Solution structure of a DNA double helix with consecutive metal-mediated base pairs

Article abstract of DOI:10.1038/nchem.512

Metal-mediated base pairs represent a powerful tool for the site-specific functionalization of nucleic acids with metal ions. The development of applications of the metal-modified nucleic acids will depend on the availability of structural information on these double helices. We present here the NMR solution structure of a self-complementary DNA oligonucleotide with three consecutive imidazole nucleotides in its centre. In the absence of transition-metal ions, a hairpin structure is adopted with the artificial nucleotides forming the loop. In the presence of Ag(i) ions, a duplex comprising three imidazoleg-Ag⁺ g-imidazole base pairs is formed. Direct proof for the formation of metal-mediated base pairs was obtained from ¹J(¹⁵ N, ^107/109 Ag) couplings upon incorporation of ¹⁵N-labelled imidazole. The duplex adopts a B-type conformation with only minor deviations in the region of the artificial bases. This work represents the first structural characterization of a metal-modified nucleic acid with a continuous stretch of metal-mediated base pairs.

Full text of DOI:10.1038/nchem.512

ARTICLES
PUBLISHED ONLINE: 17 JANUARY 2010 | DOI: 10.1038/NCHEM.512
Solution structure of a DNA double helix with
consecutive metal-mediated base pairs
1
2
2
1
2
Silke Johannsen , Nicole Megger , Dominik B o¨ hme , Roland K. O. Sigel and Jens M u¨ ller
*
*
Metal-mediated base pairs represent a powerful tool for the site-specific functionalization of nucleic acids with metal ions.
The development of applications of the metal-modified nucleic acids will depend on the availability of structural information
on these double helices. We present here the NMR solution structure of a self-complementary DNA oligonucleotide with
three consecutive imidazole nucleotides in its centre. In the absence of transition-metal ions, a hairpin structure is adopted
1
with the artificial nucleotides forming the loop. In the presence of Ag(I) ions, a duplex comprising three imidazole–Ag –
imidazole base pairs is formed. Direct proof for the formation of metal-mediated base pairs was obtained from
1
15 107/109
15
J( N,
Ag) couplings upon incorporation of N-labelled imidazole. The duplex adopts a B-type conformation with only
minor deviations in the region of the artificial bases. This work represents the first structural characterization of a metal-
modified nucleic acid with a continuous stretch of metal-mediated base pairs.
ucleic acids such as DNA are becoming increasingly popular destabilization of the B-conformation and a concomitant stabilization
as versatile building blocks in nanobiotechnology as a result of the Z-conformation. The GNA duplex on the other hand adopts a
N
of their superb, predictable self-assembling properties and conformation that is entirely different from the canonical A-, B- or
the high rigidity of their double helices on the nanoscale . Their Z-forms and is largely a result of the unnatural backbone .
applicability can be extended even further by the introduction of To fill the gap and gain structural insight into a nucleic acid
1
22
functional groups such as metal ions. One recently established double helix that actually contains metal ions along its helical axis
method for the site-specific functionalization of nucleic acids with (as shown in most cartoons representing metal-mediated base
2
–5
metal ions is based on the use of metal-mediated base pairs
.
pairs), we here determined the first structure of an oligonucleotide
Such base pairs comprise natural or artificial nucleobases and rely double helix that contains three consecutive Ag(I)-mediated base
on coordinative bonds to a central metal ion instead of (or in pairs. In this paper, we describe the solution structure of the result-
addition to) hydrogen bonds. Depending on the choice of nucleo- ing DNA double helix as determined by NMR spectroscopy.
sides, metal ions and oligonucleotide sequence, a plethora of
metal-modified double helices can be generated. Even if focusing Results
only on the use of artificial nucleosides, examples exist for DNA The modified DNA adopts a duplex structure in the presence
duplexes containing one or two metal-mediated base pairs inter- of Ag(I). For our structural investigations we used a self-
6–8
spersed between natural ones , DNA double helices with continu- complementary sequence with three consecutive artificial imidazole
ous stretches of metallated base pairs9 and DNA duplexes with nucleosides (Im) in its centre (Fig. 1). As was known from previous
–12
1
3
17,23,24
different metal-mediated base pairs at pre-defined positions . studies
When also including metal-mediated base pairs from natural or a duplex structure. However, it is difficult to differentiate
, such a sequence can in principle adopt either a hairpin
nucleosides1 , even more combinations can be envisaged.
4–16
unambiguously between a hairpin structure and a regular double
1
7
1
In addition to DNA, other nucleic acids such as RNA , GNA helix by simple [ H]-NMR experiments. To ensure a correct
19,20
(
glycol nucleic acid)18 or PNA (peptide nucleic acid)
have also assignment, we determined the solution structure of the modified
been modified with metal-mediated base pairs. Experimentally DNA both in the absence and in the presence of Ag(I) ions. The
derived structural information on such metal-modified nucleic quality of the spectra tremendously improved with the addition of
þ
acids is very scarce: only one DNA double helix comprising two Ag , which is especially true for the resonances of the imidazole
non-neighbouring metal-mediated base pairs has been structurally ligands (Fig. 2). Indeed, entirely different conformations were found,
21
characterized so far . In addition, the structure of one metal- namely a hairpin in the absence and a duplex in the presence of the
22
containing GNA duplex is known . Neither of these contains a transition-metal ions. The determination of the hairpin structure
continuous stretch of metallated base pairs despite the enormous that is adopted in the absence of Ag(I) ions (Supplementary Fig. S1)
interest in this type of modification. Interestingly, neither of the is described in detail in the Supplementary Information.
