Efficient stabilization of oligonucleotide duplexes through terpyridine metal complexes

Article abstract of DOI:10.1139/V06-080

A large increase in the melting temperature of complementary oligonucleotides was obtained through the conjugation of terpyridine ligands at their nearby 5′ or 3′ ends and the addition of stoichiometric amounts of various transition metals. We describe he

Full text of DOI:10.1139/V06-080

854
Efficient stabilization of oligonucleotide duplexes
through terpyridine metal complexes
Fabrice Freville, Nathalie Pierre, and Serge Moreau
Abstract: A large increase in the melting temperature of complementary oligonucleotides was obtained through the
conjugation of terpyridine ligands at their nearby 5′ or 3′ ends and the addition of stoichiometric amounts of various
transition metals. We describe here a short efficient synthetic route for the conjugation of terpyridine moieties and re-
port new data on the stabilizing contribution of metal-assisted hybridization of oligonucleotides through UV-monitored
melting temperature experiments.
Key words: oligonucleotides, metal complexes, terpyridine.
Résumé : Une augmentation importante de la température de fusion d’oligonucléotides complémentaires peut être
obtenue par la conjugaison des ligands terpyridine à leurs extrémités adjacentes (5′ ou 3′) et l’addition de quantités
stœchiométriques de divers métaux de transition. On décrit ici une courte voie de synthèse efficace pour la conjugaison
des groupes terpyridine. On présente également de nouvelles données expérimentales sur l’effet stabilisant des métaux
dans l’hybridation des oligonucléotides par des mesures de température de fusion soumises en spectroscopie UV.
Mots clés : oligonucléotides, complexes métalliques, terpyridine. Freville et al. 858
Introduction
new monomer was derivatized as a phosphoramidite unit
and used in a solid-supported terpyridine linker. The whole
synthetic scheme is depicted in Fig. 1.
We previously reported a high stabilization of comple-
mentary DNA strands association through the addition of a
coordination link between the oligomers (1). Such a link was
obtained using terpyridine moieties as chelating agents and
serinol as a deoxyribose mimic to allow easy conjugation of
the chelator to oligonucleotides at nearby 3′ and 5′ ends.
We report here a new way to access the efficient conjuga-
tion of terpyridine moieties on both 3′ and 5′ ends of oligo-
nucleotides. To allow structural studies, we chose to use a
pantolactone-based ribose mimic developed by Dioubankova
et al. (2). This synthetic route was used to extend our studies
on metal-assisted hybridization of oligonucleotides, provid-
ing an easy way to increase nucleic acid duplex stability.
Pantolactone 1 was treated with an excess of 1,3 diamino-
propane. The side chain aminogroup was transiently pro-
tected through trifluoroacetylation. The primary hydroxyl
group was then selectively tritylated using 4,4′-dimethoxy-
trityl chloride, yielding compound 3. 3 was purified by silica
gel flash chromatography using a dichloromethane – ethyl
acetate gradient. The overall yield from 1 was 68%. The N-
protecting group was then removed by sodium carbonate
treatment leading to 4 (yield of 74%). The terpyridine mono-
mer 5 was obtained by the reaction with the N-hydroxy-
succinimide ester 9 in high yield (82%), after chromatography
over aluminum oxide using an ethyl acetate – hexane gradi-
ent. This monomer 5 was used either to synthesize the
phosphoramidite 6 through phosphitylation with 2-cyano-
ethyl-N,N-diisopropylchloro phosphoramidite or to yield a
solid-supported reagent 7 by reaction with succinylated
LCAA-CPG. 6 was purified by reverse-phase chromatogra-
phy and elution with neat acetonitrile (yield 43%). The over-
all yield from 1 was 17%. The solid-supported reagent 7 was
obtained by known procedure (3). The loading range of the
CPG reagent was found to be 25–35 µmol/g. The terpyridine
moiety 8 was synthesized according to published procedures
(4) and the N-hydroxysucciimide ester 9 was obtained in
high yield (93%).
Results
We chose to use two short complementary DNA sequences
to evaluate the contribution of the metallic bridge on duplex
stability. This data was obtained by UV-thermal denaturation
experiments. The introduction of the terpyridine chelators at
nearby 5′ and 3′ ends required the synthesis of a phosphor-
amidite monomer bearing the terpyridine moiety and a spe-
cific solid-supported reagent for 3′ conjugation.
