Chemistry Reference
In-Depth Information
conversion in the gap-region increases with increasing gap-length, since the number
of scissile linkages increases.
In Figure 7.5(b), the relative scission-efficiency at each phosphodiester linkage is
presented by the length of arrow (the solid parts and the broken parts correspond
to the formation of 3
0
-OH and 3
0
-phosphate termini, respectively, vide infra). In all
cases, the scissions are restricted strictly to the gap-site so that the site-selectivity in-
creases monotonically with decreasing gap-length. With a 1-base gap, scission primar-
ily takes place only at both sides of the unpaired nucleotide. Scission at the 2-base gap
(as well as at the 3- and 5-gaps) is vigorous in the middle of the gap. Significantly, the
efficiency for the scission of each linkage in the gap-site is not much dependent on
either the gap-length or the sequence of the gap-site. Thus, the position for site-selec-
tive scission can be freely chosen. This is one of the most significant advantages of the
present site-selective DNA scission.
7.5.1.3
Termini Structures of Scission Fragments
The scission fragments in Figures 7.4 and 7.5 (
32
P-labelled at the 5
0
-end) are classified
into two categories. One group co-migrates with authentic samples of oligonucleotides
that have 3
0
-OH termini (in lane M). Conversely, another group is found between the
bands of authentic samples. Apparently, the first group has 3
0
-OH termini and the
second group has 3
0
-phosphate termini. In most cases, the 3
0
-OH termini [shown
by the solid parts of the arrows in Figure 7.5(b)] are preferentially formed to the
3
0
-phosphate termini (broken parts). Similarly, 5
0
-termini of the fragments mostly
have phosphate rather than OH termini. Thus, the present DNA hydrolysis mostly
proceeds via P-O(3
0
) scission of phosphodiester linkage, providing the fragments
having the same terminus structures as formed by most of the naturally occurring
nucleases. These fragments are easily ligated with other DNAs by enzymatic reactions
(vide infra).
7.5.1.4
Essential Role of Gap-structure for the Site-selective DNA Scission
The gap structures are crucially important here, since the target site must be differ-
entiated from other sites in terms of intrinsic reactivity. Thus, site-selective scission is
unsuccessful when only one oligonucleotide additive bearing a monophosphate is
used (without the second oligonucleotide additive). Here, no gap-structure is formed
in substrate DNA, and the single-stranded portion of the DNA substrate is hydrolyzed
by Ce(
IV
)/EDTA without any remarkable selectivity. Consistently, DNA scission at the
target site is never promoted when oligonucleotides bearing only the linker groups
(and no monophosphate groups) are used. The monophosphate groups are essential
to recruit the Ce(
IV
) to the target site for site-selective scission. When a small gap is
hydrolyzed by the present system, the scission efficiency depends significantly on the
length of the linker between DNA and a phosphate group. For example, scission at a
1-base gap by the DNA
(L)
-L
12
-P/P-L
12
-DNA
(R)
combination is about 3
as fast as that by
the DNA
(L)
-L
0
-P/P-L
0
-DNA
(R)
combination. However, the efficiency of scission at a
3-base gap and a 5-base gap is less dependent on linker length.
