Chemical modification of Ce(IV)/EDTA-based artificial restriction DNA cutter for versatile manipulation of double-stranded DNA.
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.
Yamamoto
Yoji
Y
Research Center for Advanced Science and Technology, The University of Tokyo, Komaba, Meguro-ku, Tokyo, Japan.
Mori
Masao
M
Aiba
Yuichiro
Y
Tomita
Takafumi
T
Chen
Wen
W
Zhou
Jing-Min
JM
Uehara
Akihiko
A
Ren
Yi
Y
Kitamura
Yoshihito
Y
Komiyama
Makoto
M
D020137:Base Pair Mismatch; D002563:Cerium; D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D004262:DNA Restriction Enzymes / Q000737:chemistry*; D004269:DNA, Bacterial / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D016680:Genome, Bacterial; D006868:Hydrolysis; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry
D004269:DNA, Bacterial; D020135:Peptide Nucleic Acids; D010710:Phosphates; D002563:Cerium; D004247:DNA; D004492:Edetic Acid; D004262:DNA Restriction Enzymes