Crystal structures of oligodeoxyribonucleotides containing 6'-α-methyl and 6'-α-hydroxy carbocyclic thymidines

Article abstract of DOI:10.1021/ja962406a

The X-ray crystal structures of two self-complementary DNA duplexes with the sequence d(CGCGAAt(Me/OH)t(Me/OH)CGCG) containing four 6'-α-methyl (t(Me)) or four 6'-α-hydroxy carbocyclic thymidines (t(OH)), respectively, have been determined. Both structure

Full text of DOI:10.1021/ja962406a

2396
J. Am. Chem. Soc. 1997, 119, 2396-2403
Crystal Structures of Oligodeoxyribonucleotides Containing
6′-R-Methyl and 6′-R-Hydroxy Carbocyclic Thymidines†
Stefan Portmann,‡,§ Karl-Heinz Altmann,| Nathalie Reynes,|, and Martin Egli*,‡
Contribution from the Department of Molecular Pharmacology and Biological Chemistry,
Northwestern UniVersity Medical School, Chicago, Illinois 60611-3008, Organic Chemistry
Laboratory, ETH Swiss Federal Institute of Technology, CH-8092 Zu¨rich, Switzerland, and
Central Research Laboratories, CIBA Ltd., CH-4002 Basel, Switzerland
ReceiVed July 12, 1996X
Abstract: The X-ray crystal structures of two self-complementary DNA duplexes with the sequence
d(CGCGAAtMe/OH Me/OHCGCG) containing four 6′-R-methyl (tMe) or four 6′-R-hydroxy carbocyclic thymidines (tOH),
t
respectively, have been determined. Both structures are isomorphous to the native Dickerson-Drew dodecamer
duplex [d(CGCGAATTCGCG)]2. The cyclopentane moieties of the modified carbocyclic thymidine residues lacking
the deoxyribose 4′-oxygen adopt either C2′-endo or C1′-exo B-DNA type puckers. The dodecamer duplex
incorporating 6′-R-methyl carbocyclic thymidines shows an enlarged minor groove relative to both the unmodified
duplex and the one with incorporated 6′-R-hydroxy carbocyclic thymidines. The pairing of oligonucleotides containing
single or multiple 6′-R-substituted carbocyclic thymidines with complementary DNA is discussed on the basis of the
structural data.
Introduction
structure that are associated with covalent modification of the
sugar-phosphate backbone of one of the component strands.3-6
Changes in the ssRNA- or DNA-binding affinity of modified
oligonucleotides do not necessarily have to reflect, or at least
not exclusively so, structural changes in the Watson-Crick
duplex but could also be related to conformational changes in
the single strand.7,8 However, it is very likely that any changes
in the duplex structure that do actually occur will also affect
duplex stability. The structural characterization of modified
DNA/DNA and DNA/RNA duplexes should, therefore, not only
improve our basic understanding of the structural properties of
nucleic acid duplexes in general but it might also lead to the
design of novel modifications with more favorable properties
for therapeutic applications.
In this context we have embarked on a comprehensive
program directed at the crystallographic investigation of nucleic
acid duplexes incorporating sugar- and backbone-modified
component strands. As part of these continuing studies, we now
report on the structural properties of two modified analogs of
the Dickerson-Drew dodecamer d(CGCGAATTCGCG), where
both thymidine residues have been replaced either by 6′-R-
Inhibition of protein synthesis by the sequence-specific
binding of an oligonucleotide to single-stranded (ss) RNA or
double-stranded (ds) DNA targets (“antisense” and “antigene”
approach, respectively) in the recent past has developed into
an important novel approach in modern drug design.1 Due to
the insufficient metabolic stability of natural DNA and RNA
under physiological conditions, the successful implementation
of such oligonucleotide-based drug design strategies depends
on the availability of chemically modified oligonucleotides or
oligonucleotide analogs. These must not only exhibit largely
increased resistance to nucleolytic degradation but also retain
the ability to bind to their complementary target nucleic acids
with high affinity and in a highly sequence-specific manner.1
As a consequence, a large variety of structurally modified
oligonucleotide analogs have become known over the last few
years and the effects of such structural modifications on nuclease
resistance and RNA- or DNA-binding affinity have been
investigated in some detail.1,2 However, in contrast to the wealth
of RNA- or DNA-binding data that have emerged from those
studies, far less information is available on the changes in duplex
(3) For NMR spectroscopic studies on duplexes incorporating formacetal-
and amide-type backbone modifications, respectively, cf.: (a) Gao, X.; Jeffs,
P. W. J. Biomol. NMR 1994, 4, 17-34 (formacetal). (b) Blommers, M. J.
J.; Pieles, U.; De Mesmaeker, A. Nucleic Acids Res. 1994, 22, 4187-4194
(amide).
(4) For structural studies on duplexes incorporating peptide nucleic acids
(PNA) strands, cf.: (a) Brown, S. C.; Thomson, S. A.; Veal, J. M.; Davis,
D. G. Science 1994, 265, 777-780. (b) Betts, L.; Josey, J. A.; Veal, J. M.;
Jordan, S. R. Science 1995, 270, 1838-1841.
(5) For X-ray crystallographic studies of self-complemenatry DNA
duplexes containing 2′-O-methyladenosine and 4′-thiothymidine, respec-
tively, cf.: (a) Lubini, P.; Zu¨rcher, W.; Egli, M. Chem. & Biol. 1994, 1,
39-45 (2′-O-methyl ribose modification). (b) Boggon, T. J.; Hancox, E.
L.; McAuley-Hecht, K. E.; Connolly, B. A.; Hunter, W. N.; Brown, T.;
Walker, R. T.; Leonard, G. A. Nucleic Acids Res. 1996, 24, 951-961 (4′-
thio modification).