helices adopts the canonical B-type structure either. The DNA
In the presence of one equivalent of Ag(I) ions—that is, the
duplex containing two pyridine-2,6-dicarboxylate–Cu2 –pyridine amount needed to insert one Ag(I) ion into every possible metal-
þ
base pairs crystallizes in the Z-conformation, probably as a result mediated base pair—the DNA adopts a duplex structure. The
2
þ
1
1
of the propensity of Cu ions to bind additional axial ligands proton resonances of the duplex system were assigned by [ H, H]-
0
(
here, furanose O4 of a neighbouring thymidine and O6 of a neigh- nuclear Overhauser enhancement spectroscopy (NOESY) on the
21
0
bouring guanine) . The structural distortion that is necessary to basis of the sequential walks along the H1 and aromatic protons.
place these ligands above and below the square planar Cu2 coordi- Three additional regions (H2 /aromatic, H2 /aromatic and
þ
0
00
nation environment provided by the artificial nucleosides leads to a aromatic/aromatic) served to cross-validate the assignments. The
1
2
Institute of Inorganic Chemistry, University of Z u¨ rich, Winterthurerstr. 190, 8057 Z u¨ rich, Switzerland, Westf a¨ lische Wilhelms-Universit a¨ t M u¨ nster, Institut
f u¨ r Anorganische und Analytische Chemie, Corrensstr. 28/30, 48149 M u¨ nster, Germany. *e-mail: roland.sigel@aci.uzh.ch; mueller.j@uni-muenster.de
NATURE CHEMISTRY | VOL 2 | MARCH 2010 | www.nature.com/naturechemistry
229
©
2010 Macmillan Publishers Limited. All rights reserved.

NATURE CHEMISTRY DOI: 10.1038/NCHEM.512
ARTICLES
a
5'
T
T
A
A
T
T
T
3'
A
A
T
T
A
A
A
5'
T
T
A
A
T
T
T
3'
A
A
T
imidazole base pairs with the N3 nitrogen atoms coordinating the
Ag(I) ions (Fig. 1c). As a result of the palindromic sequence and
1
2
17
1
the C symmetry of the double helix, resonances of 17 nucleotides
2
+3 Ag+
T
(that is, exactly half the total number) can be observed and assigned.
Interestingly, all H2 and H4 proton resonances of the imidazole
moieties display a remarkable upfield shift of 0.40–1.08 ppm on
metallation (Supplementary Table S2), which is indicative of
increased stacking interactions.
A
A
A
+
Im Im
Im
Im Ag Im
Im Ag Im
Im Ag Im
A
A
A
T
T
A
A
+
+
The resonances of the nucleotides neighbouring the imidazole
moieties show slight changes in chemical shift compared with the
hairpin, whereas the helical nucleotides display a very similar
b
T
T
T
A
A
T
T
5'
4
N
3
–O
O
5
P
1
N
Im =
2
31
pattern. All P NMR resonances were found to be in the region
O
O
25
typical for a regular B-helical structure . The hydrodynamic radius
1
7
rH as determined by diffusion-ordered spectroscopy (DOSY) and
dynamic light scattering amounts to 1.9+0.3 nm and 1.8+0.2 nm,
respectively. These values are significantly larger than the ones deter-
O
3
'
c
þ
+
Ag+
N
mined for the hairpin in the absence of Ag ions (Supplementary
Im Ag Im =
N
N
N
Table S1) and match perfectly the theoretical radius (1.81 nm),
which was calculated on the basis of the assumption of a double
helical structure again confirming duplex structure formation.
R
R'
Figure 1 | Description of the oligonucleotide system under investigation.
a, Schematic depiction of the hairpin-to-duplex transition of the
oligonucleotide. The nucleotide numbering scheme used throughout this
Ag(I)-mediated base pairs are formed in the DNA double helix.
þ
publication is colour-coded with the helical regions in green, the imidazole
To provide final proof that imidazole–Ag –imidazole base pairs
þ
15
nucleotides in gold and the Ag ions in blue (A ¼ adenine, T ¼ thymine,
are formed, we synthesized N-labelled imidazole nucleosides
1
15
Im ¼ imidazole). b, Chemical structure of the artificial imidazole nucleoside,
including atom numbering scheme. c, Chemical structure of a Ag(I)-
mediated base pair.
and incorporated these into the oligonucleotide. The [ H, N]
heteronuclear single quantum coherence (HSQC) spectra of the
oligonucleotide in the absence and presence of Ag(I) are shown
in Fig. 2 (bottom left and right panels, respectively). Prior to the
sequential walk can be followed throughout the entire oligonucleo- addition of the transition-metal ions, only a few cross peaks that
tide sequence including the central artificial imidazole nucleotides are not very well resolved are visible, indicating the presence of a
(
Fig. 2, top right). The intense and sharp resonances belonging to rather unstructured hairpin loop. The 15N chemical shifts cluster
these moieties indicate a rigid structure around the imidazole around 188 ppm for the N1 atoms (involved in the glycosidic
þ
nucleotides as is expected for the formation of imidazole–Ag – bonds) and 221 ppm for the N3 atoms (Supplementary Table S2).
