Chemistry Reference
In-Depth Information
tions. The primary reason for the suppressed efficiency is that the catalytic activity of
metal ion is notably diminished upon the conjugation with sequence-recognizing
moieties. This effect is more drastic when strong ligands are used to bind the
Ce(
IV
) tightly and place it near the target site. With weaker ligands, however, the metal
ion is fixed less strictly and the site-selectivity tends to become lower.
7.4.2
Non-covalent Strategy for the Second-generation of Artificial Restriction Enzymes
To solve the above dilemma, a “non-covalent“ strategy has been proposed [11]. Here,
target phosphodiester linkages are differentiated from other linkages in terms of in-
trinsic reactivity. DNA scission should be restricted to the target site simply because
the target phosphodiester linkages are more reactive than the others. Accordingly,
covalent fixation of Ce(
IV
) to sequence-recognizing oligonucleotides is not necessary,
and thus the intrinsic catalytic activity of this metal ion is fully utilized. As shown
below, this “non-covalent“ strategy is very successful in providing useful tools for fu-
ture molecular biology and biotechnology.
7.4.3
Chemical Basis for “Non-covalent“ Strategy
To accomplish site-selective DNA scission using the “non-covalent“ strategy, we need
both (i) molecular scissors that show sufficiently high substrate-specificity and (ii) “hot
spots“ that are formed at predetermined positions in substrate DNA and are hydro-
lyzed preferentially by these molecular scissors.
7.4.3.1
Molecular Scissors showing High Substrate-specificity
Although Ce(
IV
) ion is very active for DNA hydrolysis, it easily forms a metal hydroxide
gel at physiological pH, and the system becomes heterogeneous. This imposes
significant limitations to its practical applications. However, the Ce(
IV
)/EDTA complex
is homogeneous under physiological conditions and efficiently hydrolyzes DNA
(EDTA = ethylenediamine-N,N,N
0
,N
0
-tetraacetic acid) [12]. Homogeneous solutions
of this complex can be prepared simply by mixing stoichiometric amounts of
Ce(NH
4
)
2
(NO
3
)
6
and EDTA (4Na salt) in buffer solutions.
Notably, the Ce(
IV
)/EDTA complex shows remarkable substrate specificity. It effec-
tively hydrolyzes polynucleotides and oligonucleotides that are longer than tetranu-
cleotides. However, neither dinucleotides nor trinucleotides are hydrolyzed. Because
of this characteristic, this complex had long been believed to be inactive for DNA hy-
drolysis. In addition to this specificity with respect to substrate size, this complex
clearly differentiates between single- and double-stranded DNA. Thus, single-stranded
DNA is efficiently hydrolyzed, but double-stranded DNA is hardly cleaved [13].
