RESUMO
A monophosphate group was attached to the terminus of pseudo-complementary peptide nucleic acid (pcPNA), and two of thus modified pcPNAs were combined with Ce(IV)/EDTA for site-selective hydrolysis of double-stranded DNA. The site-selective DNA scission was notably accelerated by this chemical modification of pcPNAs. These second-generation artificial restriction DNA cutters (ARCUTs) differentiated the target sequence so strictly that no scission occurred even when only one DNA base-pair was altered to another. By using two of the activated ARCUTs simultaneously, DNA substrate was selectively cut at two predetermined sites, and the desired fragment was clipped and cloned. The DNA scission by ARCUT was also successful even when the target site was methylated by methyltransferase and protected from the corresponding restriction enzyme. Furthermore, potentiality of ARCUT for manipulation of huge DNA has been substantiated by site-selective scission of genomic DNA of Escherichia coli (composed of 4,600,000 bp) at the target site. All these results indicate promising applications of ARCUTs for versatile purposes.
Assuntos
Enzimas de Restrição do DNA/química , DNA/química , Ácido Edético/química , Ácidos Nucleicos Peptídicos/química , Pareamento Incorreto de Bases , Cério , Metilação de DNA , DNA Bacteriano/química , Escherichia coli/genética , Genoma Bacteriano , Hidrólise , Fosfatos/químicaRESUMO
We have already developed artificial restriction DNA cutter (ARCUT), which can hydrolyze double-stranded DNA site-selectively, by using Ce(IV)/EDTA in combination with two pseudo-complementary peptide nucleic acids (pcPNAs). Here, ARCUT was used to prepare a chimera protein. The gene for WW-domain containing oxidoreductase (WWOX) was clipped off by ARCUT just before its stop codon, and ligated with the gene for enhanced green fluorescent protein (EGFP). Conventional PCR for insertion of restriction enzyme site is never required.
Assuntos
DNA Recombinante/química , Desoxirribonucleases/química , Proteínas Recombinantes de Fusão/genética , Animais , Células COS , Cério , Chlorocebus aethiops , Enzimas de Restrição do DNA/química , DNA Recombinante/metabolismo , Ácido Edético/química , Proteínas de Fluorescência Verde/genética , Hidrólise , Oxirredutases/genética , Ácidos Nucleicos Peptídicos/química , Proteínas Recombinantes de Fusão/análiseRESUMO
By combining Ce(IV)/EDTA with two pseudo-complementary peptide nucleic acids (pcPNAs), both strands in double-stranded DNA were site-selectively hydrolyzed at the target site. Either plasmid DNA (4361 bp) or its linearized form was used as the substrate. When two pcPNAs invaded into the double-stranded DNA, only the designated portion in each of the two strands was free from Watson-Crick base pairing with the counterpart DNA or the pcPNA. Upon the treatment of this invasion complex with Ce(IV)/EDTA at 37 degrees C and pH 7.0, both of these single-stranded portions were selectively hydrolyzed at the designated site, resulting in the site-selective two-strand scission of the double-stranded DNA. Furthermore, the hydrolytic scission products were successfully connected with foreign double-stranded DNA by using ligase. The potential of these artificial systems for manipulation of huge DNA has been indicated.