Chemistry Reference
In-Depth Information
the context AGGA, the ratio of 5
′
to 3
′
reactivity was 1.8
±
0.1, and for GG in the
context TGGT, the ratio was 6.1
0.3. The authors propose that the accessibility of
H
2
O to the reaction site determined by the steric blocking by the methyl group plays
the dominant role for the observed sequence-selectivity, rather than electronic
effects.
23
±
1.3 NMR Spectroscopic Studies of Metal Binding to
DNA Oligonucleotides
1.3.1 NMR Methodology
Most early nuclear magnetic resonance (NMR) studies on DNA involved com-
plementary homopolymers and self-complementary, alternating copolymers, e.g.
poly(dA)/poly/dT).
24
The development of effi cient and rapid methods of large-scale
oligonucleotide syntheses has made it possible to design heteropolymeric sequences
of high purity. Dodecamer (12 base pair) sequences adopting a normal B-DNA
double-helical conformation, are assumed to complete a full turn of a right-handed
helix. The structure of such a mini-helix is probably suffi ciently close to that of real
DNA to serve as a realistic model for determining preferred metal binding sites.
The effects of adding paramagnetic metal ions to an aqueous solution of DNA
fragments may be monitored by observing the decrease in spin-lattice (T
1
) and spin-
spin (T
2
) relaxation times (related to line-broadening) for protons close to the metal
centres. Paramagnetic metal ions may be classifi ed according to their electronic
correlation times, i.e. as relaxation probes producing broad lines or as paramagnetic
shift probes producing narrow lines. Divalent manganese is a typical relaxation
probe with an estimated electronic relaxation time (
t
s
= T
1c
= T
c
) of 10
− 8
- 10
− 9
s, while
nickel, which has a shorter
t
s
in the range 10
− 10
- 10
− 12
s, is a typical chemical shift
probe. Cobalt(II) in a low-spin coordination environment has an estimated
t
s
between that of Mn
2+
and Ni
2+
in kinetically labile metal complexes. At low metal
to nucleotide ratios paramagnetic shift effects of Ni
2+
are diffi cult to detect. In this
case geometric information about metal binding sites is most effectively obtained
by measuring proton spin-lattice relaxation times (T
1
).
Paramagnetic relaxation arises in NMR spectroscopy when an unpaired elec-
tron spin interacts with a nuclear spin. The large magnetogyric ratio of the electron
compared to that of the proton makes the dipolar coupling to the electron spin a
very effective means of relaxation for the nuclear spin.
25,26
Scalar interactions
between the electron and nuclear spins have similar effects. In the simplest possible
case, a ligand molecule exchanges between a paramagnetic environment (e.g. bound
to Mn(II), S = 5/2) and a 'free' state, when the ligand is present in solution in vast
excess to the paramagnetic centre (e.g. 10
2
- 10
4
). The effect of paramagnetic metal
ions located at specifi c binding sites on DNA is observed as differential line-
broadening of proton signals close to the binding site. Often, in 1D spectra of oli-
gonucleotide molecules containing ten base pairs or more, key proton resonances
may be severely overlapped, preventing an accurate assessment of the infl uence of