Chemical synthesis
Taking into account the strategy develop by Dioubankova
et al., we designed a new synthetic route involving panto-
lactone as a precursor for a nonnucleosidic monomer. The
Duplex formation
To evaluate the stability of various metallic bridges be-
Received 30 November 2005. Published on the NRC Research Press Web site at http://canjchem.nrc.ca on 7 June 2006.
F. Freville, N. Pierre, and S. Moreau.1 INSERM U386, Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, F33076
Bordeaux, CEDEX, France.
1Corresponding author (email: serge.moreau@bordeaux.inserm.fr).
Can. J. Chem. 84: 854–858 (2006)
doi:10.1139/V06-080
© 2006 NRC Canada

Freville et al.
855
Fig. 1. Chemical synthetic pathway for the various monomer units.
tween complementary oligomers, we designed specific
strand combinations. Oligonucleotide sequences are gathered
in Table 1, together with the MS analysis of TPY-
conjugated strands by MALDI-Q-TOF in negative mode.
We thus confirmed the chemical structure of modified oligo-
mers with the monoisotopic peaks in the MS analysis. Using
the previously mentioned linkers, the terpyridine chelator
was introduced into the 3′ or 5′ ends of complementary
© 2006 NRC Canada

856
Can. J. Chem. Vol. 84, 2006
Table 1. Sequences of the duplexes used in the melting temperature experiments.
No.
I
Duplex sequences
Calcd. (m/z)
Expt. (M – H)– (m/z)
5′ CCTTTCTTG 3′
3′ GGAAAGAAC 5′
5′ CCTTTCTTG-TPY 3′
3′ GGAAAGAAC 5′
5′ CCTTTCTTG 3′
3′ GGAAAGAAC-TPY 5′
5′ CCTTTCTTG-TPY 3′
3′ GGAAAGAAC-TPY 5′
5′ CCTTTCTTG-TPY 3′
3′ TPY-GGAAAGAAC 5′
II
3237.68
3362.76
3236.68
3361.75
III
IV
V
3362.76
3361.75
Note: TPY accounts for the terpyridine ligand. Columns 3 and 4 show the results of MS analysis (MALDI-
Q-TOF, negative mode) of TPY-conjugated strands, calculated and experimental data, respectively.
Table 2. Effect of terpyridine ligands on values of Tm of oligonucleotide duplexes.
Tma (∆Tm)b
Duplexes
EDTA
Zn2+
Fe2+
Co2+
Ni2+
Cu2+
I
II
III
IV
V
26.5
30.6
32.0
37.0
34.5
26.4
30.0
33.0
61.8 (35.3)
34.0
26.4
32.3
32.6
63.5 (37)
34.4
Ndc
Nd
Nd
63.6 (37.1)
Nd
Nd
Nd
Nd
65.2 (38.7)
Nd
Nd
Nd
Nd
57.5 (31)
Nd
Note: Oligomers concentrations are 1 µmol/L.
aMelting temperature (Tm, °C, 1 °C) (mean values from at least three experiments).
b∆Tm: Tm duplex IV – Tm duplex I (∆Tm indicated inside parentheses).
cNd: not determined.
strands. Five different duplexes were studied, allowing com-
parisons between unmodified double-stranded oligomers and
either mono- or bis-terpyridine conjugated oligonucleotides.
Thermal denaturation experiments were conducted in two
different buffers. Data from nonmetalated species were ob-
tained in a buffer containing EDTA to avoid contamination
from traces of metal ions. As can be deduced from Table 2,
the addition of at least 1 equiv. of Zn2+ ions (relative to du-
plex concentration) to the terpyridine-containing duplex IV
resulted in a large increase in Tm (∆Tm = + 35.3 °C) in
comparison with that of the unmodified duplex I. All other
combinations of terpyridines moieties did not produce this
stabilizing contribution. Duplexes II and III, although reveal-
ing some slight stabilization compared with I, did not reveal
any stabilizing effect when compared with the data obtained
from an unmetallated incubation medium (see column 2, Ta-
ble 2). The presence of the two nearby terpyridine ligands is
a prerequisite for the stabilization through added metal ions;
duplex V, which exhibits noncontiguous terpyridine ligands,
was not stabilized. The expected stoichiometry of the liga-
tion reaction was obtained from a titration experiment with
Zn2+ ions using the specific absorption band (320 nm) of the
metalated terpyridine determined in a previous study
(1) (Fig. 2).