* Author to whom correspondence should be addressed: MP&BC
Department, Northwestern University Medical School; Phone (312) 503-
0845; Fax (312) 503-0796; E-mail m-egli@nwu.edu.
† Coordinates for both crystal structures have been deposited in the
Nucleic Acid Data Base; entry codes NDBS79 (MEME) and NDBS80
(OHOH).
‡ Northwestern University Medical School.
§ ETH Swiss Federal Institute of Technology.
| CIBA, Ltd.
Summer Student from April to July 1994.
X Abstract published in AdVance ACS Abstracts, February 15, 1997.
(1) (a) Uhlmann, E.; Peyman, A. Chem. ReV. 1990, 90, 543-584. (b)
Milligan, J. F.; Matteucci, M. D.; Martin, J. C. J. Med. Chem. 1993, 36,
1923-1937. (c) Antisense Research and Applications; Crooke, S. T., Lebleu,
B., Eds.; CRC Press, Inc.: Boca Raton, FL, 1993. (d) Altmann, K.-H.;
Dean, N. M.; Fabbro, D.; Freier, S. M.; Geiger, T.; Ha¨ner, R.; Hu¨sken, D.;
Martin, P.; Monia, B. P.; Mu¨ller, M.; Natt, F.; Nicklin, P.; Phillips, J.; Pieles,
U.; Sasmor, H.; Moser, H. E. Chimia 1996, 50, 168-176.
(2) (a) Varma, R. S. Synlett 1993, 621-637. (b) De Mesmaeker, A.;
Ha¨ner, R.; Martin, P.; Moser, H. E. Acc. Chem. Res. 1995, 28, 366-374.
(c) De Mesmaeker, A.; Altmann, K.-H.; Waldner, A.; Wendeborn, S. Curr.
Opin. Struct. Biol. 1995, 343-355.
(6) Recent X-ray crystal structure determinations of chemically modified
oligonucleotide analogs have been reviewed Egli, M. Angew. Chem., Int.
Ed. Engl. 1996, 35, 1894-1909.
(7) Schmit, C.; Be´vierre, M.-O.; De Mesmaeker, A.; Altmann, K.-H.
Bioorg. Med. Chem. Lett. 1994, 4, 1969-1974.
(8) Altmann, K.-H.; Imwinkelried, R.; Kesselring, R.; Rihs, G. Tetra-
hedron Lett. 1994, 35, 7625-7628 and references cited therein.
S0002-7863(96)02406-7 CCC: $14.00 © 1997 American Chemical Society

Oligodeoxyribonucleotides Containing Carbocyclic Thymidines
J. Am. Chem. Soc., Vol. 119, No. 10, 1997 2397
Scheme 1a
Figure 1. Structures and atom-numbering systems for 6′-R-substituted
carbocyclic thymidine building blocks: MCT (A) and HCT (B).
methyl (MCT) or 6′-R-hydroxy (HCT) carbocyclic thymidines
(Figure 1). Both of these modified building blocks have been
previously investigated in the context of possible antisense
applications. In these studies, oligonucleotides containing
stretches of contiguous MCT residues were found to exhibit
slightly reduced RNA-binding affinity (as compared to the
unmodified wild-type parent sequences), while the incorporation
of HCT led to increased DNA/RNA duplex stability.9,10
However, in both cases the degree of nuclease resistance
conferred to an adjacent phosphodiester bond does not suffice
to produce significant biological effects with phosphodiester-
based antisense oligonucleotides.9,10 When the possible origins
of the observed effects of MCT and HCT on RNA-binding
affinity as well as the structural consequences associated with
incorporation of these residues (at the single strand as well as
at the duplex level) are contemplated, it should be remembered
that the replacement of the furanose oxygen O4′ by a carbon
atom (designated C6′, Figure 1) leads to the loss of the
stereoelectronic effects influencing the conformation of the sugar
moiety and renders the five-membered ring more flexible.11 In
addition, the solvation of oligonucleotides containing carbocyclic
residues can be expected to be significantly different (at least
at the site of the modification) from the unmodified parent
duplex.
a Key: i. BzCl, Et3N, CH2Cl2, rt, 18 h, 85%. ii. H2, Lindlar’s catalyst,
MeOH, rt, 6 h, 98%. iii. CH3OCHdC(CH3)CONCO, CH2Cl2, -60°
f rt, 1.5 h, 97%. iv. 0.2 N HCl EtOH/H2O 9:1, reflux, 24 h, 74%. v.
H2, 10% Pd-C, AcOEt/MeOH 1:1, rt 3 h, 87%. vi. DMTrCl, Et3N,
DMAP (cat), pyridine, rt, 3 h, 67%. vii. [(i-C3H7)2N]2POCH2CH2CN,
(i-C3H7)2NH2+CHN4, CH2Cl2, rt, 2 h, 97%.
Here, we describe the crystal structures of the modified
duplexes [d(CGCGAAtMe Me
t CGCG)]2 (MEME; tMe ) MCT)
and [d(CGCGAAtOH OHCGCG)]2 (OHOH; tOH ) HCT) and
t
efficiency was observed under these conditions. After completion of
chain assemblage, the 5′-O-DMTr-protected product was released from
the support with concomitant cleavage of all protecting groups by
treatment of the support-bound oligonucleotide with concentrated
aqueous NH3 for 16 h at 55 °C. The 5′-O-DMTr-protected products
were purified by RP-HPLC, and the 5′-O-DMTr group was subse-
quently removed by treatment with 80% aqueous acetic acid for 30
min at room temperature (rt). For UV-melting studies, the deprotection
mixture was evaporated to dryness and the residue was twice evaporated
with EtOH/H2O 2:1. It was then redissolved in 2 mL of water and
extracted twice with ether, and the solution was lyophilized. According
to gel electrophoresis or capillary electrophoresis (CE), the fully
deprotected oligonucleotides were at least 95% pure.
compare them with the structure of the unmodified wild-type
duplex. In addition, we describe the effects of MCT and HCT
on DNA binding and try to analyze the DNA-binding affinity
of HCT- and MCT-containing oligodeoxyribonucleotides in light
of the structural properties of the two modified dodecamers.
