Chemistry Reference
In-Depth Information
the added metal ions. In these cases, 2D NOESY experiments may be used to obtain
suffi cient resolution.
For diamagnetic metal ions (no unpaired spin) the formation of a chemical
bond is usually found to cause changes in the chemical shifts of proton resonances
of hydrogen atoms in the proximity of the metal binding site. However, the coordi-
nation of Hg
2+
ions to single nucleobases induces only rather insignifi cant
1
H shift
changes, as shown for thymidine and guanosine.
27
This could be explained by a
down - fi eld chemical shift change, induced by metal binding, being cancelled by an
up - fi eld chemical shift caused by changes in ring current effects due to altered
nucleobase stacking.
28
The heteronuclei
13
C,
15
N and
31
P may experience large shift-
effects when a metal ion binds to nucleobase or phosphate groups.
1.3.2 Model Systems
One of the fi rst oligodeoxynucleotides studied by X-ray crystallography
29
and
subsequently by NMR spectroscopy
30
was the famous Dickerson-Drew sequence,
d(C
1
G
2
C
3
G
4
A
5
A
6
T
7
T
8
C
9
G
10
C
11
G
12
) (seq. I). The 1D and 2D NMR spectra used as
bases for the three-dimensional NMR structure showed an unexpectedly strong
line - broadening of the G
4
-H8 resonance. As a consequence, the G
4
- H8 to C
3
- 1
H
cross-peak was barely detectable in the 2D NOESY spectra, and the corresponding
interproton distance was scored as
′
4 Å by using the usual r
6
dependency to calcu-
late the proton-proton distance from the intensity of the cross-peaks.
31
Structure
determination based on protom-proton distance constraints showed a distinct bend
of the helix axis at the G
4
residue.
30
However, at a later stage, when EDTA was
added to the solution the G
4
-H8 resonance was found to exhibit normal line width.
7
Since trace amounts of paramagnetic impurities are often found to be present in
nucleotide preparations it is recommended to always run samples through a
CHELEX column to remove such impurities before NMR metal ion titration
experiments are performed.
>
1.3.3 Oligionucleotide - Transition Metal Adducts
Mn
2+
Ions
The fi rst metal ion titration NMR experiment on a DNA oligonucleotide was
carried out by Frøystein and Sletten on the Dickerson-Drew duplex d(C
1
G
2
C
3
G
4
A
5
A
6
T
7
T
8
C
9
G
10
C
11
G
12
) (seq. I) which contains the
Eco
- RI recognition sequence
GAATTC.
7
Since most of the base proton signals are well resolved in the 1D spec-
trum, the effect of adding aliquots of a solution of MnCl
2
was easily observed. A
plot of metal-induced paramagnetic line-broadening of G-H8 showed a clear prefer-
ence for G
4
compared to G
2
, G
10
and G
12
(Figure 1.5 ). At a Mn
2+
: duplex ratio of
1 : 10
4
substantial line-broadening was already detected for the G
4
- H8 signal, while
insignifi cant effects were observed for the other base proton resonances in the
duplex. Of particular importance was the observation that the central adenine A-H2
protons showed no broadening effect, indicating that Mn
2+
ions were not located in
