Chemistry Reference
In-Depth Information
enhanced in each round by reducing reaction time and cofactor concentration, so
that highly active DNAs can be enriched gradually. This process is repeated for a
certain number of cycles until DNA sequences with very high activity and selectivity
can be isolated and identifi ed. The same strategy has also been successfully employed
in the fi eld of aptamer research, where DNAs with high binding affi nities and selec-
tivities for the targets of interest, instead of high catalytic activities, are produced.
The selection process has allowed the discovery of aptamers for a wide variety of
targets, ranging from metal ions
33
and small organic molecules,
34,35
to proteins
36
and
even cancer cells.
37,38
Theoretically, the selection of DNA/RNAzymes can also be
carried out in the presence of many different cofactors, thus allowing the isolation
of DNA/RNAzymes with very specifi c requirements for various cofactors, including
any metal ions of interest. Indeed, efforts in this direction have yielded DNA/
RNAzymes that are catalytically active in the presence of some less common metal
ion cofactors, such as Pb
2+
,
1,39
Cu
2+
,
12,17
Zn
2+
,
40,41
Co
2+
,
42
and
UO
2+
.
43
It is expected
that DNA/RNAzymes dependent on other metal ions can be generated by a similar
approach with well - controlled selection conditions.
An inherent problem with the
in vitro
selection strategy described above is that
even though aptamers or DNA/RNAzymes can be obtained under controlled condi-
tions, it does not exclude the possibility that these selected products can also func-
tion under different conditions or with different cofactors. This problem obviously
results in lower-than-ideal selectivity of the aptamers or DNA/RNAzymes. In some
cases, the activity of the nucleic acid enzymes in the presence of the target metal
ion is even lower than with certain other metal ions. For example, the Zn(II)-binidng
aptamer binds some other metal ions equally well, including Ni(II) and Co(II).
44 - 46
Another DNAzyme, termed '10-23' DNAzyme was selected with Mg(II) present,
but its activity with Mn(II) is even higher.
15,47,48
Furthermore, the same ' 8 - 17 ' motif
of a DNAzyme was obtained by four different laboratories with different cofactor
conditions.
15,49,40,50
It has a metal-dependent activity in the order Zn(II) >>
Ca(II)
Mg(II) under similar conditions. Interestingly, further evaluation of this
enzyme showed even higher activity with Pb(II).
39
The apparent
K
d
values for
Pb(II), Zn(II) and Mg(II) are 13.5 mM (at pH 6.0), 0.97 mM (at pH 6.0) and 10.5 mM
(at pH 7.0), respectively.
39
The lack of specifi city in these DNAzymes poses major
problems in studying metal DNA/RNA interactions and in applying DNAzymes as
metal ion sensors.
To solve this problem and improve metal ion specifi city, a negative selection
strategy can be introduced into the selection process by incubating the DNA pool
with nontarget metal ions following positive selection with the target ion. In this
step, any DNA sequences that have activity in the presence of other metal ions will
be discarded, leaving the sequences that only function with the target ion present.
This negative selection can be conducted multiple times as needed to greatly elimi-
nate nonspecifi c sequences. As a demonstration of improved selectivity by negative
selection, two parallel selections of Co(II)-dependent DNAzymes were carried out
with and without negative selections (shown in Figure 14.3).
42
In the absence of the
negative selection, the resulting DNAzymes were more active with Pb(II) and
Zn(II) than with Co(II). In comparison, when negative selection was incorporated,
the obtained DNAzymes were much more active with Co(II) than with other metal
ions. No detectable cleavage activity was observed with several other metal ions
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