8
.0
7.5
7.0
6.5
6.0
8.0
7.5
7.0
6.5
Im10
6.0
5.4
5.6
5.8
6.0
6.2
6.4
5.4
5.6
5.8
6.0
6.2
6.4
T2
Im9
T15
T2
T15
T5
Im8
T7
A11
A16
T14
A11 A12
A16
A3
T14
A12
A13
T5
T1
T1
T6
T7
A3
T6
A17
A13
A4
A17
A4
N1 {
N3 {
{
{
N1
1
2
2
2
90
00
10
20
190
200
210
220
4
3
5
N
1
N
R
2
N3
8
.0
7.5
7.0
6.5
6.0
8.0
7.5
7.0
δ [ H] (ppm)
6.5
6.0
1
1
δ [ H] (ppm)
1
1
1
15
Figure 2 | Comparison of sections of the [ H, H]-NOESY and [ H, N]-HSQC spectra of the hairpin (left) and metallated helix (right). It can be seen that
1
1
more and sharper resonances are visible for the metallated duplex compared to the hairpin. Top: the sequential walk through the [ H, H]-NOESY spectra is
indicated by solid and dotted lines. Blue, green and red lines represent nucleotides 1–7 (prior to the artificial nucleotides), 8–10 (artificial nucleotides) and
1
1–17 (after the artificial nucleotides), respectively. The labels represent the bases using the numbering scheme introduced in Fig. 1 (A ¼ adenine, T ¼ thymine,
Im ¼ imidazole). A sequential walk can only be followed in regions adopting a helical structure. Thus, a comparison of the left and right panels clearly shows
1
15
that the imidazole nucleotides are included within the helical structure only on formation of metal-mediated base pairs. Bottom: sections of the [ H, N]-
HSQC spectra show the upfield shift of the endocyclic imidazole nitrogen atoms N1 and N3 (for atom numbering scheme, see right panel) that is indicative of
the formation of Ag(I)-mediated base pairs. The black dotted vertical lines interconnect cross peaks of identical protons within the different panels.
230
NATURE CHEMISTRY | VOL 2 | MARCH 2010 | www.nature.com/naturechemistry
2010 Macmillan Publishers Limited. All rights reserved.
©

NATURE CHEMISTRY DOI: 10.1038/NCHEM.512
ARTICLES
a
180
As a result of the C symmetry of the double helix, only three dis-
2
tinguishable imidazole N3 atoms are observed per duplex, which all
show the same coupling within the resolution limits. It was not poss-
δ [ H] (Hz×10–3)
1
5
.10
5.05
1
15 107
1 15 109
ible to differentiate between the J( N, Ag) and J( N, Ag)
1
90
1
1
4.5
1
5
couplings because of heavy overlap of the N3 signals in the [ N]
insensitive nuclei enhancement by polarization transfer (INEPT)
spectrum (Supplementary Fig. S2) and because of relatively large
4.6
200
1
15
line widths of the N3 signals of 17 Hz in the [ H, N]-HSQC spec-
trum. However, from the large linewidth it is obviousthat both coup-
lings must be present because the N1 signals display a width of only
5
.10
5.05
210
7
Hz (Fig. 3a). One can assume that the observed 86 Hz corresponds
8
.0
7.5
δ [ H] (ppm)
7.0
1
15 107
1 15 109
1
to an average value of J( N, Ag) and J( N, Ag) because of
the almost identical natural abundance of 1 Ag and 109Ag (51.8%
versus 48.2%). Hence the individual coupling constants can be calcu-
lated based on the gyromagnetic ratios that differ by only 15%
07
b
c
3
+
N3
N
Ag
1
N
1
1
09
107
1 15 107
N
(g( Ag)/g( Ag) ¼ 1.15) (ref. 27), yielding J( N, Ag) ꢀ
R
Rʹ
1 15 109
1J
80 Hz and
J( N, Ag) ꢀ 92 Hz (ref. 28). In general, the
N,Ag
1
15 107/109
J( N,
Ag) value of 86 Hz represents the largest coupling (by
about 20 Hz) of this type reported as yet either in solution or the
2
8–30
solid state
.
The solution structure of the resulting double helix is shown in
Fig. 4. The structure was determined based on 848 conformationally
restrictive nuclear Overhauser enhancement (NOE) distance
restraints (Table 1). During structure refinement a non-crystallo-
graphic symmetry term was applied to accomplish perfect C sym-
2
metry as based on the NMR spectra (see above). The overall root
mean square deviation (r.m.s.d.) of all heavy atoms from the 20
lowest energy structures is 1.3+0.4 Å. The individual superposition
of the artificial nucleotides results in an r.m.s.d. of 0.5+0.2 Å.
1
Figure 3 | Direct evidence for the formation of imidazole–Ag –imidazole
1
15
base pairs. a, Section of the [ H, N]-HSQC (recorded at higher
resolution than that displayed in Fig. 2) with the magnification of the Im8
N3–H2 and N3–H5 cross peaks as an example to illustrate the
1
15
J( N,107/109Ag) coupling of 86 Hz. b, The chemical structure shows the
connectivities within the metal-mediated base pair, highlighting the bond
1
along which the J coupling is observed. c, Top view on the three
consecutive metal-mediated base pairs along the helix axis. The
backbone ribbon is displayed for the whole duplex. It can clearly be
discerned that the metal ions align along the helical axis. This panel has
been prepared with MOLMOL based on the lowest-energy structure40
.
