Site-selective scission

Current molecular biology and biotechnology are based on two key processes (i) site-selective scission of DNA by restriction enzymes and (ii) recombination of DNA fragments by another enzyme, known as ligase. These methods, first reported in the 1970s, are almost perfect as long as we manipulate small DNA such as the plasmid DNA of bacteria and genome DNA of virus, which are composed of several thousand... 

Figure 7.3 DNA substrates and oligonucleotide additives used for site-selective scission of single-stranded DNA.

Figure 7.5 (a) Site-selective scission of gaps of different lengths by Ce(iv)/... 

Enzymatic Ligation of the Fragments Obtained by Site-selective Scission... 

The present site-selective scission proceeds by an hydrolytic pathway, as is the case in DNA scission by nucleases. Thus, the scission fragments can be recombined with various oligonucleotides by using DNA ligase. Figure 7.6 depicts a typical example. 

Site-selective Scission of Double-stranded DNA by Combining Ce(iv 169... 

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... 

Both linear and supercoiled double-stranded DNA can be used as substrate for site-selective scission. Typical methods for the scission of linear double-stranded DNA are given in Sections 7.6.2.1 and 7.6.2.2. The substrate is obtained by treating supercoiled PBR322 plasmid DNA by a restriction enzyme EcoRI. Here, the plasmid DNA is cut at one-site, providing a linear DNA (this DNA is known as form III). Section 7.6.2.3 describes the method for site-selective hydrolysis of supercoiled DNA. Figure 7.9(a)... 

For the present site-selective scission of double-stranded DNA, both pcPNA additives must have flanking portions so that gap-like structures are formed in the double-stranded DNA. Thus, no site-selective scission was observed when pcPNA 3 and pcPNA 4 were combined as additives [Figure 7.10(a), lane 4], These two pcPNAs are completely complementary (their duplex is not much formed due to mutual steric repulsion under the conditions employed), and thus no gap-like structures are produced in the invasion complex [see structure at the bottom of Figure 7.9(a)], No scission occurred in the absence of pcPNA additives, as expected (lane 2). 

Direct Site-selective Scission of Supercoiled Plasmid DNA... 

Non-covalent strategy has been used also for site-selective scission of RNA (a) D. Hiisken, G. Goodall, M. J. J. Blommers, W. Jahnke,... 

Evidence for the hydrolytic scission of 11. Covalent strategy for site-selective scission of ... 

As described in the introduction section, one of the goals of non-enzymatic hydrolysis of DNA and RNA is to prepare artificial enzymes for site-selective scission of them. With these new tools, we can develop novel biotechnology and therapy, which are impossible with the use of naturally occurring enzymes. Now that we have lanthanide ions as efficient molecrrlar scissors on DNA and RNA, what we should do next for the site-selective scission is to let these scissors work at the desired site in DNA and RNA substrates. 

Fig. 16. Two strategies for site-selective scission of DNA and RNA (A) covalent strategy in which lanthanide complexes are covalently linked near the target phosphodiester linkages by using oligonucleotides that are complementary with the DNA and RNA substrates, and (B) non-covalent strategy , in which the target phosphodiester linkage is activated by some non-covalent interactions and dilferentiated from the others in the substrate in terms of intrinsic reactivity. The black ribbons show the oligonucleotides (or their equivalents) used for the artificial enzymes...

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