Chemistry Reference
In-Depth Information
Figure 7.8 Proposed method for gene manipulation of single-stranded DNA.
7.6
Site-selective Scission of Double-stranded DNA by Combining Ce(
IV
)/EDTA Complex with
Pseudo-complementary PNA
7.6.1
Design of Artificial Restriction Enzymes for Double-stranded DNA Scission
Despite many attempts, there have been few reports on artificial restriction enzymes
for site-selective hydrolysis of double-stranded DNA [15]. Since most of DNA in nature
is double-stranded, these tools are quite important for practical applications. The
following tactics, developed only recently, also take advantage of the remarkable
substrate-specificity of the Ce(
IV
)/EDTA complex (preferential hydrolysis of single-
stranded DNA over double-stranded DNA). Thus, gap-like structures are formed in
both strands of double-stranded DNA by using the invasion of pseudo-complementary
PNA (pcPNA), and these gap-like sites are used as “hot spots“ for selective hydrolysis
by the complex. In pcPNA as DNA analog, phosphodiester linkages are replaced with
amide linkages. Because of the absence of negative charges, pcPNA forms a more
stable duplex with its complementary DNA than does the corresponding DNA (there
is no electrostatic repulsion between negative charges) [16]. Furthermore, both ade-
nine and thymine groups are chemically modified in pcPNA so that the formation
of A-T base pairs in the duplex between two “complementary“ pcPNAs is suppressed.
Thus, the pcPNA/pcPNA duplex is only inefficiently formed, whereas formation of the
pcPNA/DNA duplex is less affected by the chemical modification. Accordingly,
pcPNA easily invades its complementary part in double-stranded DNA without
much interference from the formation of pcPNA/pcPNA duplexes [17].
The two pcPNA additives used are designed so that a single-stranded portion is
formed at predetermined sites in both strands of double-stranded DNA substrate
when they invade the substrate; the structure is shown in the bottom of Figure
7.9(a). These gap-like sites are the “hot spots“ that are selectively hydrolyzed by
Ce(
IV
)/EDTA, resulting in the site-selective scission of both strands at the target
