Chemistry Reference
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of Ce(
IV
), since the DNA fragments obtained by the scission can be, when necessary,
recombined with other fragments by using ligase (this enzyme is effective for the
recombination of hydrolytic scission products but cannot recombine the products
of oxidative scission). The second key step in molecular biology and biotechnology
is successfully achievable. Thus, Ce(
IV
) and its complexes are satisfactorily compatible
with the current technology, and are very promising for the catalytic center of artificial
restriction enzymes. Other tetravalent metal ions, Th(
IV
) [7] and Zr(
IV
) [8], are also
active for DNA hydrolysis. However, their activities are considerably smaller than
that of Ce(
IV
). Even today, Ce(
IV
) is still the sole catalyst that can promptly hydrolyze
linear DNAs irrespective of DNA sequences.
7.4
Molecular Design of Artificial Restriction Enzymes (Covalent vs. Non-Covalent Strategy)
7.4.1
Covalent Strategy for the First-generation of Artificial Restriction Enzymes
Artificial restriction enzymes were first prepared by conjugating “molecular DNA scis-
sors“ with a “sequence-recognizing moiety“. The “molecular DNA scissors“ are con-
centrated near the target phosphodiester linkages, and preferentially hydrolyze them
because of favorable activation-entropy term. For example, the Ce(
IV
) ion was com-
bined with an oligonucleotide (sequence-recognizing moiety) that is complementary
with the substrate DNA near the target site and bears an iminodiacetate group at its
5
0
-end. The substrate DNA was selectively hydrolyzed at the target-site [9] [Figure
7.1(a)]. Similarly, tris(hydroxymethyl)aminomethane-modified peptide nucleic acid
(PNA: sequence-recognizing moiety) was combined with Zr(
IV
) ion as molecular
DNA scissors [10].
This “covalent“ strategy is very successful for site-selective DNA scission. However,
the scission is not sufficiently efficient and must be promoted for practical applica-
Figure 7.1 Site-selective hydrolysis of DNA using (a) covalent and (b)
non-covalent strategies.