Fig. 2. Titration experiment showing the variation of the
absorbance at 320 nm as a function of added ZnCl2 (equiv.
refers to the duplex concentration (1 µmol/L)).
Table 2 also showed data from various chelating metals.
All produced large increases in the Tm values of metalated
duplexes; ∆Tm from the unmodified duplex I ranged from
31 to 38.7 °C. The observed variations in the stabilizing con-
tributions of the various chelated metals might arise from the
coordination properties of the metals. All are known to form
octahedral terpyridine complexes (5), however, other coordi-
nation numbers of 4 and 5 are also observed for copper (6,
Data from the nonmetalated buffer revealed a slight con-
tribution from the terminal terpyridine in the stabilization of
the duplexes (see column 2, Table 2). This stabilizing effect
might arise from the formation of stacked aromatic pyridines
on double-stranded oligonucleotide ends. The intercalation
abilities of the terpyridine nucleus was discarded from previ-
ously observed data.
© 2006 NRC Canada

Freville et al.
857
7) and zinc–nickel complexes (8, 9). We cannot, therefore,
exclude the occurrence of different coordination patterns in
the metal-assisted hybridization of the oligonucleotides with
coordination links between the nearby terpyridines varying
from one to three.
(CH2NHCOCF3) 35.4 (CH2NHCO), 29.0 (CH2CH2CH2),
22.0 (CH3a), 20.9 (CH3b).
Compound 4
Compound 3 (3 g, 5.9 mmol) was dissolved in MeOH
(40 mL), then an aqueous solution of Na2CO3 (10%, 11 mL)
was added. The reaction mixture was stirred overnight at
65 °C, evaporated, diluted with 200 mL of EtOAc, washed
with 1.5% NaHCO3 (3 × 150 mL), then dried (Na2SO4),
evaporated, and co-evaporated with toluene (3 × 30 mL) and
CH2Cl2 (2 × 30 mL) to yield 2.2 g of 4 (74% yield). This
Experimental section
General
Thin layer chromatography was performed on Merck sil-
ica gel 60F254 aluminum-backed plates. Flash chromatogra-
phy refers to column chromatography performed with a
Biotage system. NMR spectra were recorded with a Bruker
1
compound was used without further purification. H NMR
(300 MHz, CDCl3, ppm) δ: 7.36–6.82 (m, 14H, HDMTr and
3
1
NHCO), JNH−CH = 6 Hz), 4.08 (s, 1H, CHOH), 3.78 (s,
Avance 300 spectrometer working at 300 MHz for H, 75.45
2
6H, OCH3), 3.16 (m, 3H, CH2NHCO and CH2aODMTr),
for 13C, and 121.49 for 31P. The chemical shifts are ex-
3
1
3.01 (d, 1H, CH2bODMTr), 2.62 (t, 2H, CH2NH2, JH–H
=
pressed in ppm using TMS as internal standard (for H and
6.6 Hz), 1.57 (m, 2H, CH2CH2CH2), 1.12 (s, 3H, CH3a),
1.02 (s, 3H, CH3b). 13C NMR (75.45 MHz, CDCl3, ppm) δ:
172.4 (CONH), 158.6 (COCH3), 144.1–113.4 (7C, DMTr),
86.4 (OCDMTr), 77.1 (CHOH), 70.8 (CH2ODMTr), 55.2
(OCH3), 39.3 (CH2NH2), 39.0 (C(CH3)2), 36.4 (CH2NHCO),
33.0 (CH2CH2CH2), 22.0 (CH3a), 20.9 (CH3b).
13C NMR data) and 85% H3PO4 as external standard for
31P NMR data. Mass spectra (MALDI-Q-TOF, negative
mode) were recorded on a Waters Ultima spectrometer. A
special preparation technique was designed to analyse the
TPY-modified oligonucleotides. The stainless steel target
was first covered with a thin layer of matrix (0.5 µL of
2,4,6-trihydroxyacetophenone (THAP), 10 mg/mL in ace-
tone) and this layer was washed with a saturated EDTA solu-
tion (2 × 1 µL) and water (2 × 1 µL). The oligonucleotides
(0.5 µL of a 20 µmol/L solution in water) were then depos-
ited on this matrix layer and allowed to dry. The final prepa-
ration step was the application of a second layer of matrix
(0.5 µL of a 1:1 solution of THAP (30 mg/mL in ethanol)
and 100 mmol/L aqueous ammonium citrate) that was al-
lowed to dry at room temperature.
