Sequence-specific protection of duplex DNA against restriction and methylation enzymes by pseudocomplementary PNAs

TY - JOUR

T1 - Sequence-specific protection of duplex DNA against restriction and methylation enzymes by pseudocomplementary PNAs

AU - Izvolsky, K I

AU - Demidov, V V

AU - Nielsen, P E

AU - Frank-Kamenetskii, M D

PY - 2000/9/5

Y1 - 2000/9/5

N2 - 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.

AB - 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.

KW - Adenoviridae

KW - Base Pair Mismatch

KW - Base Pairing

KW - Base Sequence

KW - Binding Sites

KW - DNA

KW - DNA Modification Methylases

KW - DNA Restriction Enzymes

KW - DNA, Viral

KW - Deoxyribonucleases, Type II Site-Specific

KW - Nucleic Acid Heteroduplexes

KW - Peptide Nucleic Acids

KW - Plasmids

M3 - Journal article

C2 - 10978178

SN - 0006-2960

VL - 39

SP - 10908

EP - 10913

JO - Biochemistry

JF - Biochemistry

IS - 35

ER -