts (p.p.m) for iso d(GGGGCCCC)
2
non-
exchangeable protons.
Residue H6/H8 H1¢ H2¢ H3¢ H3¢¢ H4¢ H5¢/H5¢¢ H5
G1 7.97 5.98 5.18 2.53 2.37 4.58 3.88,3.69 –
G2 7.78 5.83 4.7 2.45 2.31 4.61 4.18,4.08 –
G3 7.62 5.96 4.91 2.56 2.48 4.78 4.51,4.14 –
G4 7.58 5.99 4.61 2.48 2.38 4.77 – –
C5 7.5 6.08 4.62 2.44 – 4.76 4.39,4.07 5.10
C6 7.88 5.94 4.5 2.31 – 4.71 4.10 5.48
C7 7.73 6.03 4.66 2.49 2.31 4.04 4.27 5.52
C8 7.84 5.66 4.26 1.84 1.82 4.56 4.39,4.0 5.61
Table 4 . NMR restraints for iso d(GGGGCCCC)
2
.

NOE distance restraints (per strand)
Non-exchangeable NOE restraints 140
Exchangeable NOE restraints 22
Total restraints 162
Intra-residue 115
Inter-residue 47
Sugar dihedral restraints (per strand) 40
Hydrogen bond restraints 36
BFI (model obtained when iso BDNA is used as the starting duplex)
Number of convergent structures 39
rmsd from the average structure 0.5 A
˚
)1.0 A
˚
NOE violation > 0.2 A
˚
1
Dihedral angle violation > 5° Nil
BFII (modelobtained when iso BDNA is used as the starting duplex)
Number of convergent structures 20
rmsd from the average structure 0.3 A
˚
)1.0 A
˚
NOE violation > 0.2 A
˚
1
Dihedral angle violation > 5° Nil
AFI (model obtained when ADNA is used as the starting duplex)
Number of convergent structures 85

rmsd from the average structure 0.1 A
˚
)0.6 A
˚
NOE violation > 0.2 A
˚
1
Dihedral angle violation > 5° Nil
AFII (model obtained when ADNA is used as the starting duplex)
Number of convergent structures 15
rmsd from the average structure 0.1 A
˚
)0.5 A
˚
NOE violation > 0.2 A
˚
1
Dihedral angle violation > 5° Nil
2960 B. J. Premraj et al. (Eur. J. Biochem. 271) Ó FEBS 2004
minimal stacking between cytosines. Likewise, stacking at
the GC s tep, which links the G stretch w ith the C stretch, i s
minimal o wing to a larger s lide ()3.32 A
˚
). Superposition of
thebasepairsofthe(GGGCCC)
2
fragment of the iso
d(G
4
C

4
) duplex with the ideal iso BDNA (Fig. 7), demon-
strates a strong resemblance in the stacking patterns.
An estimate of the dimensions of major and minor
grooves is obtained by generating a 12mer duplex using t he
central hexamer of the average structure (AFI) as the repeat
using the program
FREEHELIX
[31]. The groove topologies of
AFI show significantly different features f rom the ideal
duplex models. T he major groove is wide (17 A
˚
), while its
minor groove is narrow (10.3 A
˚
).
The 3¢ deoxy sugars i n iso d(G
4
C
4
) favor N-type pucker,
corresponding to the C4¢ exo conformational domain ( P ¼
38–64°), except in the residue C7, which favors C4 ¢ exo/O4¢
endo pucker, corresponding to P ¼ 54–90° (Table 1) in
AFI. In any case, none of the sugars shows a tendency for
S-type sugar conformation.
Base pairs in A FI are slightly overwound, an d the duplex
has 9 bp per t urn, with an average helical twist o f 38.4° and
a rise of 3.76 A
˚

(Table 5). The average helical twist at the
GG and CC steps is  41 °, w hile it is 28° at the GC step.
Slight underwinding at this step is accompanied by a higher
slide of )3.32 A
˚
. Base p airs are nearly perpendicular t o the
helix axis (inclination angle 3 °).Thetwocentralbasepairs
of the duplex are p ractically planar and they do not exhibit
significant propeller twist (Table 6), while the base pairs
flanking them possess a larger value of )22°. Phosphodi-
ester conformations at all the GG steps, as well as at the GC
step, conform to t he (g
–
,g
–
) domain, while they correspond
to the (t,g
–
) at the CC steps (Table 7 ).
Discussion
It is now well established that nucleic acids, even with 2 ¢,5¢
linkages, associate to form Watson and Crick paired
duplexes [2–5,10,11,32–37]. They also selectively associate
with DNA and RNA with a v arying degree of stability.
Interestingly, it has b een shown recently that 2¢,5¢ RNA
fragments form even hairpins with a stability comparable to
RNA hairpins [38]. In an effort to obtain a comprehensive
understanding of the stereochemistry that govern the
structures of 2 ¢,5¢ nucleic acids, we recently reported the
Fig. 5. Stereo plot of the average structure of is o d(G