In the presence of one equivalent of Ag(I), the resolution of the
HSQC spectrum improves dramatically indicating a more rigid struc-
ture and thus being well in line with the formation of a double helix.
All nitrogen resonances experience an upfield shift: the N1 resonances
shift to about 185 ppm (Dd ꢀ 23 ppm) and the N3 resonances shift
to about 206 ppm (Dd ꢀ 215 ppm). The significant upfield shift
2
6
clearly indicates a metal coordination to the N3 positions and is
also indicative of increased stacking interactions as expected in a
helix. Most importantly, the 15N resonances of the N3 atoms show
a splitting of 86 Hz in the presence of Ag(I) ions (Fig. 3a). This split-
ting is attributed to a direct coupling between the nitrogen and the
1
15 107/109
Ag(I) atoms—that is, a J( N,
Ag) coupling (Fig. 3b)—
thereby providing the first direct proof for the formation of an
þ
azole–Ag –azole base pair. To the best of our knowledge this is
also the first time that such a coupling has been measured in
1
15 107/109
aqueous solution at ambient temperature. J( N,
Ag) coupling
constants are observed rarely because Ag(I) usually binds to nitrogen
donors in a highly labile fashion. Under those conditions, the rapid Figure 4 | Lowest-energy structure of the duplex containing the Ag1-
ligand exchange removes the coupling between silver and any other mediated imidazole base pairs in its centre. The natural adenine–thymine
magnetic nucleus on the ligand. The observed 1J( N,
15
107/109
Ag) base pairs are coloured in green, the imidazole nucleobases in gold, and
þ
coupling therefore shows that the Ag(I) ions are tightly bound to the Ag ions are shown as blue spheres. This figure has been prepared
the artificial nucleosides and exchange reactions are slow.
with MOLMOL40
.
NATURE CHEMISTRY | VOL 2 | MARCH 2010 | www.nature.com/naturechemistry
231
©
2010 Macmillan Publishers Limited. All rights reserved.

NATURE CHEMISTRY DOI: 10.1038/NCHEM.512
ARTICLES
change could be used to design small molecules that specifically
target this type of metal-mediated base pair.
Table 1 | NMR restraints and structural statistics for the
hairpin and metal-containing duplex structures. All statistics
are given for the respective 20 lowest energy structures out
of 200 calculated structures.
Along the same line, the helical rise h is typically in the range of
3.4 Å, again with significant deviations from this value in the central
region of the double helix (Fig. 5b): h increases to 4.3(1) Å between
þ
Hairpin
Duplex
adjacent adenine–thymine and imidazole–Ag –imidazole base
NOE-derived distance restraints
Natural base pairs
Imidazole nucleotides Im8–Im10/Im25–Im27
Intra-nucleotide
Inter-nucleotide (ji 2 jj ¼ 1)
Long-range (ji 2 nj ꢂ 2)
Repulsive
NOE restraints per residue
NOE violation . 0.2 Å
Dihedral restraints
Dihedral violations . 58
Hydrogen-bond restraints
Imidazole–imidazole restraints
r.m.s.d. (for all heavy atoms to the best
structure, Å)
354
316
38
147
185
22
0
20.8
0
148
0
848
650
198
366
442
40
0
24.9
0
218
0
pairs and to 4.1(3) Å between consecutive metal-mediated base
pairs. Interestingly, the rise between a natural and an artificial
base pair is slightly larger than that in between two artificial base
a
4
2
40
38
36
34
32
30
28
26
0
28
-
56
18
–2
–
–
–
4
6
8
Overall
Helix
Im8–Im10/Im25–Im27
1.6+0.4
0.9+0.3
2.7+0.8
1.3+0.4
0.6+0.1
0.5+0.2
–
10
TT TA AA AT TT TT TIm ImIm ImIm ImA AA AA AT TT TA AA
AA AT TT TA AA AA AIm ImIm ImIm ImT TT TT TA AA AT TT
A total of 198 NOE-derived distance constraints were obtained just
for the artificial nucleotides, including 6 interstrand and 36 direct
stacking NOEs of the imidazole rings. The large number of distance
restraints obtained for these residues helps tremendously to pre-
cisely define the orientation of the artificial bases. Overall, the metal-
Base pair step
b
1.4
.2
1
4.6
4.4
4.2
4.0
3.8
3.6
3.4
þ
lated DNA retains its B-form helix upon formation of Ag -
mediated base pairs in its centre.
1.0
0.8
0.6
Discussion
0.4
The solution structure shown in Fig. 4 provides the first structural
insight into a nucleic acid with consecutive metal-mediated base
pairs. It also proves that a DNA double helix can adopt the
regular B-type conformation while at the same time arranging
metal ions along its helical axis (Fig. 3c), as is evident from the
base pair parameters shown in Fig. 5 and Supplementary Fig. S3,
0
0
.2
.0
3
.2
.0
–
–
–
0.2
0.4
0.6
3
2.8
2.6
31
calculated with the programme 3DNA . In both figures, the abso-
–0.8
–1.0
lute values of the respective parameters are shown on the right
2.4
whereas the values relative to those of average B-DNA32 are given
TT TA AA AT TT TT TIm ImImImIm ImA AA AA AT TT TA AA
AA AT TT TA AA AA AIm ImImImIm ImT TT TT TA AA AT TT
on the left. Tilt t (that is, the dihedral angle for rotation of a base
pair around its short axis, with respect to its neighbour), roll r
Base pair step
(that is, the dihedral angle for rotation of a base pair around its Figure 5 | According to the base-pair parameters, the Ag(I)-containing
long axis, with respect to its neighbour), inclination h (that is, the double helix adopts a regular B-DNA structure with only slight deviations
angle between the long axis of a base pair and a plane perpendicular (see also Supplementary Fig. S3). Parameters that show a slight distortion
to the helix axis) and global displacement (that is, translation of a around the metal-mediated base pairs are helical twist V (the twist angle
base pair within its mean plane with respect to the helix axis) of between two successive base pairs) and helical rise h (the distance between
0
0
all nucleobases adopt values close to those of B-DNA two successive base pairs). Both are global parameters based on C1 –C1
Supplementary Fig. S3).