Materials and Methods
Synthesis of Modified Nucleosides. The synthesis of MCT together
with that of the corresponding 5′-O-(4,4′-dimethoxytrityl) (DMTr) 3′-
(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite as required for
oligonucleotide synthesis has been previously described.10 The synthesis
of HCT is based on the known azido alcohol 1 and is summarized in
Scheme 1. Experimental details for the synthesis of 6′-OBz-HCT 5
and its derived 5′-O-(4,4′-dimethoxytrityl) (DMTr) 3′-(2-cyanoethyl-
N,N-diisopropylamino) phosphoramidite 7 are contained in the Sup-
porting Information.
Cystallization of Modified Oligonucleotides. For crystallization
experiments, four d(CGCGAATTCGCG)-type dodecamers with 6′-R-
substituted carbocyclic residues were synthesized and subjected to
crystallization trials. Rhombohedral crystals of the dodecamers
d(CGCGaOHaOH OH OH
t t CGCG) and d(CGCGaOHATtOHCGCG) were dis-
Oligonucleotide Synthesis. Oligonucleotides were synthesized on
an ABI 390b DNA synthesizer using long-chain alkylamino controlled
pore glass (CPG, 500Å) and standard phosphoramidite chemistry (1.5
µmol scale; 10 µmol scale for modified Dickerson-Drew dodecam-
ers).12 Bases were protected by benzoyl (dC, dA) and isobutyryl (dG)
groups. Extended coupling times of 10-12 min were employed for
modified phosphoramidites, and no significant reduction in coupling
ordered and diffracted only to low resolution. However, the ortho-
rhombic crystals obtained with the MEME and OHOH dodecamers
with two modified thymidines per single strand were isomorphous to
the native DNA dodecamer and both diffracted X-rays to better than
2.5 Å resolution. They were grown from 1.0 mM DNA (1.2 mM for
OHOH), 20 mM sodium cacodylate buffer pH 6.9, 18.8 mM Mg(OAc)2,
and 0.7 mM spermine tetrahydrochloride, equilibrated against 40%
2-methyl-2,4-pentanediol (MPD), using the sitting drop vapor diffusion
method. Data sets for both crystals were collected at room temperature
with Cu KR radiation for the MEME dodecamer (crystal size 1.5 ×
(9) Altmann, K.-H.; Be´vierre, M.-O.; De Mesmaeker, A.; Moser, H. E.
Bioorg. Med. Chem. Lett. 1995, 5, 431-436.
(10) Altmann, K.-H.; Kesselring, R.; Pieles, U. Tetrahedron 1996, 52,
12699-12722.
(11) For a comprehensive discussion of nucleic acid conformation, see:
Saenger, W. Principles of Nucleic Acid Structure; Springer Verlag: New
York, 1984.
(12) (a) Sinha, N. D.; Biernat, J.; McManus, J.; Ko¨ster, H. Nucleic Acids
Res. 1984, 12, 4539-4557. (b) Oligonucleotide synthesis - a practical
approach; Gait, M. J., Ed.; IRL Press: Oxford, 1984.

2398 J. Am. Chem. Soc., Vol. 119, No. 10, 1997
Portmann et al.
Table 1: Selected Crystal and Refinement Data
parameter
d(CGCGAAtMe Me
t
CGCG)
d(CGCGAAtOHtOHCGCG)
temperature
a (Å)
b (Å)
c (Å)
space group
resolution (Å)
rt
rt
25.20
40.80
66.27
P212121
2.50
24.88
40.27
66.49
P212121
2.14
R
merge (%)
3.5
2072
5.1
3605
no. of reflections at a 2 σ(Fobs) level used for
refinement (10 Å to full resolution)
strands per asymmetric unit
vol./base pair (Å3)
no. of waters
R-factor (%)
2
2
1420
65
17.0
29.2
0.018
3.70
1388
66 + 1Mg2+
20.7
Rfree (%)
32.9
0.017
3.36
rms from ideality, bond lengths (Å)
rms from ideality, bond angles (deg)
Figure 2. Stereodrawing of an omit electron density map contoured at 1.2σ around the sugar-phosphate backbone portions of residues OCT7, -8,
-19, and -20 for OHOH (including all data to a resolution of 2.14 Å). The map was calculated with duplex coordinates minus the sugar portions
of the four modified thymidines and the phosphates between them.
0.17 × 0.1 mm) on a Marresearch 300 mm image plate mounted on
an Enraf-Nonius rotating anode generator and for the OHOH dodecamer
(crystal size 0.5 × 0.2 × 0.2 mm) on an R-AXIS-II image plate
mounted on a Rigaku rotating anode generator. Data were processed
with the DENZO/SCALEPACK13 program package and details con-
cerning resolution and quality of the data sets are listed in Table 1.
Mg2+ ion with six coordinated water molecules. Refinement data are
summarized in Table 1, and an omit density map around the modified
residues in the OHOH duplex at the final stage is depicted in Figure 2.
