Chemistry Reference
In-Depth Information
Other methods have also been developed to study nucleic acid-metal ion inter-
actions. Pyle and coworkers used terbium(III) to identify metal ion binding sites of
a family of group II intron ribozymes.
61
Tb
3+
and related ions are able to cleave RNA
effi ciently under physiological conditions. In this study, various concentrations of
Tb
3+
were incubated with the ribozymes in the presence of Mg
2+
. The cleaved frag-
ments of the enzymes were analysed by polyacrylamide gel electrophoresis (PAGE).
Fragments at low Tb
3+
concentrations revealed strong metal ion binding sites, while
fragments produced by high concentrations of Tb
3+
corresponded to lower-affi nity
binding sites. Comprehensive analysis of the gel data was successfully used to map
most metal ion binding sites of the ribozymes. These Tb
3+
- binding sites were found
to colocalize with Mg
2+
-binding sites based on two results. First, addition of Mg
2+
at
low pH effectively eliminated Tb
3+
cleavage. Second, Mg
2+
was shown to cleave at
the same Tb
3+
cleavage sites, only at much slower rates.
In addition to mapping metal ion binding sites by metal-ion-mediated hydroly-
sis, NMR and related techniques have been useful in studying nucleic acid enzyme-
metal ion interactions. Sigel, Pyle and coworkers employed NMR and two - dimensional
nuclear overhauser enhancement spectroscopy (NOESY) to reveal the solution
structure of the central core of a group II intron ribozyme and its interactions with
Mg
2+
.
62
It was found that binding to Mg
2+
had most impact on the nucleotides in the
bulge region of Domain 5. Using NMR and other techniques, Sigel and coworkers
reported the solution structure of a catalytically active Domain 6 (D6) of a self-
splicing Group II intron ribozyme. A single unpaired adenosine was found to reside
within the helix of D6 and was partially stacked between two fl anking GU wobble
pairs. A novel prominent Mg
2+
binding site was identifi ed in the major groove of
this site. In a recent report, Sigel and coworkers performed paramagnetic line-
broadening experiments with Mn
2+
and titration studies with Mg
2+
using NMR to
determine the affi nity constants for Mg
2+
binding to fi ve distinct sites in Domain 6
of Group II introns.
63
Even though most of the aforementioned methods have been developed and
used for ribozymes and RNAs, adaptation of these techniques to DNAzymes is not
likely to cause major diffi culties. Many efforts are underway and possibilities are
being explored along this line of research.
Compared to many other techniques, methods based on fl uorescence resonance
energy transfer (FRET) have been shown to be able to provide unique information
on structural alterations of the DNA/RNAzymes upon metal ion binding in real
time with minimum perturbation of the interactions. FRET is the energy transfer
between two fl uorophores when they have inherent spectrum overlap and are in
close proximity (
10 nm). The result is that when excited, the donor fl uorophore
generates less fl uorescence output compared to when there is no FRET, while fl uo-
rescence emission from the acceptor fl uorophore is observed even though it is not
usually excited by the donor excitation. Since the FRET effi ciency is tightly related
to the distance between the two fl uorophores, it can often be used to report subtle
intramolecular distance changes or structure alterations. To study DNAzyme-metal
ion interactions in real time, an initial FRET study was carried out with a trifl uoro-
phore - labelled DNAzyme,
64
followed by a more comprehensive study of the metal-
ion-induced folding of DNAzymes using FRET (Figure 14.4).
65
In the latter report,
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