Interestingly, small deviations from the B-type structure are the left and absolute values are given on the right. a, The helical twist V
observed for helical twist V (that is, the twist angle between two suc- drops from a regular ꢁ368 to ꢁ288 at the central two base-pair steps,
(
vectors. Values relative to those of average B-DNA are given on the scale on
þ
cessive base pairs) and helical rise h (that is, the distance between indicative of a slight unwinding of the helix at consecutive imidazole–Ag –
two successive base pairs) (Fig. 5): the helical twist V adopts imidazole base pairs (see also Supplementary Fig. S4). b, Interestingly, the
values of about 368, with the exception of the two central base maximum helical rise h is observed for contiguous natural and artificial base
pair steps between the three metal-mediated base pairs. The pairs (h ¼ 4.3(1) Å) rather than for contiguous artificial base pairs
helical twist of these three metal-mediated base pairs amounts to (h ¼ 4.1(3) Å). It is therefore unlikely that this structural deviation is related
only ꢁ288, indicating a slight unwinding of the double helix. This to the introduction of positive charges along the helix axis. The relatively large
unwinding seems to be caused by an opening of the minor standard deviation of the latter value indicates that the energy differences
groove, whereas the major groove remains largely unchanged between conformations with differing metal–metal distances are small. The
(
Supplementary Fig. S4). As this slight distortion of the B-type error bars in a and b represent the standard deviation of the 20 lowest
DNA conformation occurs only at the centre of the duplex, it energy structures. The standard deviation (as calculated with Origin 7.0) is
appears as if intrinsic structural parameters of the artificial base defined as the standard error of the mean multiplied by the square root of
pairs are the main contributors. The small but significant structural the sample size (20).
232
NATURE CHEMISTRY | VOL 2 | MARCH 2010 | www.nature.com/naturechemistry
2010 Macmillan Publishers Limited. All rights reserved.
©

NATURE CHEMISTRY DOI: 10.1038/NCHEM.512
ARTICLES
pairs. The increased rise therefore appears to be an intrinsic prop- prepared using an Expedite 8909 synthesizer in the DMT-off mode and purified by
þ
high-performance liquid chromatography with a Nucleogen 60-7 DEAE column
following standard procedures. For details see Supplementary Information.
erty of the imidazole–Ag –imidazole base pair rather than a
result of a potential interaction of adjacent metal ions along the
helical axis. In this context, the metal–metal distances between NMR spectroscopy. All oligonucleotide NMR spectra were recorded on a Bruker
neighbouring Ag(I) ions along the helical axis amount to 3.92 Å AV700 MHz spectrometer equipped with a CP-TXI z-axis pulsed-field gradient
CryoProbe, except for the 31P spectra, which were obtained on a Bruker AV2-
and 3.97 Å for the lowest-energy structure, and vary between
4
00 MHz spectrometer equipped with a QNP probe. Non-exchangeable resonances
3
4
.79 and 4.51 Å for the 20 best structures. The value of almost
Å for the lowest-energy structure appears to rule out direct
1
1
were assigned from [ H, H]-NOESY (250 ms mixing time, 293, 298 and 303 K),
1
1
1
[
H, H]-total correlation spectroscopy (TOCSY) (50 ms mixing time, 298 K),
13
1
15
1
metal–metal interactions. This distance is unexpectedly large, [ H, C]-HSQC (298 K) and long-range [ H, N]-HSQC ( J ¼ 20 Hz, 298 K)
NH
especially considering that attractive argentophilic interactions are spectra in D O. Only very few exchangeable proton resonances could be assigned
2
33
owing to large overlap of the thymine N3H resonances. DOSY spectra (298 K) were
frequently observed between closely spaced Ag(I) ions . On the
other hand, the high variation—also manifested in the relatively
large standard deviation of the helical rise h for the central two
1
7
1
1
1
13
acquired and processed as previously described . All [ H, H] and [ H, C] spectra
were recorded at natural isotope abundance, whereas [ H, N] spectra were recorded
1
15
with DNA containing 98% enriched 15N imidazole moieties. NMR data were
base pair steps—shows that the energy differences between confor- processed with TOPSPIN 1.3 and 2.0 (Bruker) and analysed with Sparky (http://
mations with differing metal–metal distances are small. As a result, www.cgl.ucsf.edu/home/sparky/). NOE peak volumes were integrated with the
Gaussian peak-fitting function in Sparky.
only minor energetic changes can lead to structures with a significant
metal–metal interaction. For example, a recent theoretical inspection
of DNA containing Cu(II)-mediated base pairs shows that metal–
Structure calculations. NOE distances were estimated from the integrated peak
1
1
volumes obtained from the [ H, H]-NOESY spectra acquired at 298 K with a mixing
3
7
metal distances down to 3.22 Å are possible within the structural time of 250 ms. Distances were calibrated using the CALIBA macro in DYANA . The
34
NOEs were grouped into four categories, corresponding to strong (1.8–3.0 Å),
medium (1.8–4.5 Å), weak (3.0–6.0 Å) and very weak (4.0–7.0 Å). Structure
calculation of the hairpin was then performed following standard procedures using
DYANA 1.5 (ref. 37) and Xplor-NIH 2.15.0 (ref. 38). The imidazole residue was
inserted into the parameter and topology files using the structural parameters from
context of a DNA double helix . At this point it remains unclear
whether the observed metal–metal distances are merely the result
of an overestimation of the electrostatic repulsion during the structure
calculations and whether they might actually be a bit shorter.
