Chemistry Reference
In-Depth Information
nucleotide base-pairs. Soon, however, we will certainly want to manipulate DNAs of
higher animals and plants, and widen the scope of technological applications. Can we
directly use the present methods there? The answer is absolutely “no“, as clearly shown
by the following simple statistical calculation. Naturally occurring restriction enzymes
have rather poor site-specificity, although the selectivity is almost 100%. Most of them
recognize a specific sequence composed of 4 or 6 DNA-bases and cut the DNA there.
On average, the scission site of these restriction enzymes should come out at every 4
4
(256) or 4
6
(4096) DNA-bases (assuming that four kinds of nucleotides, A, G, C, and T,
are randomly distributed in the DNA). These site-specificities are sufficient for site-
selective scission of small DNA (e.g., plasmid DNA), but apparently too low to cut the
huge DNAs of higher life forms at the desired site. For example, the DNA of human
beings is composed of more than 10
9
DNA-bases. If this DNA is treated with a 6-base
recognizing restriction enzyme, the scission should occur at more than 10
5
sites. Pre-
cise gene manipulation is impossible. Thus, artificial restriction enzymes, which have
much higher site-specificity and can selectively hydrolyze huge DNA at a predeter-
mined position, are crucially important for further developments. To cut only one-
site in human DNA, for example, we have to recognize a 16- or longer base sequence
(4
16
10
9
).
The importance of artificial restriction enzymes has been well understood by many
chemists, and several challenging attempts have been made already. Although many
difficulties remain, elegant work by many people is paving the way. This chapter deals
mainly with the recent work of our laboratory on site-selective DNA scission and its
applications to biotechnology.
>
7.3
Non-enzymatic Catalysts for DNA Hydrolysis
One of the most crucial obstacles for the preparation of artificial restriction enzymes is
the enormous stability of phosphodiester linkages in DNA. Nucleophilic attack of OH
-
towards phosphodiester linkage in DNA for its hydrolytic scission is prevented by elec-
trostatic repulsion between these two negatively charged species (the half-life of one
linkage for the hydrolytic scission at pH 7 and 25
8
C is estimated to be 200 million
years [2]). For a long time, DNA could not be hydrolyzed without using naturally
occurring nucleases. However, superb catalysts have been developed and this obstacle
has been gradually overcome. In the 1990s, it was found that rare earth metal ions are
remarkably active for the hydrolysis of phosphoesters and efficiently hydrolyze DNA at
reasonable rates under physiological conditions [3] (rare earth metal ions are also
highly active for the hydrolysis of RNA [4]). For DNA hydrolysis, the Ce(
IV
) ion is espe-
cially active. For example, 10m
M
Ce(
IV
) decreases the half-life of dinucleotides to only
several hours [5]. Compared with their uncatalyzed hydrolysis, an almost 10
12
-fold
acceleration has been achieved. When oligo- or polynucleotides are hydrolyzed, scis-
sion takes place almost randomly without specific-base preference. Although Ce(
IV
)is
a well-known oxidant, no concurrent oxidative scission of DNA occurs, and the DNA
scission proceeds totally via a hydrolytic pathway [5, 6]. This is a significant advantage
