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X-ray crystal structure of a locked nucleic acid (LNA) duplex composed of a

palindromic 10-mer DNA strand containing one LNA thymine monomer†

Martin Egli,*a George Minasov,b Marianna Teplova,a Ravindra Kumarc and Jesper Wengelc
a Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235, USA,
E-mail: martin.egli@vanderbilt.edu
b Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School,
Published on 14 March 2001. Downloaded by University of California - Santa Cruz on 24/10/2014 00:18:14.

Chicago, Illinois 60611, USA

c Department of Chemistry, University of Southern Denmark, DK-5230 Odense M, Denmark

Received (in Cambridge, UK) 27 November 2000, Accepted 22nd February 2001
First published as an Advance Article on the web 14th March 2001

Locked nucleic acid (LNA), a recently introduced nucleic The mixed DNA–LNA decamer was synthesized‡ using
acid analogue with a bicyclic 2A-O,4A-C-methylene linked phosphoramidite chemistry as previously described1 and puri-
furanose sugar, exhibits enhanced affinities for DNA and fied to > 98%. The modified decamer crystallizes§ in the A-
RNA relative to the corresponding oligodeoxyribonucleo- form, the right-handed duplex geometry presumably preferred
tides and oligoribonucleotides; we report the first crystal by an LNA–LNA duplex. The crystallographic model was
structure of an LNA unit incorporated in an oligonucleotide refined¶ with simulated annealing and force field methods,
duplex. The structure at 1.4 Å resolution of the DNA–LNA using an initial orientation from Molecular Replacement.¶ The
decamer duplex with one LNA thymine monomer per strand decamer duplex exhibits an average helical rise of 2.95 Å and
provides a detailed view of the conformation and hydration the average values for helical twist and inclination are 31.2 and
of locked nucleic acid residues in a duplex A-form. 17.8°, respectively. An overview of the crystal data and
refinement parameters is shown in Table 1 and final electron
LNA (Fig. 1), exhibits stability of self-pairing that significantly density maps around LNA residues are depicted in Fig. 2.
exceeds those observed with DNA and RNA.1–3 The UV The sugar conformations of the two locked thymines 6 and 16
melting temperatures of mixed DNA–LNA1,4 and RNA–LNA5 are both C3A-endo (Fig. 3) and the respective values for the
strands paired to DNA or RNA are increased by between 4 to pseudorotation17 angle P are 16.9 and 18.3° (calculated with the
9 °C per modified residue compared to the corresponding program CURVES18). All deoxyriboses in the decamer exhibit
unmodified duplexes. Increased RNA affinity, higher nuclease C3A-endo puckers and the average value of their P angles (17.9°;
resistance and the observation that RNAs targeted by mixed excluding the two LNA residues) is very similar to those of the
DNA–LNA oligonucleotides are degraded by RNase H render modified thymines. Thus, the bicyclic sugar moieties fit
LNA a promising third-generation antisense modification.6 seamlessly into an A-form double helix and the additional
restraints appear to lock them in an A-type pucker.
In addition to the sugar–phosphate backbone torsion angle d
that is a characteristic of the ribose conformation (Fig. 1), the
five other backbone torsions adopt values that are also
consistent with the standard sc2, ap, sc+, sc+, ap, sc2 genus (a
to z) of A-form double helices. A comparison of the backbone

Table 1 Crystal data and refinement parameters

Fig. 1 Structure of LNA; torsion angles are labeled. Space group orthorhombic P212121
Unit cell constants [Å] a = 26.14, b = 43.96,
c = 45.80
NMR solution structures of DNA–LNA7–9 and RNA–LNA10
Temperature [K] 110
duplexes containing either single or multiple LNA residues in Wavelength [Å] 0.93218
one strand demonstrated the preference of the locked sugar for Beamline /detector APS DND-CAT 5-ID/
a C3A-endo conformation. Based on these experiments it was MARCCD
concluded that enthalpic (improved stacking) and entropic Resolution [Å] 1.40
factors (conformational preorganization) may account for the No. of unique reflections 10,950
unprecedented gains in the thermodynamic stabilities of LNA- Data completeness (all/last shell) [%] 99.7/96.7
modified duplexes. In order to examine the detailed conforma- Rsym (all/last shell) [%] 4.9/26.0
tion of LNA residues and the hydration of the bicyclic sugar No. of nucleic acid atoms 408
moiety, we determined the single crystal structure of the DNA– No. of water molecules 127
R-work/R-free [%] 16.7/17.4
LNA decamer duplex [GCGTATLACGC]2 with a single LNA r.m.s for bonds/angles from standards Å/°] 0.009/1.51
thymine TL at high resolution.