3
9
In summary, we have presented the first structural insight into a previous DFT calculations and was then patched into the oligonucleotide like a
B-type DNA double helix containing consecutive metal-mediated
standard nucleotide by the DYANA and Xplor program, respectively. For details see
Supplementary Information.
Structure calculation of the duplex was performed with Xplor-NIH . Torsion
angles and sugar pucker restraints were set on the basis of NMR data as described for
base pairs. The solution structure of a self-complementary oligonu-
3
8
þ
cleotide comprising three central imidazole–Ag –imidazole base
pairs shows that the metal ions are located along the helical axis. the hairpin. The backbone torsion angles were set to exclude the trans range and
In the sole previous structure of a DNA duplex with metal-mediated cover the B-DNA range. The sugar pucker was restrained to S-type and the
glycosidic torsion angle x was set to 2120+208 (anti) for all residues apart from the
base pairs, the metal ions were located at the periphery of the helix
2
1
imidazole moieties, which were left unrestrained. First, an extended structure was
due to the Z-type conformation that was adopted in that structure .
þ
generated including the geometrical parameters of the imidazole–Ag –imidazole
1
15 107/109
The observation of J( N,
Ag) couplings in aqueous solution
39
base pair obtained from DFT calculations . Starting from the extended strand a set
at room temperature now provides direct proof for the formation of of 2,000 structures was calculated using NOE distances and dihedral restraints. In
the metal-mediated base pairs.
An analysis of the base-pair parameters shows that the incorpo-
ration of the artificial metal-containing base pairs proceeds without
addition to the planarity and hydrogen-bond distance restraints of the natural base
pairs, planarity and distance restraints for the imidazole–Ag –imidazole base pairs
were also included to maintain the coordinative-bond properties. Based on the
þ
perfect C symmetry observed in the NMR spectra showing only one half of the
2
major conformational distortions. A slight unwinding of the helix in
duplex, a non-crystallographic symmetry term was introduced. The 200 structures
þ
the region of the imidazole–Ag –imidazole base pairs provides the with lowest energies were used for further refinement using additional RAMA
opportunity for the development of small molecules that selectively and ORIE database terms. The 20 lowest-energy structures of the 200
calculated structures of the hairpin and the duplex were visualized and
recognize this structural motif. Ag–Ag distances between consecu-
tive metal-mediated base pairs amount to 3.92 Å and 3.97 Å for
the lowest-energy structure. Nevertheless, direct Ag–Ag interactions
4
0
analysed using MOLMOL .
Structure coordinates are deposited at the Protein Data Bank (hairpin: 2K68,
duplex: 2KE8), and the NMR chemical shift assignments at the BioMagResBank
cannot be ruled out completely because the duplex structure seems (hairpin: 15860, duplex: 16138).
to be rather dynamic, thereby allowing shorter metal–metal dis-
Received 13 July 2009; accepted 3 December 2009;
published online 17 January 2010
tances without large energy barriers.
Methods
Preparation of 3,5-Di-O-p-toluoyl-1,2-dideoxy-b-1-(15N-imidazol-1-yl)-D-
References
1
5
ribofuranose 1. N imidazole was synthesized according to a modified literature
1. Niemeyer, C. M. & Mirkin, C. A. Nanobiotechnology (Wiley-VCH, 2004).
2. M u¨ ller, J. Metal-ion-mediated base pairs in nucleic acids. Eur. J. Inorg. Chem.
3749–3763 (2008).
35
procedure . To a solution of glyoxal (40 wt% solution in water, 11.3 mmol, 1.29 ml)
and formaldehyde (36.5 wt% solution in water, 11.3 mmol, 851 ml) was added
15
15
NH Cl (1.02 g, 18.7 mmol, 98% N), and the pH was adjusted to zero by addition
3. Tanaka, K. & Shionoya, M. Programmable metal assembly on bio-inspired
templates. Coord. Chem. Rev. 251, 2732–2742 (2007).
4
of concentrated HCl. After stirring at 95 8C for 2 h, the reaction mixture was cooled
to ambient temperature and treated with solid KOH until pH 10 was reached. The
4. Clever, G. H., Kaul, C. & Carell, T. DNA-metal base pairs. Angew. Chem. Int. Ed.
46, 6226–6236 (2007).
5. He, W., Franzini, R. M. & Achim, C. Metal-containing nucleic acid structures
based on synergetic hydrogen and coordination bonding. Prog. Inorg. Chem. 55,
545–611 (2007).
6. Weizman, H. & Tor, Y. 2,2’-Bipyridine Ligandoside: A novel building block for
modifying DNA with intra-duplex metal complexes. J. Am. Chem. Soc. 123,
3375–3376 (2001).