Two Crystal Forms. Replacing Mg2+ with Ca2+ in crystallization
solutions of both the MEME and OHOH dodecamers yields a new
rhombohedral crystal form. Such crystals typically diffract X-rays to
a resolution of 2.8 Å or less. Several other dodecamer strands with
identical sequence, but containing either four 6′-unsubstituted or 6′-
R-hydroxy carbocyclic residues at positions 6-9 or with the carbocyclic
residues incorporated at locations other than T7 and T8, also crystallized
in this rhombohedral form, even in the presence of Mg2+. However,
crystals grown of the unmodified d(CGCGAATTCGCG) DNA dodecam-
er uniformly belong to the orthorhombic space group P212121,
independent of whether the crystallization solutions are supplemented
with Mg2+ or Ca2+. In the lattice of the OHOH structure, a hydrated
Mg2+ ion is located between two adjacent symmetry-equivalent
duplexes, whereby all six coordinated water molecules form direct
contacts to DNA atoms. Three of them are hydrogen bonded to
phosphate oxygens of a first duplex (O1P MCT7, O2P A6, and O1P
A6), and the remaining ones form hydrogen bonds to major groove
base atoms of a second duplex (N7 G2, O6 G2, and O6 G22).
Structure Determinations and Refinements. The unit cell con-
stants and the space group of the MEME and OHOH crystals suggested
14
isomorphism with the native DNA duplex [d(CGCGAATTCGCG)]2
(NDB15 Code BDL001). After initial rigid body refinement and several
cycles of positional refinement with X-PLOR,16 using the unmodified
dodecamer duplex as a model for both structures, the additional
exocyclic substituents appeared clearly in 2Fobsd - Fcalcd sum and Fobsd
- Fcalcd difference electron density maps. The models were modified
and the standard DNA restraints dictionary was adjusted. Following
several cycles of refinement including a slow cooling procedure, water
molecules were assigned to peaks appearing in both the sum and
difference density maps. Water molecules that were accepted had to
display good hydrogen-bonding geometry, be surrounded by 2Fobsd
-
Fcalcd sum density at a 2σ level, and have a temperature factor of below
60 Å2 after refinement. The latter two criteria were handled less strictly
than the first. Refinement was terminated once no new water molecules
could be added. In the OHOH structure a high-density region with
octahedral shape in the sum and difference maps was interpreted as a
UV Melting Experiments. With the exception of self-complemen-
tary sequences, melting temperatures (Tm values) were determined in
10 mM phosphate buffer, pH 7, 100 mM Na+, 0.1 mM EDTA, at 4
µM strand concentration. The Tm values for the modified Dickerson-
Drew dodecamers as well as the wild-type oligonucleotide were
recorded in 10 mM cacodylate buffer, pH 7, 100 mM NaCl, 0.1 mM
EDTA, at 10 µM strand concentration, and the Tm values are (0.5°.
For further experimental details, see ref 17.
(13) (a) Otwinowski, Z. Oscillation Data Reduction Program. In Pro-
ceedings of the CCP4 Study Weekend: Data Collection and Processsing;
Sawyer, L., Isaacs, N., Bailey, S., Eds.; SERC Daresbury Laboratory:
England, January 1993; pp 56-62. (b) Minor, W. XDISPLAYF Program;
Purdue University, 1993.
(14) (a) Wing, R.; Drew, H.; Takano, T.; Broka, C.; Tanaka, S.; Itakura,
K.; Dickerson, R. E. Nature 1980, 287, 755-758. (b) Dickerson, R. E.;
Drew, H. R. J. Mol. Biol. 1981, 149, 761-786.
(17) Lesnik, E. A.; Guinosso, C. J.; Kawasaki, A. M.; Sasmor, H.;
Zounes, M.; Cummins, L. L.; Ecker, D. J.; Cook, P. D.; Freier, S. M.
Biochemistry 1993, 32, 7832-7838.
(18) (a) Holbrook, S. R.; Dickerson, R. E.; Kim, S.-H. Acta Crystallogr.
B, 1985, 41, 255-262 (NDB code BDLB05). (b) Westhof, E. J. Biomol.
Struct. Dyn. 1987, 5, 581-600 (NDB code BDL020).
(15) Berman, H. M.; Olson, W. K.; Beveridge, D. L.; Westbrook, J.;
Gelbin, A.; Demeny, T.; Hsieh, S.-H.; Srinivasan, A. R.; Schneider, B.
Biopys. J. 1992, 63, 751-759.
(16) Bru¨nger, A. T.; Kuriyan, J.; Karplus, M. Science 1987, 235, 458-
460.

Oligodeoxyribonucleotides Containing Carbocyclic Thymidines
J. Am. Chem. Soc., Vol. 119, No. 10, 1997 2399
Figure 3. Helical rise, twist, slide, and minor groove widths of the [d(CGCGAAtMetMeCGCG)]2 (MEME), [d(CGCGAAtOHtOHCGCG)]2 (OHOH),
and [d(CGCGAATTCGCG)]2 (native, NDB code BDL001) duplexes. Helical parameters were calculated with the program NEWHEL93 distributed
by R. E. Dickerson. The groove width is defined as the shortest distance between pairs of phosphorus atoms across the minor groove minus 5.8 Å,
the sum of van der Waals radii for two phosphate groups.
Results and Discussion
residues G10 and HCT19, which is slightly longer in OHOH
than in the native duplex. In the minor groove of the OHOH
duplex, the hydroxy substituents form the corners of a paral-
lelogram (Figure 4B). The distances between the 6′-oxygens
within strands are 4.65 and 4.80 Å for strands 1 and 2,
respectively, and they are 4.70 and 4.82 Å between oxygen
atoms from opposite strands. The shortest distance of 4.26 is
observed along the short diagonal of the parallelogram between
O6′ of residue HCT8 and O6′ of residue HCT20. Thus, the
smaller and more polar 6′-R-substituents of HCT residues in
the OHOH duplex are accommodated more smoothly within
the B-DNA dodecamer structure and consequently do not alter
the minor groove topology, while the more bulky methyl
substituents of MCT residues in the MEME duplex distort the
groove topology more severely.