Compound 5
4 was carefully dried by three co-evaporations with
pyridine and then dissolved in anhydrous pyridine. The NHS
ester 9 was added and the solution stirred at RT and moni-
tored by TLC. The reaction medium was evaporated and
purified by chromatography over aluminum oxide with a
gradient of ethyl acetate in hexane (50%–80%) in the pres-
1
ence of 0.25% triethylamine (1.35 g, yield 82%). H NMR
3
(300 MHz, CDCl3, ppm) δ: 8.68 (ddd, 2H, Jortho = 4.8 Hz,
5
4Jmeta = 1.8 Hz, and Jpara = 0.9 Hz, H6 and H6′′), 8.62 (ddd,
Chemical synthesis
3
4
5
2H, Jortho = 7.5 Hz, Jmeta = 1.0 Hz and Jpara = 0.9 Hz, H3
and H3′′ ), 8.02 (s, 2H, H3′ and H5′), 7.85 (ddd, 2H,
Compound 3
3Jortho = 7.8 Hz, Jortho = 7.5 Hz, and Jmeta = 1.5 Hz, H4 and
3
4
A solution of (R)-(–)-pantolactone (1 g, 7.68 mmol) and
1,3-diaminopropan (1.67 mL, 38.42 mmol) in 6 mL EtOH
was kept at 55 °C for 48 h, to which was added 13.7 mL
(115.3 mmol) of ethyl trifluoroacetate. The mixture was kept
at room temperature overnight, evaporated, then co-evaporated
with toluene and dry pyridine. The residue was dissolved in
dry pyridine (40 mL), cooled in an ice bath, and 4,4′-
dimethoxytrityl chloride (2.86 g, 8.45 mmol) was slowly
added. The reaction was stopped after 2 h by the addition of
0.5 mL of water and then diluted with 200 mL of CHCl3.
The organic phase was washed with 2.5% NaHCO3 (3 ×
60 mL), water (2 × 60 mL), and 20% NaCl (60 mL), then
dried (MgSO4). The residue was purified by flash chroma-
tography using a stepwise gradient of EtOAC (0%–15%) in
CH2Cl2–Et3N (0.25%) to yield 3 as a colorless oil (3.14 g,
H4′′ ), 7.41–6.85 (m, 15H, HDMTr, H5 and H5′), 6.64 (t, 1H,
3
3JH–H = 6 Hz, NHCO), 6.52 (t, 1H, JH–H = 5.7 Hz, NHCO),
3
4.29 (t, 2H, JH–H = 5.9 Hz, CH2CH2O), 4.09 (s, 1H,
CHOH), 3.80 (s, 6H, CH3O), 3.19 (m, 5H, CH2NHCO and
CH2aODMTr), 3.02 (d, 1H, CH2bODMTr), 2.45 (t, 2H,
3JH–H = 7.2 Hz, CH2CO), 2.22 (m, 2H, NHCH2CH2CH2NH),
1.50 (m, 2H, CH2CH2CH2), 1.07 (s, 3H, CH3a), 1.00 (s, 3H,
CH3b). 13C NMR (75.45 MHz, CDCl3, ppm) δ: 172.8
(CONH), 172.2 (CONH), 166.9 (C4′), 158.5 (COCH3),
156.9 (C2′, C6′), 155.9 (C2,C2′′ ), 148.9 (C6, C6′′ ), 144.3–
113.2 (12C, DMTr), 136.6 (C4, C4′′ ), 123.7 (C5, C5′′ ),
121.2 (C3, C3′′ ), 107.4 (C3′, C5′), 86.4 (OCDMTr), 77.3
(CHOH), 70.8 (CH2ODMTr), 66.2 (OCH2), 55.2 (OCH3),
38.8 (C(CH3)2), 35.8 (CH2NHCO) 35.6 (CH2NHCO), 32.8
(OCH2CH2CH2CONH), 29.6 (NHCH2CH2CH2NH), 25.1
(NHCH2CH2CH2O), 22.0 (CH3a), 20.9 (CH3b). MS (FAB–)
calcd.: 823.97; found: 822 (M – H)–.