Fig. 2 Stereo drawing of the simulated annealing omit electron density (3s-level) around modified residues TL6 and TL16 in the [(dA5pTL6)- (dA15pTL16]
base pair step. Atoms are colored yellow, red, blue and magenta for carbon, oxygen, nitrogen and phosphorus, respectively.

DOI: 10.1039/b009447l Chem. Commun., 2001, 651–652 651

This journal is © The Royal Society of Chemistry 2001
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torsions in the modified thymines with those in the rest of the Britta M. Dahl for oligonucleotide synthesis and Dr
duplex does not manifest any notable deviations. Therefore the Christopher J. Wilds for discussions. The DNDCAT Synchro-
bicyclic modification in LNA is fully compatible with the A- tron Research Center at the APS, Argonne, IL, is supported by
form geometry adopted by both DNA and RNA. Similarly, the E. I. DuPont de Nemours & Co., The Dow Chemical Company,
glycosidic torsion angles in both LNA residues are in the the NSF and the State of Illinois.
standard anti geometry and no significant changes appear to
result from the chemical modification in the local helical
parameters, such as slide and x- and y-displacement (Fig. 3). Notes and references
Our findings here based on X-ray crystallographic data are † Coordinates and structure factors have been deposited in the Protein Data
consistent with those obtained from NMR solution studies of Bank (pdb code 1i5w).
DNA and RNA duplexes with either single or multiple LNA ‡ The decamer was synthesized on a 2 mmole scale and the trityl-on strand
residues in one of the strands.7–10 According to those experi- was purified by RP-HPLC (C4, triethylammonium acetate pH 7–acetonit-
Published on 14 March 2001. Downloaded by University of California - Santa Cruz on 24/10/2014 00:18:14.