Duplex Conformations. Isomorphism of the two modified
duplex crystals with those of the native B-DNA dodecamer
duplex14 is consistent with relatively small geometrical devia-
tions between the three structures. The rms deviations between
the Dickerson-Drew dodecamer and MEME and OHOH are
0.80 Å and 0.48 Å, respectively. The root mean square (rms)
deviation between the MEME and OHOH structures is 0.64 Å.
The smaller conformational difference between the OHOH and
Dickerson-Drew duplexes, compared with the difference
between the MEME duplex and the native one, are also apparent
in a comparison of the helical and topological parameters of
the three duplexes (Figure 3).
In accordance with the location of the modifications, the
major geometrical differences between the duplexes occur in
their central portions, particularly at the A6pT7 (A18pT19) step.
There, the rise of MEME relative to the native duplex is
increased by 0.68 to 3.80 Å and its twist is lowered by 5.1° to
26.7°. In addition, the base pairs are shifted in a positive
direction (slide) in the three central base pair steps of the MEME
duplex. Taken together, these changes result in an enlargement
of the central minor groove by about 2.3 Å on average (Figure
3). The 7′-methyl substituents of the carbocyclic thymidines
are directed into the central section of the minor groove and
form the corners of an approximate square (Figure 4A). The
distances between the C7′-carbon atoms within strands are 4.53
and 5.03 Å for strands 1 and 2, respectively, and they are 4.87
and 5.21 Å between carbon atoms from opposite strands. Thus,
in terms of both their intra- and interstrand interactions, the
methyl groups are in near van der Waals contact with one
another. The minor groove widening is most likely caused by
the bulky methyl groups.
Conformations and Interactions of Carbocyclic Residues.
All 6′-R-substituted carbocyclic thymidines in the MEME and
OHOH duplexes adopt either C2′-endo- or C1′-exo-puckers,
resulting in pseudoequatorial positions of the 6′-R-substituents.
The deoxyriboses of thymidines in the native structure have
the lowest pseudorotation angle on average with many of them
adopting C1′-exo-type pucker. The cyclopentane rings of
carbocyclic thymidines in MEME display the highest pseudoro-
tation angle values, indicative of C2′-endo-puckers, whereas
those in OHOH lie between the P values of the native and the
MEME duplexes (Figure 5). The two modified structures
demonstrate that the cyclopentane ring of 6′-R-substituted
carbocyclic residues can adjust smoothly to the geometrical
constraints exerted by the furanosyl phosphate backbones and
that they are fully compatible with B-form DNA.
The most drastic changes in the MEME backbone angles
relative to those in the unmodified dodecamer duplex are
observed in residues MCT7, MCT8, and C9 (Figure 5). The
latter residue adopts a C3′-endo-pucker instead of the C1′-exo
one in the native duplex. Its angle R is changed from -sc to
ap, angle ꢀ from ap to +sc, and angle ú from -sc to +sc. In
The helical parameters of the OHOH duplex are more similar
to those of the native duplex, and their minor grooves display
roughly the same widths (Figure 3). The only exception is
constituted by the distance between the phosphorus atoms of

2400 J. Am. Chem. Soc., Vol. 119, No. 10, 1997
Portmann et al.
Table 2. Geometry of the Hydrogen Bonds between HCT
6′-R-Hydroxyl Groups and N3 or O2 Atoms of Adjacent A or HCT
Residues, Respectively, in the Minor Groove of the OHOH Duplexa
HCT7-
A6
HCT19-
A18
HCT8-
HCT7
HCT20-
HCT19
interactionb
O6′‚‚‚ N3
2.89 Å
112.2°
132.9°
3.04 Å
100.4°
141.3°
C6′-O6′-N3
O6′-N3-C4
O6′-N3-C4-N9 -26.8°
O6′‚‚‚O2
-24.6°
2.64 Å
113.9°
151.4°
2.75 Å
106.8°
147.9°
C6′-O6′-O2
O6′-O2-C2
O6′-O2-C2-N1
-40.5°
-46.1°
a See also Figure 6. b Atoms of HCT residues are underlined.
structures. The only notable deviations from the native
backbone geometry there are seen in residues MCT19 and
HCT19, where the ꢀ angles are converted from ap to -sc and
the ú angles from -sc to ap. This corresponds to a switch from
the BI backbone conformational state to the BII one that had
already been observed in the structure of a Dickerson-Drew
dodecamer brominated at position C9 (NDB code BDLB04).19
Packing forces in the orthorhombic crystal lattice are the most
likely reason for the asymmetric geometrical changes in the
backbone conformations of the MEME and OHOH structures.
It is possible that such forces constrain the second strand to a
larger extent, causing the duplex to adapt to the structural
perturbations induced by the carbocyclic modifications mainly
by altering its more flexible first strand.
In the OHOH structure, the 6′-R-hydroxyl groups of car-
bocyclic thymidines lie roughly in the planes defined by the
bases of preceding residues and form hydrogen bonds to O2
and N3 atoms of T and A residues, respectively. In the first
strand, the O6′ of HCT7 is hydrogen bonded to N3 of A6 and
the O6′ of HCT8 is hydrogen bonded to the O2 of HCT7.
Similarly, in the second strand, the O6′ of HCT19 is hydrogen
bonded to N3 of A18 and the O6′ of HCT20 is hydrogen bonded
to O2 of HCT19 (Figure 6). Hydrogen bonds between HCT
and A residues display almost ideal geometries (Table 2).
