Chemistry Reference
In-Depth Information
A
C
5
3
E
FRET
k
obs
(min
−
1
)
0.25
0.25
4
2
3
0.20
0.20
2
1
1
0.15
0.15
E
FRET
k
obs
(min
−
1
)
0
0
0.0
0.5 1.0
[ZnCl
2
] (mM)
1.5
2.0
0 0 0 0 0 0
[Pb(OAc)
2
] (
M)
µ
B
D
0.3
0.25
0.2
0.20
0.1
0.15
E
FRET
k
obs
(min
−
1
)
0.0
Stem II
8-17 DNAzyme
0 0 0 0
[MgCl
2
] (mM)
40
50
Figure 14.4
FRET effi ciency and catalytic activity (
k
obs
) of the 8-17 DNAzyme in the pres-
ence of Zn(II) (A), Mg(II) (B) and Pb(II) (C), and proposed folding model (D). (Reprinted
with permission from
J. Am. Chem. Soc
., 2007,
129
, 6896-6902. Copyright 2007, American
Chemical Society.)
the Cy3/Cy5 FRET pair was labelled each time on two of the three stems of the
Pb
2+
- dependent ' 8 - 17 ' DNAzyme. The FRET effi ciency between Cy3 and Cy5 was
monitored as an indication of the distance change between the two stems when
metal ions were introduced into the DNAzyme solution. FRET results indicated
that, in the presence of Zn
2+
and Mg
2+
, the DNAzyme folds into a compact structure,
in which Stem III approached a confi guration defi ned by Stems I and II, without
changing the angle between Stems I and II. Correlations between metal-induced
folding and activity were also studied. For Zn
2+
and Mg
2+
, the metal ion with higher
affi nity for the DNAzyme in global folding (
K
d
(Zn) = 52.6 m M and
K
d
(Mg) = 1.36 mM)
also displayed higher affi nity in activity (
K
d
(Zn) = 1.15 mM and
K
d
(Mg) = 53 mM)
under the same conditions. Global folding was saturated at much lower concentra-
tions of Zn
2+
and Mg
2+
than the cleavage activities, indicating the global folding of
the DNAzyme occurs before the cleavage activity of the metal ions. Surprisingly, no
Pb
2+
-dependent global folding was observed. These results suggest that for Pb
2+
,
global folding of the DNAzyme may not be a necessary step in its function, which
may contribute to the DNAzyme having the highest activity in the presence of Pb
2+
.
While FRET studies in bulk solutions offer useful insights into DNAzyme
folding and activity, some valuable information may be lost due to the averaging
effect of the large quantity of molecules present in the solution. Moreover, higher
temporal resolution is needed in order to reveal the real underlying mechanism of
the metal-ion-dependent catalytic activities. To overcome these limitations, single-