4
C
4
). (A) AFI an d
(B) BFI.
Fig. 4. Stereo plot of th e families of conv erged structures of is o
d(GGGGCCCC)
2
. (A) B FI (39 structures), (B) BFII (20 structures),
(C) AFI (85 structures), a nd (D) AFII ( 15 structures).
Table 5. Base-step parameters i n the average structure (AFI) of the iso
d(GGGGCCCC) duplex.
Base step Slide (A
˚
) X-disp (A
˚
) Twist (°) Rise (A
˚
)
G2-G3 )1.53 )3.36 42.3 3.68
G3-G4 )1.71 )3.13 39.67 3.61
G4-C5 )3.32 )3.19 28.02 4.23
C5-C6 )1.71 )3.13 39.63 3.61
C6-C7 )1.53 )3.23 42.37 3.68
Average )1.96 )3.20 38.39 3.76
Ó FEBS 2004 2¢,5¢ DNA with hybrid features of A and BDNA
1
(Eur. J. Biochem. 271) 2961
NMR structure of a 2¢,5¢ RNAfragment[11]thatexhibited
interesting features which supported our predictions from

modeling studies [8,9]. We re port here the results o f high-
resolution NMR structure of a 2¢,5¢-linked D NA fragment
d(GGGGCCCC).
The structural model, AFI, that emerged from N OE
and other NMR data, exhibit slide ()1.96 A
˚
)and
intermediate X-displacement ()3.32 A
˚
)forthebasepairs,
a feature normally seen only in ADNA duplexes.
However, the magnitude of X-displacement observed here
is lower () 4.7 A
˚
) than that found in ADNA. Interest-
ingly, the slide ()3.32 A
˚
) at the lone GC step, linking the
G stretch with the C stretch, is found to be nearly twice
that found at the GG steps ()1.62 A
˚
), indicating possible
sequence effects. A comparison of the stacking pattern
observed at the GG steps of the present structure with
those in ideal ADNA, iso ADNA and iso BDNA
duplexes (Fig. 6B) brings out a strong similarity. It is
interesting t hat the similarity in stacking p ersists, notwith-
standing different values f or X displacement that c harac-
terizes these duplexes (Table 5). However, it should be
noted that all of them possess nearly the same slide

()1.7 A
˚
). Thus, the base stacking pattern in iso d(G
4
C
4
)is
like that in ADNA, except at the GC step where a large
slide causes adjacent b ases to move aw ay, resu lting in
minimal overlap between them.
Another unusual feature is the predominance of N-type
pucker i n nearly all the 3 ¢ deoxy s ugars in 2¢,5¢ d(G
4
C
4
).
This is in sharp contrast to the S -type puckers preferred
by 2¢ deoxy sugars in DNA duplexes. This has been
Fig. 6. Base stacking at different steps in the AFI dup lex of is o (G
4
C
4
) and the GG steps of is o BDNA: iso ADNA and ADN A . Note the identic al base
stacking at the GG s teps of AFI a nd ideal duplex es. Figures were d rawn using 3
DNA
v1.5 [ 47].
2962 B. J. Premraj et al. (Eur. J. Biochem. 271) Ó FEBS 2004
anticipated in view of certain stereochemical arguments
[8,9]. Exclusive preference for the N-type sugar puckers
has, in fact, been indicated by the early NMR studies on

2¢,5¢-AAA [39] and crystal structures of 3¢ deoxynucleo-
sides [25–27]. Such preference for N-type pucker has also
been confirmed by recent
1
H N MR analysis on a number
of 3¢ d eoxynucleosides and stereo-electronic arguments
[40,41]. Unconstrained molecular dynamics simulations of
a2¢,5¢ DNA duplex, l asting a few nanoseconds, have also
demonstrated the retention of N-type pucker for the sugar
[42]. It should be recognized at this juncture that the
consequence of N-type sugar pucker is to render the
preferred nucleotide conformation to b e extended in 2¢,5¢
DNA a nd compact in 3 ¢,5¢ DNA [8,9]. It is well known
that the extended nucleotide repeats lead to an extended
BDNA, and the compact nucleotide repeat leads to a
compact ADNA type of duplexes (Fig. 8). The 2 ¢,5¢ DNA
fragment d (G
4
C
4
) is thus composed of extended nucleo-
tide repeats that are normally part of BDNA but with a
distinct X-displacement, slide and base stacking like in
ADNA. Thus, the duplex model AFI of 2¢,5¢ d(G
4
C
4
),
possesses composite features of both A and BDNA. In
view of these, it is perhaps appropriate to regard the