7. Meggers, E., Holland, P. L., Tolman, W. B., Romesberg, F. E. & Schultz, P. G.
A novel copper-mediated DNA base pair. J. Am. Chem. Soc. 122,
10714–10715 (2000).
23
solvent was then removed in vacuo. A Kugelrohr distillation (100 8C, 10 mbar)
1
5
15
was performed to separate N imidazole from the side product, KCl. This crude
N
imidazole (375 mg, 5.35 mmol assuming 100% purity) was dissolved in MeCN
(
40 ml), and NaH (60% in oil, 257 mg, 6.42 mmol) was added at 0 8C. After stirring
3
6
for 30 min at 0 8C, 3,5-di-O-p-toluoyl-a-D-erythro-pentofuranosyl chloride (2.5 g,
.4 mmol) was added in four portions every 20 minutes. The solvent was removed in
6
vacuo after stirring for an additional 2 h, and the remaining solid was dissolved
in CH Cl (50 ml), washed with H O (30 ml) and dried (Na SO ). The solvent
2
2
2
2
4
was removed, and the crude product purified by column chromatography
SiO , cyclohexane:CH Cl :Et N 50:30:8) yielding 1.2 g (2.9 mmol, 54%) of 1.
(
2
2
2
3
For the characterization of 1 and further synthetic steps towards the
8. Heuberger, B. D., Shin, D. & Switzer, C. Two Watson-Crick-like metallo base-
pairs. Org. Lett. 10, 1091–1094 (2008).
0
4
,4 -dimethoxytrityl (DMT)- and phosphoramidite-protected imidazole
nucleoside, see Supplementary Information.
9. Polonius, F.-A. & M u¨ ller, J. An artificial base pair, mediated by hydrogen
bonding and metal-ion binding. Angew. Chem. Int. Ed. 46, 5602–5604 (2007).
10. Clever, G. H. & Carell, T. Controlled stacking of 10 transition-metal ions inside a
DNA duplex. Angew. Chem. Int. Ed. 46, 250–253 (2007).
Oligonucleotide preparation. Phosphoramidites of the natural nucleosides as
well as CPGs were purchased from Glen Research. The oligonucleotides were
NATURE CHEMISTRY | VOL 2 | MARCH 2010 | www.nature.com/naturechemistry
233
©
2010 Macmillan Publishers Limited. All rights reserved.

NATURE CHEMISTRY DOI: 10.1038/NCHEM.512
ARTICLES
1
1
1
1
1
1
1
1. Switzer, C., Sinha, S., Kim, P. H. & Heuberger, B. D. A purine-like nickel(II) base
pair for DNA. Angew. Chem. Int. Ed. 44, 1529–1532 (2005).
and silver(I) and copper(I) coordination properties in solution ( H, 109Ag, and
N NMR). Inorg. Chem. 24, 1367–1375 (1985).
15
2. Tanaka, K., Tengeiji, A., Kato, T., Toyama, N. & Shionoya, M. A discrete self-
assembled metal array in artificial DNA. Science 299, 1212–1213 (2003).
3. Tanaka, K. et al. Programmable self-assembly of metal ions inside artificial DNA
duplexes. Nature Nanotech. 1, 190–194 (2006).
31. Lu, X.-J. & Olson, W. K. 3DNA: a software package for the analysis, rebuilding
and visualization of three-dimensional nucleic acid structures. Nucleic Acids Res.
31, 5108–5121 (2003).
32. Chandrasekaran, R. & Arnott, S. The structure of B-DNA in oriented fibers.
J. Biomol. Struct. Dyn. 13, 1015–1027 (1996).
33. Liu, X. et al. Three novel silver complexes with ligand-unsupported argentophilic
interactions and their luminescent properties. Inorg. Chem. 45,
3679–3685 (2006).
34. Mallajosyula, S. S. & Pati, S. K. Conformational tuning of magnetic interactions
in metal–DNA complexes. Angew. Chem. Int. Ed. 48, 4977–4981 (2009).
35. Gridnev, A. A. & Mihalteva, I. M. Synthesis of 1-alkylimidazoles. Synth.
Commun. 24, 1547–1555 (1994).
36. Rolland, V., Kotera, M. & Lhomme, J. Convenient preparation of 2-deoxy-3,5-di-
O-p-toluoyl-a-D-erythro-pentofuranosyl chloride. Synth. Commun. 27,
3505–3511 (1997).
37. G u¨ ntert, P., Mumenthaler, C. & W u¨ thrich, K. Torsion angle dynamics for NMR
structure calculation with the new program DYANA. J. Mol. Biol. 273,
283–298 (1997).
38. Schwieters, C. D., Kuszewski, J. J., Tjandra, N. & Clore, G. M. The Xplor-NIH
NMR molecular structure determination package. J. Magn. Reson. 160,
65–73 (2003).
39. M u¨ ller, J., B o¨ hme, D., Lax, P., Morell Cerd a` , M. & Roitzsch, M. Metal ion
coordination to azole nucleosides. Chem. Eur. J. 11, 6246–6253 (2005).
40. Koradi, R., Billeter, M. & W u¨ thrich, K. MOLMOL: A program for
display and analysis of macromolecular structures. J. Mol. Graphics 14,
51–55 (1996).
15
15
II
II
4. Tanaka, Y. et al. N– N J-coupling across Hg : Direct observation of Hg -
mediated T–T base pairs in a DNA duplex. J. Am. Chem. Soc. 129, 244–245 (2007).