1
68%). H NMR (300 MHz, CDCl3, ppm) δ: 8.03 (t, 1H,
NHCOCF3), 7.36–6.82 (m, 13H, HDMTr), 6.66 (t, 1H,
3
NHCOCH(OH), JNH−CH = 6 Hz), 4.07 (s, 1H, CHOH),
3.77 (s, 6H, OCH3), 3.272(m, 2H, CH2NHCOCF3), 3.16 (m,
3H, CH2NHCO and CH2aODMTr), 3.01 (d, 1H,
CH2bODMTr), 1.57 (m, 2H, CH2CH2CH2), 1.12 (s, 3H,
CH3a), 1.02 (s, 3H, CH3b). 13C NMR (75.45 MHz, CDCl3,
ppm) δ: 173.8 (CONH), 158.7 (COCH3), 157.5 (q, COCF3,
2JC–F = 36.5 Hz), 144.1–113.4 (7C, DMTr), 116.0 (q,
COCF3, 1JC–F = 285.9 Hz), 86.8 (OCDMTr), 77.8 (CHOH),
71.2 (CH2ODMTr), 55.3 (OCH3), 38.9 (C(CH3)2), 36.0
Compound 6
5 (1 g, 1.2 mmol) was dissolved in anhydr. CH2Cl2
(10 mL). Diisopropylethylamine (520 µL, 3 mmol) and
2-cyanoethyl diisopropylchlorophosphoramidite (1.8 mmol,
430 mg) were then added to the previous solution. The reac-
tion was stopped after 1 h at RT with 500 mL of CH3OH
and diluted with 100 mL of EtOAc, washed with 2.5%
© 2006 NRC Canada

858
Can. J. Chem. Vol. 84, 2006
NaHCO3 (200 mL), and then dried over Na2SO4. The resi-
due was purified over reverse phase silica gel (Lichroprep
RP-18 (40–63 µmol/L)) and eluted with CH3CN–Et3N
(0.25%) to yield 534 mg of solid 5 (43%). 1H NMR
was stirred at 40 °C overnight. The crude mixture was con-
centrated and crystallization from methanol yielded 9
(2.17 g, 93%).
3
(300 MHz, CDCl3, ppm) δ: 8.67 (d, 2H, Jortho = 4.2 Hz, H6
Oligonucleotide synthesis
3
All oligonucleotides were synthesized on a 0.2 µmol scale
with a Millipore Expedite 9809 synthesizer using conven-
tional β-cyanoethyl phosphoramidite chemistry. The standard
bases were dissolved in anhydrous acetonitrile (0.1 mol/L fi-
nal concentrations). The modified phosphoramidites were
dissolved in anhydrous acetonitrile and coupled manually
with a coupling time of 15 min. The coupling efficiency was
the same as that of unmodified amidites. All oligomers were
synthesized “trityl off”. The solid support was treated over-
night at 55 °C with fresh concd. NH4OH (1 mL). The crude
oligomers were purified by electrophoresis on denaturated
polyacrylamide gels (20%) and visualized by UV shadow-
ing. The pure samples were desalted through reverse-phase
set-pak cartridges (C18, Waters)
and H6′′ ), 8.60 (d, 2H, Jortho = 7.8 Hz, H3 and H3′′ ), 8.01
3
(s, 2H, H3′ and H5′), 7.85 (td, 2H, Jortho = 7.5 Hz, and
4Jmeta = 0.9 Hz, H4 and H4′′ ), 7.41–6.85 (m, 15H, HDMTr
,
3
H5 and H5′′ ), 6.56 (t, 1H, JH–H = 6.0 Hz, NHCO), 6.43 (t,
3
3
1H, JH–H = 5.7 Hz, NHCO), 4.28 (t, 2H, JH–H = 6.0 Hz,
2
COCH2CH2CH2O), 4.09 (d, 1H, JH–P = 12.6 Hz, CHOP),
3.78 (s, 6H, CH3O), 3.70–3.4 (m, 4H, CH2NHCO), 3.4–2.9
(m, 6H, CH2ODMTr, CH2CH2OP, NCH), 2.43 (m, 4H,
OCH2CH2CH2CO, POCH2CH2CN), 2.21 (m, 2H,
NHCH2CH2CH2NH), 1.55 (m, 2H, OCH2CH2CH2CO), 1.11
(m, 15H, CH3), 1.00 (s, 3H, CH3b). 13C NMR (75 MHz,
CDCl3, ppm) δ: 171.4 (CONH), 170.0 (CONH), 167.0
(C4′), 158.6 (COCH3), 157.1 (C2′, C6′), 156.1 (C2, C2′′ ),
149.0 (C6, C6′′ ), 145.2–112.9 (7C, DMTr), 136.7 (C4,