structure of iso d(G
4
C
4
) as a hybrid structure of A and B
forms.
It is grati fying that the more populated AFI family of iso
d(G
4
C
4
) resembles the ideal iso BDNA-like duplex [9],
which is also characterized by similar values of slide,
intermediate displacement, base stacking p attern and
extended nucleotide repeat formed out of N-type sugar
puckers (Table 5). Furthermore, the overall groove topol-
ogies of iso d(G
4
C
4
) resemble BDNA, with the widths of the
major groove and the minor groove having values of 17 A
˚
and 10.3 A
˚
, respectively (Table 8 ).
It has been demonstrated from modeling investigations
that 2¢,5¢ isomers, even with 3¢ deoxyriboses, cannot form
duplexes without base pair displacements [9]. Results of CD
and FTIR investigations on iso DNA fragments comprising

a variety of base sequences also seem to converge to suggest
that they favo r A-type r ather than B -type duplexes (S. Raja
& N. Y athindra, unpublished observation). F urthermore, it
has been found [43] that iso d(CGCGCG) does not associate
to form left-handed ZDNA. T his has been attributed to the
inaccessibility [42] to form t he well-known water-mediated
hydrogen bond stabilization i nteraction between the amino
group of the syn guanine and the anion oxygen of the
phosphate group [44]. These clearly point to the constraint
on the r ange of duplex helical structures possible for nucleic
acids with 2¢,5¢ linkages.
The lateral slide of the sugar-phosphate chain from the
periphery (as in 3¢,5¢ links) towards the helix interior in
Fig. 7. Superposition of the G
3
C
3
fragment of
AFI with ideal iso BDNA. Root mean square
deviation with respect to base pairs is 0.6 A
˚
.
Table 6. Propeller twist (°) of base pairs in th e average structure (AFI)
of the iso d(GGGGCCCC) duplex.
Base pair Propeller twist (°)
G2–C15 )22.4
G3–C14 )19.6
G4–C13 1.0
C5–G12 1.0
C6–G11 )19.4

C7–G10 )22.4
Average )14.3
Table 7. Conformation angles (°) in the av erage structure (AFI) of th e iso d(GGGGCCCC)
2
duplex.
Residue
a
(P-O5¢)
b
(O5¢-C5)
c
(C4¢-C5¢)
n
(C2¢-O2¢)
f
(P-O2¢)
v
(C1¢-N) P
G1 – – 29 )115 )86 86.2 49
G2 )50 167 46 )78 )122 )137 37.6
G3 )31 138 36 )69 )131 )137 56.4
G4 )45 143 32 )87 )89 )142 49.5
C5 )68 )177 27 )76 )161 )138 62.7
C6 )25 128 35 )60 )133 )151 37.4
C7 )28 131 34 )90 )144 )140 70.7
C8 )44 158 39 – – )151 34.1
Ó FEBS 2004 2¢,5¢ DNA with hybrid features of A and BDNA
1
(Eur. J. Biochem. 271) 2963
2¢,5¢ nucleic acids causes t he base pairs t o s lide, resulting i n

the intrinsic requirement of slide, and hence X-displace-
ment, that manifest in all 2¢,5¢ nucleic acid duplexes. This
limits the access to a lower range of values of both s lide
(< )1.5 A
˚
) and X-displacement (< )2.5 A
˚
)in2¢,5 ¢ nucleic
acids. In contrast, nucleic acids with 3¢,5¢ links have a w ider
range of access for both slide (0–2.5 A
˚
) and X-displacement
(0–4.7 A
˚
) that includes ranges forbidden for the 2¢,5¢ isome r.
This enables 3¢,5¢-linked nucleic acids to assume a variety of
duplexes with distinct topological features and also afford
other capabilities, such as b ending, kinking and curvature,
which form the basis for nucleic acid compaction and
specificity of interaction with proteins. It is therefore
anticipated that the restricting factors in 2¢,5¢ n ucleic ac ids,
Fig. 8. Shape and dimension (adjacent P–P separations) of the repeating nucleotide units in 2¢,5¢- and 3¢,5¢-linked nucleic acids. An equatorial (e) link
renders the adjacent phosphates to be proximal, leading to a compact nucleotide (P–P  5.9 A
˚
), while an axial (a) link renders them to be distal,
leading to an e xtended nucleotide (P–P  7.0 A
˚
).
Table 8. Comparison of structural features of the iso d(GGGGCCCC)
2

