Sequence-Specific Protection of Duplex DNA against Restriction and Methylation Enzymes by Pseudocomplementary PNAs

A new generation of PNAs, so-called pseudocomplementary PNAs (pcPNAs), which are able to target the designated sites on duplex DNA with mixed sequence of purines and pyrimidines via double-duplex invasion mode, has recently been introduced. It has been demonstrated that appropriate pairs of decameri...

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Bibliographic Details
Published in:Biochemistry (Easton) 2000-09, Vol.39 (35), p.10908-10913
Main Authors: Izvolsky, Konstantin I, Demidov, Vadim V, Nielsen, Peter E, Frank-Kamenetskii, Maxim D
Format: Article
Language:English
Subjects:
Adenoviridae - chemistry
Adenoviridae - genetics
Base Pair Mismatch
Base Pairing
Base Sequence
Binding Sites
deoxyribonuclease BclI
Deoxyribonucleases, Type II Site-Specific - chemistry
DNA - chemistry
DNA - genetics
DNA methyltransferase
DNA Modification Methylases - chemistry
DNA Restriction Enzymes - chemistry
DNA, Viral - chemistry
DNA, Viral - genetics
Nucleic Acid Heteroduplexes - chemistry
Nucleic Acid Heteroduplexes - genetics
peptide nucleic acids
Peptide Nucleic Acids - chemistry
Peptide Nucleic Acids - genetics
Plasmids - chemistry
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Summary:A new generation of PNAs, so-called pseudocomplementary PNAs (pcPNAs), which are able to target the designated sites on duplex DNA with mixed sequence of purines and pyrimidines via double-duplex invasion mode, has recently been introduced. It has been demonstrated that appropriate pairs of decameric pcPNAs block an access of RNA polymerase to the corresponding promoter. Here, we show that this type of PNAs protects selected DNA sites containing all four nucleobases from the action of restriction enzymes and DNA methyltransferases. We have found that pcPNAs as short as octamers form stable and sequence-specific complexes with duplex DNA in a very salt-dependent manner. In accord with a strand-invasion mode of complex formation, the pcPNA binding proceeds much faster with supercoiled than with linear plasmids. The double-duplex invasion complexes selectively shield specific DNA sites from BclI restriction endonuclease and dam methylase. The pcPNA-assisted protection against enzymatic methylation is more efficient when the PNA-binding site embodies the methylase-recognition site rather than overlaps it. We conclude that pcPNAs may provide the robust tools allowing to sequence-specifically manipulate DNA duplexes in a virtually sequence-unrestricted manner.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi000675e