RESUMEN
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.
Asunto(s)
Enzimas de Restricción del ADN/química , ADN/química , Ácido Edético/química , Ácidos Nucleicos de Péptidos/química , Disparidad de Par Base , Cerio , Metilación de ADN , ADN Bacteriano/química , Escherichia coli/genética , Genoma Bacteriano , Hidrólisis , Fosfatos/químicaRESUMEN
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.
Asunto(s)
Cerio/metabolismo , ADN/metabolismo , Ácido Edético/análogos & derivados , Ácido Edético/metabolismo , Ácidos Nucleicos de Péptidos/química , Secuencia de Bases , ADN/química , ADN Ligasas/metabolismo , ADN Superhelicoidal/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Hidrólisis , Datos de Secuencia Molecular , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , Especificidad por SustratoRESUMEN
Through the invasion of pseudo-complementary PNA (pePNA) to double-stranded DNA, gap-like structures were formed at predetermined sites in both strands of PBR322 plasmid DNA. These gap-like sites were selectively hydrolyzed by Ce(IV)/EDTA complex, and two designed fragments were obtained. Furthermore, the scission fragment by this artificial restriction enzyme was successfully ligated with foreign DNA.