There, the N3 lone pairs of adenosines are directed more or
less at the 6′-hydroxyl groups of carbocyclic thymidines. The
O6′-N3-C4 angles are 132.9° and 141.3° for the HCT7-A6
and HCT19-A18 interactions, respectively, and the small values
for the improper torsion angles O6′-N3-C4-N9 indicate that
the 6′-R-hydroxyl groups are practically positioned within the
planes defined by the adenine bases (Table 2, Figure 6). By
comparison, the hydrogen bonds between 6′-R-hydroxyl groups
and exocyclic O2 atoms from thymidines are shorter, but the
geometry at the acceptors is not ideal. The O6′-O2-C2 angles
are 151.4° and 147.9° for the HCT8-HCT7 and HCT20-HCT19
interactions, respectively, and the larger values for the improper
torsions O6′-O2-C2-N1 indicate that the 6′-R-hydroxyl
groups are located further away from the planes defined by the
thymine bases.
Figure 4. Views into the central portion of the minor grooves for the
[d(CGCGAAtMetMeCGCG)]2 (A) and [d(CGCGAAtOHtOHCGCG)]2 (B)
duplexes. The included water molecules (cyan) have direct contacts to
either DNA atoms (carbon green, oxygen red, nitrogen blue, phosphorus
orange) or to first hydration-shell waters (distance criteria 3.4 Å). The
exocyclic C7′ methyl carbon and O6′ hydroxyl oxygen atoms are
highlighted in yellow and magenta, respectively. In both drawings, the
strands comprising residues 19 (bottom) and 20 (top) are on the left,
and the strands comprising residues 7 (top) and 8 (bottom) are on the
right. Rotation of Figure 7 by 90°, such that the labels A-C are at the
bottom, will generate an orientation of the duplexes that is identical to
the one depicted here. Thus, the water molecule in the center of Figure
4A and the yellow carbon sphere to its immediate left represent the
short C-H‚‚‚O contact between W67 and the C7′ methyl group of
residue MCT19 in MEME (see Figure 7A). Similarly, the water
molecule located between the two bottom magenta spheres in Figure
4B represents W67 bridging the 6′-R-hydroxyl groups of residues
HCT19 and HCT8 across the minor groove of OHOH (see Figure 7B).
residue MCT8, angle ꢀ switches from ap to -sc and angle ú
from -sc to +sc. In residue MCT7 angle ꢀ switches from ap
to -sc and angle ú from -sc to ap. These alterations in the
geometry of the sugar-phosphate backbone appear necessary
to accommodate the 6′-R-methyl groups of the carbocyclic
thymidines in the B-type duplex and together result in the
opening of the minor groove.
As expected from the smaller changes in the overall structure
of the OHOH duplex relative to the native one, their sugar-
phosphate backbone geometries deviate only slightly, with the
most notable change occurring at residue C9 adjacent to HCT8
(Figure 5). There, the R and γ torsion angles are altered in a
concerted way, the former angle from -sc to +sc and the latter
from +sc to -sc. This crankshaft motion around torsion angle
â may help to readjust the relative position of stacked base pairs
at that site as a consequence of a subtle local conformational
change induced by the 6′-R-OH modification. An interesting
coincidence is the virtual lack of geometrical changes in the
backbone of the second strands in the MEME and OHOH
Hydration. In the central portion of the minor groove in
the native B-DNA dodecamer duplex, a spine of hydration
involving seven first hydration-shell water molecules was found
to link guanine N3 or N2 and adenine N3 with cytosine and
thymine O2 from opposite strands (Figure 7C).20 Six second
hydration-shell water molecules (not all shown in Figure 7C)
then connect those with direct DNA contacts, leading to an
uninterrupted network of water molecules. The 4′-oxygens
(19) Fratini, A. V.; Kopka, M. L.; Drew, H. R.; Dickerson, R. E. J. Biol.
Chem. 1982, 257, 14686-14707 (NDB codes BDLB03 and BDLB04).
(20) Drew, H. R.; Dickerson, R. E. J. Mol. Biol. 1981, 151, 535-556.

Oligodeoxyribonucleotides Containing Carbocyclic Thymidines
J. Am. Chem. Soc., Vol. 119, No. 10, 1997 2401
Figure 5. Backbone torsion angles and sugar pseudorotation phase angles P (in place of torsion angle δ) in [d(CGCGAAtMetMeCGCG)]2 (MEME),
[d(CGCGAAtOHtOHCGCG)]2 (OHOH), and [d(CGCGAATTCGCG)]2 (native, NDB code BDL001). The statements in the text concerning the deviations
in the sugar-phosphate geometry between this native unmodified duplex and the duplexes carrying the MCT and HCT modifications, respectively,
are independent of the restraint parameters that were used for the refinements of the original structure15 and subsequent ones.18,19 Thus, the average
backbone torsion angles (in degrees, omitting terminal base pairs) for the dodecamer structures with NDB codes BDL001, BDLB03, BDLB04,
BDL005, and BDL020 are the following (standard deviations in parentheses): R ) 298.5(13.6), â ) 170.4(15.1), γ ) 52.7(12.8), δ ) 123.2(21.4),
P ) 130.4(28.9), ꢀ ) 189.6(28.7), ú ) 254.2(37.2), and ø ) 244.1(15.3). By comparison, the average angles and standard deviations when omitting
BDL001 (our standard) are the following: R ) 298.6(14.8), â ) 170.2(15.2), γ ) 52.3(13.8), δ ) 123.2(22.2), P ) 130.5(29.9), ꢀ ) 189.7(29.6),
ú ) 254.2(38.0), and ø ) 244.2 (15.7).
