Chemistry Reference
In-Depth Information
ma's DNA conjugate, probably due to the use of the less reactive Zr(IV) ion.
Moreover, the employment, under the conditions used, of a large amount of free
Zr(IV), due to the low metal affi nity of the ligand (TRIS) subunit, leads to a sub-
stantial nonspecifi c random cleavage. At any rate, the fragments formed are consist-
ent with the anticipated selectivity and the scission occurrs with remarkable
regiospecifi city at the P-O3
′
bond.
13.7 The ARCUT System
Very recently, Komiyama and coworkers described a new approach to the selective
hydrolytic cleavage of DNA, based on the selectivity of the Ce(IV)-EDTA complex.
5
As mentioned before, this complex cleaves ssDNA with such preference that bulges
or gap-sites in dsDNA are selectively cleaved.
17,18
In the ARCUT protocol, gap-like
structures are formed at the desired sites of dsDNA or plasmid DNA by invasion
of two pseudo-complementary PNA strands (pcPNA), whose sequences are selected
in order to bind laterally shifted base sequences in the target (Figure 13.17).
49
The
two single-stranded portions formed in the invaded DNA are hence cleaved by
incubation with Ce(IV)-EDTA. The sequence selectivity can be programmed
without limitations and cleavage does not occur even in the presence of a single
base mismatch.
50
Cleaving effi ciency is greatly enhanced by attaching a serine phos-
phate monoester at the pcPNA termini close to the gap site, since the dianionic
terminal phosphate induces the accumulation of the Ce(IV) complex.
50
However
the reaction still requires, in many cases, high temperature (50 °C) and prolonged
reaction times (64 h). DNA fragments obtained can be religated and connected with
foreign double-stranded DNA by using DNA ligase and a ligation joint to provide
recombinant DNA (Figure 13.17).
18,49,51
The method has been applied by Komiyama
and coworkers to the site-selective scission of genomic DNA of
Escherichia coli
(4.6
10
6
bases),
50
to the site-selective scission of enzymatically methylated DNA
(which is resistant to restriction enzymes)
50
, to the preparation of recombinant
DNA
18,49,51
and even of fusion proteins.
52
×
gap sites
Ligation
joint
pcPNAs
C
e (IV)/E
DTA
Ligase
Figure 13.17
Schematic representation of the ARCUT protocol for the preparation of recom-
binant DNA