duplex (AFI) with the ideal A and B types of duplexes formed by 3¢,5¢ and 2¢,5¢
links.
Features/parameters BDNA ADNA iso BDNA iso ADNA AFI
X-disp (A
˚
) )0.1 )4.7 )2.5 )4.7 )3.2
Slide (A
˚
) 0.4 )1.6 )1.7 )1.67 )1.96
Twist (°) 36 32.7 36 32.7 38.4
Rise (A
˚
) 3.4 2.56 3.4 2.56 3.76
No: res./turn 10 11 10 11  9.4
Inclination (°) 3.4 20.0 0 19.3 3
P–P separation (A
˚
) 7 5.9 7.5 5.9 7.4
Sugar pucker S type N type N type S type N type
C2¢endo C3¢endo C3¢endo C2¢endo C4¢exo
Major groove (A
˚
) 17 8.2 19.8 10.7 17
Minor groove (A
˚
) 11.7 16.9 10.5 14.8 10.3
2964 B. J. Premraj et al. (Eur. J. Biochem. 271) Ó FEBS 2004
which are mentioned above, probably impose additional
constraints limiting these capabilities. Also, it has been
shown f rom modeling c onsideration t hat the lateral slide

of the sugar-phosphate chain leads to overwinding of the
2¢,5¢-linked single-stranded helix to enhance the adjacent
base–base or s ugar–base stabilizing i nteractions [9,42,45].
Tighter winding of the 2¢,5¢ single-stranded DNA helix,
compared with 3¢,5¢ DNA, probably offers restrictions to
the folding abilities of even single-stranded 2 ¢,5¢ DNA.
Hence, it may be argued that topological restrictions
inherent to the 2¢,5¢-linked helical duplexes might have a lso
contributed towards their rejection. It is worth mention ing
that the inherent low thermal stability of 2¢,5¢ links might
have been another factor involved in nature’s selection of
the 3¢,5¢ links. T hus, optimization o f the topology o f duplex
helix, besides the optimization o f b ase p air s tability [46],
must have been important in the chemical etiology of
nucleic acid structures.
Conclusions
Systematic investigations of 2¢,5¢ nucleic acids h ave provided
new p erspectives on the stereochemical d etails pertaining to
their ability, or lack of it, to form dup lex structures akin to
their naturally occurring 3 ¢,5¢ isomers. In parallel t o our
finding [8,9] of the critical features that distinguish the
shapes and dimensions of the r epeating nucleotides of 3¢,5¢
and 2 ¢,5¢ isomers, we have provided structural details of an
iso RNA [11] and an iso DNA duplex fragment ( present
work) from NMR studies. T ogether, these should p rovide a
structural basis for understanding much of the experimental
data from solution stu dies concerning the associations of
2¢,5¢ nucleic acids a nd also with DNA and R NA. C ompar-
ison of the structure deduced for iso d(GGGGCCCC), from
the current study, and that of iso d(CGGCGCCG) [10]

suggestthateven2¢,5¢ DNAs are pr one t o s equence e ffects,
as evidenced by some differences seen in structures of the
two sequences. The former sequence a ssumes a hybrid
structure of A and BDNA duplexes, while the latter assumes
an ADNA-like duplex with mixed C 2¢ endo and C3¢ endo
sugar puckers for t he central hexamer. The fact that both
these sequences, s tudied by NMR, point to a non-BDNA
duplex structure, suggest a constrained nature o f base p air
movements i n 2 ¢,5¢ nucleic acids vis-a
`
-vis their 3¢,5¢ isomers.
This is in complete conformity with the modeling studies
[8,9] which indicate that slide and X-displaceme nt of base
pairs lower than )1.7 A
˚
and )2.5 A
˚
, respectively, are
inaccessible owing to the inherent chemistry o f the 2 ¢,5¢-
linked sugar-phosphate backbone. It seems, then, that a
need for greater topological flexibility of DNA helices might
have had a bearing on the selection of 3¢,5¢ links over 2¢,5¢
links during the course of evolution.
Acknowledgements
NMR a nd c omputational facilities, provided by the National F acility
for High R esolution NMR at the Tata Institute of Fundamental
Research, Mumbai, are gratefully a cknowledged. N.Y. a nd B.J.P.
thank DST and CSIR f or a research g rant and senior f ellowship,
respectively. S.R. thank s CSIR for a Senior Research Fellowship. UGC
and DST are thanked for the financial support to the Department

under DSA (UGC) and FIST (DST) programs.
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