of 6′-R-methyl-substituted carbocyclic residues in the central
portion of the duplex. If the furanose 4′-oxygens indeed
contributed to the stabilization of the spine, their absence in
portions of both strands in the MEME and OHOH duplexes
should affect the geometry of the hydration spine. Comparison
between the water positions in the native B-DNA duplex and
MEME reveals that two second-shell water molecules are
replaced by the four methyl groups in the latter structure (Figures
4A and 7A,C). In the terminology used by Drew and Dicker-
son,20 these are water molecules w33 and w35, which bridge
first-shell water molecules (throughout this section, water
molecules in the native structure are in small font and water
molecules in MEME or OHOH are in capital font). The first-
shell water molecule W67 (w27 in the wild-type structure) is
thereby isolated from the spine of hydration. This water is
Figure 6. Hydrogen bonds between the 6′-R-hydroxyl groups (labeled)
of carbocyclic thymidines and N3 or O2 atoms of 3′-adjacent A or
located in the center of the approximate square formed by the
methyl groups, half-way between the periphery of the minor
groove and its floor (Figure 4A). It forms hydrogen bonds to
oxygens O2 of MCT7 and MCT19 and has a close contact of
3.12 Å to one of the 7′-methyl carbons (calculated [C]H‚‚‚O
distance of 2.4 Å, Figure 7A). In the native structure, this water
(w27) forms a weak hydrogen bond to the 4′-oxygen of T20
(3.38 Å) in addition to the above exocyclic keto oxygens; while
in the modified structure, the corresponding W67 is slightly
moved away from its native position, with an increased distance
to the 6′-carbon of MCT20 (Figure 7A). Relative to the
symmetrical arrangement of hydration spine water molecules
HCT residues (bold and underlined), respectively, in the central portion
of the minor groove of the [d(CGCGAAtOHtOHCGCG)]2 duplex. 6′-R-
Hydroxyl oxygens are solid black, hydrogen bonds are dashed with
their lengths given in italics, and darker portions of the duplex are
further away from the reader.
contribute to a polar environment within the minor groove and
are believed to stabilize the water spine.21
In MEME, the polarity near the floor of the minor groove is
expected to change significantly as a result of the introduction
(21) Kopka, M. L.; Fratini, A. V.; Drew, H. R.; Dickerson R. E. J. Mol.
Biol. 1983, 163, 129-146.

2402 J. Am. Chem. Soc., Vol. 119, No. 10, 1997
Portmann et al.
Figure 7. Schematic representation of the minor groove hydrations in the [d(CGCGAAtMe Me
t
CGCG)]2 (A), [d(CGCGAAtOH OH
t
CGCG)]2 (B), and
wild-type [d(CGCGAATTCGCG)]2 duplexes (C). DNA atoms are thin circles, water molecules with direct contacts to DNA are bold circles, and
bridging water molecules without direct contacts to DNA are solid black. Squares are exocyclic methyl groups C7′ or hydroxyl groups O6′ of
6′-R-methyl carbocyclic thymidines (MCT) or 6′-R-hydroxy carbocyclic thymidines (HCT), respectively.
in the native duplex, with three water molecules coordinating
to bases in both outer portions of the minor groove (Figure 7C),
several waters have shifted their positions in the MEME minor
groove. In the bottom half of the MEME minor groove (left in
Figure 7A), four water molecules constitute the hydration spine
instead of the three in the Dickerson-Drew dodecamer, each
shifted away somewhat more from the center of the duplex
compared with the native structure (Figure 7A,C, left). Thus,
around base pair G10‚C15, water molecules W89 and W50
replace w71 and water molecules W51 and W84 replace w98.
W84 is hydrogen bonded to the phosphate group of G10, and
at that site, the hydration spine is shifted away from the base
edges toward the backbone. At the other end of the minor
groove, only two first-shell water molecules remain in MEME,
as compared to the three in the native duplex (Figure 7A,C,
right). The third and fourth layers of ordered waters are not
present in the modified structure. This may be a consequence
of the presence of the methyl groups, which render this region
more hydrophobic, but may also be related to the lower
resolution of the modified structure compared with the native
one. At several such locations, water molecules had been
assigned to difference density peaks, but had shown high
B-factors after refinement and had consequently been deleted.
The observed water molecules in OHOH do not form an
extended network comparable to those seen in the two other
structures. However, there are some similarities. As in MEME,
water w71 is replaced by two water molecules (W62 and W64).
W65 (w98) is shifted toward strand 1 (O2 of C9) and looses its
contact to N3 of A17. W36 (w41) bridges O2 of HCT8 and
O6′ of HCT19 instead of O2 of T8 and N3 of A18. w27 is
replaced by the two most closely spaced O6′ atoms (O6′ of
HCT8 and O6′ of HCT20), and W72 (w34) is shifted toward
the second strand (O2 of HCT20) and looses contact to strand
1. Remarkably, only water molecule W67 is involved in direct
hydrogen bonds to two 6′-R-hydroxyl oxygens from carbocyclic
residues (residues HCT8 and HCT19, O‚‚‚O distance criteria
e3.4 Å). However, as mentioned above, all 6′-R-hydroxyl
oxygens are engaged in intrastrand internucleotide hydrogen
bonds to either O2 of HCT or N3 of adenosine residues (Figure
6).
