Chemistry Reference
In-Depth Information
essential for catalytic function at physiological conditions. However, most DNA/
RNAzymes found to date cannot match protein enzymes in terms of either the
diversity of metal ions they employ or the specifi city of the metal-binding sites. For
example, protein enzymes use a wide variety of metal ions, including even the
second and third-row transition-metals such as Mo and W, while metal ions used by
DNA/RNAzymes are often limited to Mg
2+
, Ca
2+
and Mn
2+
. Furthermore, it is well
known that many protein enzymes possess remarkable metal-binding affi nity and
specifi city, and they are commonly classifi ed by the metal ions they specifi cally bind
(e.g., copper proteins or zinc proteins). DNA/RNAzymes have also been shown to
bind certain metal ions selectively, although the number of different metal ions that
DNA/RNAzymes can bind is much fewer and the corresponding metal binding
affi nity is generally lower than those in metalloproteins.
26 - 32
These weaknesses in
DNA/RNAzymes are being overcome by using
in vitro
selection (see below).
14.3 Selection of DNA zymes Using
in vitro
Evolution
Since DNAzymes are almost exclusively generated via
in vitro
selection, the key to
more diverse metal ion cofactors, as well as higher binding affi nities and selectivities
is improving the selection process. Illustrated in Figure 14.2, generally the selection
starts with a pool containing 10
14
to 10
15
random DNA sequences. Besides a random
region in the middle, every DNA in the pool also contains two common priming
regions at two ends for polymerase chain reaction (PCR). Under given conditions
and cofactors, the DNA sequences that can carry out desired catalytic functions are
separated from inactive ones. These active sequences are then amplifi ed by PCR for
the next round of reaction and selection. The stringency of the selection can be
Figure 14.2
(Plate 12)
Schematic representation of the
in vitro
selection process (See colour
plate section)