5. Ono, A. et al. Specific interactions between Ag(I) ions and cytosine–cytosine
pairs in DNA duplexes. Chem. Commun. 4825–4827 (2008).
6. Wettig, S. D., Wood, D. O., Aich, P. & Lee, J. S. M-DNA: A novel metal ion
complex of DNA studied by fluorescence techniques. J. Inorg. Biochem. 99,
2
093–2101 (2005).
1
7. Johannsen, S., Paulus, S., D u¨ pre, N., M u¨ ller, J. & Sigel, R. K. O. Using in vitro
transcription to construct scaffolds for one-dimensional arrays of mercuric ions.
J. Inorg. Biochem. 102, 1141–1151 (2008).
1
1
2
2
2
2
8. Schlegel, M. K., Zhang, L., Pagano, N. & Meggers, E. Metal-mediated base pairing
within the simplified nucleic acid GNA. Org. Biomol. Chem. 7, 476–482 (2009).
9. Franzini, R. M. et al. Metal binding to bipyridine-modified PNA. Inorg. Chem.
4
5, 9798–9811 (2006).
0. K u¨ sel, A. et al. Metal binding within a peptide-based nucleobase stack with
tuneable double-strand topology. Eur. J. Inorg. Chem. 4317–4324 (2005).
1. Atwell, S., Meggers, E., Spraggon, G. & Schultz, P. G. Structure of a copper-
mediated base pair in DNA. J. Am. Chem. Soc. 123, 12364–12367 (2001).
2. Schlegel, M. K., Essen, L.-O. & Meggers, E. Duplex structure of a minimal
nucleic acid. J. Am. Chem. Soc. 130, 8158–8159 (2008).
3. B o¨ hme, D., D u¨ pre, N., Megger, D. A. & M u¨ ller, J. Conformational change
induced by metal-ion-binding to DNA containing the artificial 1,2,4-triazole
nucleoside. Inorg. Chem. 46, 10114–10119 (2007).
2
4. Kuklenyik, Z. & Marzilli, L. G. Mercury(II) site-selective binding to a DNA
hairpin. Relationship of sequence-dependent intra- and interstrand cross-linking to
the hairpin–duplex conformational transition. Inorg. Chem. 35, 5654–5662 (1996).
5. Varani, G., Aboul-ela, F. & Allain, F. H.-T. NMR investigation of RNA structure.
Prog. Nucl. Magn. Reson. Spectrosc. 29, 51–127 (1996).
Acknowledgements
Financial support by the European ERAnet-Chemistry, the Swiss National Science
Foundation (20EC21-112708 and 200021-124834 to R.K.O.S.), the Swiss State Secretariat
for Education and Research (R.K.O.S.), the Deutsche Forschungsgemeinschaft
2
2
(
MU1750/1-3 and MU1750/2-1 to JM), COST D39 and the Fonds der Chemischen
6. Tanaka, Y. & Ono, A. Nitrogen-15 NMR spectroscopy of N-metallated nucleic
Industrie (J.M.) is gratefully acknowledged. We also thank T. van der Wijst for providing us
with the partial charges of protonated and unprotonated 1-methylimidazole, as well as R.
Micura and K. Breuker, University of Innsbruck, for recording mass spectra.
1
5
acids: insights into N NMR parameters and N–metal bonds. Dalton Trans.
965–4974 (2008).
7. Zangger, K. & Armitage, I. M. Silver and gold NMR. Met. Based Drugs 6,
4
2
2
2
2
39–245 (1999).
8. Bowmaker, G. A. et al. Solid-state 109Ag CP/MAS NMR spectroscopy of some
diammine silver(I) complexes. Magn. Reson. Chem. 42, 819–826 (2004).
9. van Stein, G. C., van Koten, G., Vrieze, K., Brevard, C. & Spek, A. L. Structural
Author contributions
J.M. and R.K.O.S. designed research, N.M. and D.B. performed syntheses, S.J. performed
the NMR experiments and structure calculations, S.J. and R.K.O.S. analysed data, and J.M.,
R.K.O.S., S.J. and N.M. wrote the manuscript.
investigations of silver(I) and copper(I) complexes with neutral N donor
4
ligands: X-ray crystal and molecular structure of the dimer [Ag fm-(R, S)-1,2-
2
1
13
109
15
(
py-2-CH¼N) Cyg ](O SCF ) and H, C, and INEPT Ag and N NMR
Additional information
2
2
3
3 2
solution studies. J. Am. Chem. Soc. 106, 4486–4492 (1984).
0. van Stein, G. C. et al. Group 11 metal ions in poly(donor atom) environments:
The authors declare no competing financial interests. Supplementary information
accompanies this paper at www.nature.com/naturechemistry. Reprints and permission
information is available online at http://npg.nature.com/reprintsandpermissions/.
Correspondence and requests for materials should be addressed to R.K.O.S. and J.M.
3
X-ray crystal and molecular structure of [M((R, S)-1,2-(5-R-thio-2-CH¼N) -c-
2
Hx) ](O SCF ) (M ¼ Ag(I), R ¼ Me, thio ¼ Thiophene, c-Hx ¼ Cyclohexane)
2
3
3
234
NATURE CHEMISTRY | VOL 2 | MARCH 2010 | www.nature.com/naturechemistry
©
2010 Macmillan Publishers Limited. All rights reserved.