C4′′ ), 123.8 (C5, C5′′ ), 121.3 (C3, C3′′ ), 117.9 (CH2CN),
107.4 (C3′, C5′), 85.8 (OCDMTr), 77.3 (CHOP), 70.8
(CH2ODMTr), 67.3 (OCH2CH2CH2CO), 58.2 (NCCH2CH2OP),
55.2 (OCH3), 43.4 (NCH), 38.8 (C(CH3)2), 35.9
(CH2NHCO) 35.7 (CH2NHCO), 32.8 (OCH2CH2CH2CONH),
29.6 (NHCH2CH2CH2NH), 25.1 (COCH2CH2CH2O), 24.6
(NCH(CH3)2), 22.7 and 22.3 (C(CH3)2), 20.4 (CH2CN). 31P
NMR (120 MHz, CDCl3, ppm) δ: 156.0, 152.5. MS (FAB–)
calcd.: 1023.19 (M – H)–; found: 1022 (M – H)–.
UV-monitored melting experiments
Purified oligonucleotides (each strand was 1 µmol/L)
were diluted in 200 mL of the appropriate buffer. The mix-
ture was boiled for 2 min and the hybridization was assured
by low-temperature cooling of the sample. Melting experi-
ments were performed with a Cary 1E UV–vis spectro-
photometer with a temperature controller unit. Samples were
kept at 5 °C for at least 30 min and then heated from 4 to
90 °C at a rate of 0.4 °C/min. The absorbance (λ = 260 nm)
was measured every minute. The melting temperature was
determined from the maxima of the first derivative. Two dif-
ferent buffers were used. The studies on the metalated spe-
cies were performed in a buffer containing 10 mmol/L
sodium phosphate (10 mmol/L, pH 7) and sodium chloride
(150 mmol/L). The metal-free experiments were measured in
an EDTA-containing buffer: EDTA (200 mmol/L), sodium
phosphate (10 mmol/L, pH 7), and sodium chloride
(150 mmol/L).
Compound 7
A solution of succinic anhydride (300 mg, 3 mmol) and
DMAP (80 mg, 0.66 mmol) in 10 mL of dry pyridine was
added to 3000 mg of LCAA-CPG (500 Å) and the mixture
was left at RT for 24 h with occasional swirling. After filtra-
tion, successive washes with 10 mL portions of pyridine,
CH2Cl2, Et2O, and drying, the succinylated LCAA-CPG was
suspended in 4 mL of DMF pyridine (1:1 v/v); compound 5
(206 mg, 0.25 mmol), 1,3-diisopropylcarbodiimide (0.28 mL,
1.8 mmol)), and DMAP (20 mg) were added. The suspen-
sion was left for 48 h at RT. To block the remaining
carboxylic groups, a solution of pentachlorophenol (100 mg)
was added and the mixture was kept at RT for further 12 h.
The support was filtered, resuspended in 5% v/v solution of
piperidine (3 mL), reacted for 10 min, filtered again and
washed successively with 10 mL portions of CHCl3, CH3OH,
CH3CN, and dried in vacuo. Loading was determined by
treating a portion (5 mg) of 7 with 1 mL of 3% w/v
CCl3CO2H in 1,2-dichloroethane and measuring the absorb-
ance of DMTr cation at 504 nm (ε = 75 000 L mol–1 cm–1).
Loading found: 25–35 µmol/g.
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Compound 9
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formamide (40 mL), and N,N-diisopropylcarbodiimide
(1.1 equiv., 5.9 mmol, 923 mL) and N-hydroxysuccinimide
(1.1 equiv., 5.9 mmol, 678 mg) were added. The reaction
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© 2006 NRC Canada