ments, the exceptional thermodynamic stability gains seen with rile). Following detritylation the unprotected oligonucleotide was HPLC-
LNA-modified duplexes (both in the DNA and the RNA purified a second time and then desalted.
§ Crystallization conditions were screened with a commercial sparse matrix
contexts) are a result of the conformational preorganization of kit (Nucleic Acid Miniscreen, Hampton Research, Laguna Niguel, CA),
modified single strands for the duplex state (entropic contribu- using the hanging drop vapor diffusion technique. Crystals suitable for
tion) as well as of the improved stacking both in the single- and diffraction experiments were obtained under the following conditions: 2 ml
double-stranded states (enthalpic contribution). of a 2.8 mM decamer solution (single strand) were mixed with 2 ml buffer
Unlike 2A-deoxyribose sugars which lack a functionality for solution (10% 2-methylpentane-2,4-diol (MPD), 40 mM sodium cacodylate
hydrogen bond formation at the C2A-position, the locked sugars pH 6, 12 mM spermine tetrahydrochloride and 80 mM potassium chloride)
contain a hydrogen bond acceptor in the form of O2A (Fig. 1). In and equilibrated against 1 ml of a 35% (v/v) MPD reservoir solution. A
the crystal structure both 2A-oxygens are engaged in hydrogen crystal was mounted in a nylon loop and shock-frozen in liquid nitrogen.
bonds to water molecules (Fig. 3). Extensive hydration of Diffraction data were collected on the insertion device beamline (5-ID) of
the DuPont-Northwestern-Dow Collaborative Access Team at the Ad-
individual hydrogen bond acceptors and donors in oligonucleo- vanced Photon Source (APS), Argonne National Laboratory (Argonne, IL).
tides is often accompanied by an increased thermodynamic A total of 300 frames at high and low resolution ranges were recorded and
stability of the corresponding duplexes (see, for example, reflections were integrated and merged in the DENZO/SCALEPACK
references 19 and 20). However, it is difficult to draw suite.11 A summary of amount and quality of the data is given in Table 1.
conclusions as to the role of sugar hydration in the overall ¶ The structure of the LNA-modified decamer was determined by the
stability of LNA-modified DNA duplexes or all-LNA duplexes. Molecular Replacement method using an A-form search model and the
The arrangement of water molecules around locked sugars program AMORE.12 The initial model was refined with the program CNS13
observed here is reminiscent of the solvation in the case of the setting aside 10% of the reflections for calculating the free R-factor.14
2A-oxygen in 2A-O-methyl RNA.21,22 Duplexes of 2A-O-methyl- Standard bond lengths and angles constraints15 were employed for the DNA
portion of the model and the geometric parameters for LNA residues were
ated oligoribonucleotides exhibit thermodynamic stabilities that calculated with the program CHEM3D (CambridgeSoft Corporation,
are increased by about 1 °C per modified residue relative to Cambridge, MA). The individual duplex models and the resulting Fourier
RNA. However, the precise contribution of hydration to the electron density maps were visualized with the program TURBO-FRODO16
stability increase in the case of 2A-O-methyl RNA is not on Silicon Graphics computers. Final refinement parameters and average
understood. root mean square (r.m.s.) deviations for bonds and angles from standard
Our study provides a first look at the conformational values are listed in Table 1. CCDC 156032.
properties of LNA in a crystal structure at relatively high
resolution. The main characteristics of the structure are the 1 A. A. Koshkin, S. K. Singh, P. Nielsen, V. K. Rajwanshi, R. Kumar, M.
standard A-type conformation induced by LNA residues and the Meldgaard, C. E. Olsen and J. Wengel, Tetrahedron, 1998, 54, 3607.
2 J. Wengel, Acc. Chem. Res., 1999, 32, 301.
capacity of the 2A-oxygen that is part of the bicyclic sugar 3 S. Obika, D. Nanbu, Y. Hari, J. Andoh, K. Morio, T. Doi and T.
framework to engage in a least two hydrogen bonds to water Imanishi, Tetrahedron Lett., 1998, 39, 5401.
molecules. Circular dichroism spectra (CD) of LNA–LNA 4 S. K. Singh, P. Nielsen, A. A. Koshkin and J. Wengel, Chem. Commun.,
duplexes in solution indicated that such duplexes appear to 1998, 455.
adopt a conformation that closely resembles the A-form 5 S. K. Singh and J. Wengel, Chem. Commun., 1998, 1247.
geometry of RNA–RNA duplexes. However, these spectra also 6 C. Wahlestedt, P. Salmi, L. Good, J. Kela, T. Johnsson, T. Hökfelt, C.
manifested subtle differences between the two species (data not Broberger, F. Porreca, J. Lai, K. Ren, M. Ossipov, A. Koshkin, N.
shown). The present analysis of a duplex with only a single Jacobsen, J. Skouv, H. Oerum, M. H. Jacobsen and J. Wengel, Proc.
LNA residue per strand does not provide any insight into Natl. Acad. Sci. U.S.A., 2000, 97, 5633.
7 C. B. Nielsen, S. K. Singh, J. Wengel and J. P. Jacobsen, J. Biomol.
potential conformational differences between LNA and RNA Struct. Dyn., 1999, 17, 175.
duplexes. Attempts to determine a crystal structure of a 8 M. Petersen, C. B. Nielsen, K. E. Nielsen, G. A. Jensen, K.
completely modified LNA–LNA duplex are underway. Bondensgaard, S. K. Singh, V. K. Rajwanshi, A. K. Koshkin, B. M.
This work was supported by the NIH (GM-55237 to M. E.). Dahl, J. Wengel and J. P. Jacobsen, J. Mol. Recogn., 2000, 13, 44.
We thank the Danish Natural Science Research Council and the 9 K. E. Nielsen, S. K. Singh, J. Wengel and J. P. Jacobsen, Bioconj.
Danish Technical Research Council for financial support, Ms Chem., 2000, 11, 228.
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13 A. T. Brünger, Crystallography & NMR System (CNS), Version 0.9,
Yale University, New Haven, CT, 1998.
14 A. T. Brünger, Nature, 1992, 355, 472.
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Université Aix-Marseille II, Marseille, France, 1997.
17 C. Altona and M. Sundaralingam, J. Am. Chem. Soc., 1972, 94, 8205.
Fig. 3 The [TLpdA]2 base pair step viewed approximately along the helical 18 R. Lavery and H. Sklenar, J. Biomol. Struct. Dyn., 1989, 6, 655.
axis. Atoms of the upper and lower base pairs are shown in yellow and gray, 19 M. Egli, N. Usman and S. Portmann, Biochemistry, 1996, 32, 3221.
respectively. Atoms of the sugar–phosphate backbone are colored green, red 20 V. Tereshko, S. Gryaznov and M. Egli, J. Am. Chem. Soc., 1998, 120,
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6 (left) and 16 (right) are drawn as cyan spheres and hydrogen bonds are 22 D. A. Adamiak, J. Milecki, M. Popenda, R. W. Adamiak, Z. Dauter and
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652 Chem. Commun., 2001, 651–652