Duplex Stability. The effects of MCT and HCT on the
DNA-binding affinity of the corresponding oligonucleotides are
summarized in Table 3 (UV-melting temperatures of modified
DNA/DNA duplexes). RNA-binding data for MCT10 as well
as HCT9 have been previously published. As indicated by the
decrease in melting temperature of the corresponding DNA/
DNA duplexes of -1.6 °C per modification (Table 3, sequence
II), the incorporation of isolated 6′-R-methyl carbocyclic thy-
midines into oligodeoxyribonucleotides reduces their affinity
for complementary DNA. This is similar to the previously
observed, reduced stability of duplexes between DNA strands
comprising isolated MCT residues and complementary RNArel-
ative to the native hybrids.10 The reduced affinity for DNA
would be consistent with the observed distortion of the global

Oligodeoxyribonucleotides Containing Carbocyclic Thymidines
J. Am. Chem. Soc., Vol. 119, No. 10, 1997 2403
Table 3. Effect of 6′-R-Methyl Carbocyclic Thymidine (MCT) and
6′-R-Hydroxy Carbocyclic Thymidine (HCT) on the DNA Binding
Affinities of Oligodeoxyribonucleotides (∆Tm values/modificationa)
bridging of 6′-R-hydroxyl groups in OHOH by water molecules
(e.g., water W67, Figure 7B).
a
sequenceb
∆Tm/mod
Tm(WT)c
Conclusions
t
The two crystal structures demonstrate that the cyclopentane
moieties of 6′-R-substituted carbocyclic nucleotides can mimic
the furanosyl conformation in a B-type DNA double helix and
do not induce drastic changes in the overall duplex conforma-
tion. This is in agreement with the ability of modified DNA
strands to form stable Watson-Crick base-paired duplexes with
either complementary DNA or RNA strands. The modification
that causes the smaller conformational deviations from the wild-
type structure (i.e., 6′-R-hydroxy carbocyclic thymidine) gener-
ally leads to duplexes of higher stability. Perhaps surprisingly,
the polar 6′-R-hydroxy substituent distorts the minor groove
hydration more severely than the nonpolar 6′-R-methyl sub-
stituent. However, this adverse effect is compensated for by
the 6′-R-hydroxyl groups constituting a covalent “first hydration-
sphere” themselves and by their acting as hydrogen-bonding
acceptors for additional water molecules at the groove periphery.
It is noteworthy that the formation of these hydrogen bonds is
sequence-independent, as all bases can in principle accept
hydrogen bonds via either their N3 or O2 atoms from the 6′-
R-hydroxyl groups. The presented work offers a structural
rationale for the differences in thermodynamic stability between
DNA/DNA duplexes incorporating either MCT or HCT modi-
fications in one or both strands. Furthermore, OHOH and
MEME add to the small database of crystal structures of
oligonucleotides containing building blocks with chemically
modified sugar moieties.5
MCT
HCT
MCT
HCT
MCT
HCT
MCT
HCT
MCT
HCT
I
I
II
II
III
III
IV
IV
Vd
Vd
-1.7
-1.9
-1.6
-0.6
-1.1
-0.6
-1.3
-0.1
-1.5
-0.25
43.0
43.0
62.5
62.5
58.2
58.2
54.1
54.1
55.0
55.0
a Difference in melting temperature (∆Tm) between the modified
DNA/DNA duplex and the respective unmodified wild-type (WT)
duplex divided by the number of modified building blocks (∆Tm ) Tm
- Tm(WT)); Tm values were determined in 10 mM phosphate buffer,
pH 7, 100 mM NaCl, 0.1 mM EDTA, at 4 µM strand concentration.
b Sequences are the following: I 5′-TTTTtCTCTCTCTCT-3′; II 5′-
tCCAGGtGtCCGCAtC-3′; III 5′-CTCGTACttttCCGGTCC-3′; IV 5′-
GCGttttttttttGCG-3′; V 5′-CGCGAAttGCGC-3′. c The Tm of the
corresponding wild-type duplex in degrees Celcius. d Self-complemen-
tary sequence, Tm determined in 10 mM sodium cacodylate buffer, pH
7, 100 mM NaCl, 0.1 mM EDTA, at 10 µM strand concentration.
hydration motif in our structure. Duplex destabilization appears
to be slightly less pronounced for stretches of contiguous
modified nucleotide units (Table 3, sequences III and IV; ∆Tm
) -1.1 and -1.3 °C per modification, respectively), which
might be related to the release of water molecules in the vicinity
of the methyl substituents combined with hydrophobic interac-
tions between methyl groups in near van der Waals contacts.
However, the subtleness of the effect makes it difficult to
determine its precise structural origin.
With the exception of sequence I, DNA/DNA duplexes
incorporating 6′-R-hydroxy-substituted nucleotide units in one
of the component strands generally exhibit significantly higher
thermodynamic stabilities (higher Tm values) than those contain-
ing the 6′-R-methyl carbocyclic modification. Hydrogen-
bonding interactions between O6′ atoms from HCT residues
and O2 or N3 atoms from bases of adjacent residues as observed
in the minor groove of the OHOH duplex could account for
these differences. The 6′-R-hydroxyl groups of the HCT
residues may thus be considered covalently bound water
molecules, donating hydrogen bonds to base acceptor atoms in
the minor groove. In addition, the difference in thermodynamic
stability between the two self-complementary OHOH and
MEME duplexes may also be attributed to the interstrand
Acknowledgment. We thank Dr. Fritz K. Winkler (Hoff-
mann LaRoche Ltd., Basel) for data collection with the MEME
crystals, Dr. Uwe Pieles and Werner Zu¨rcher (CIBA Ltd., Basel)
for oligonucleotide synthesis, and Dr. Dieter Hu¨sken (CIBA
Ltd., Basel), Dr. S. M. Freier (ISIS Pharmaceuticals), and Dr.
E. A. Lesnik (ISIS) for Tm measurements. We are also grateful
to Dr. Sandro Hollenstein (University of Zu¨rich) for help during
the initial crystallization experiments, Dr. Valya Tereshko for
producing Figure 2, and the two reviewers for helpful comments
on the manuscript.
Supporting Information Available: Experimental and
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