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 be en introduced. It has been demonstrated that appropriate pairs of decameric pcPNAs block an access of RNA polymerase to the corresponding promoter. He re, we show that this type of PNAs protects selected DNA sites containing a ll four nucleobases fram the action of restriction enzymes and DNA methyltr ansferases. 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 bin ding proceeds much faster with supercoiled than with linear plasmids. The d ouble-duplex invasion complexes selectively shield specific DNA sites from BclI restriction endonuclease and dam methylase. The pcPNA-assisted protect ion against enzymatic methylation is more efficient when the PNA-binding si te embodies the methylase-recognition site rather than overlaps it. We conc lude that pcPNAs may provide the robust tools allowing to sequence-specific ally manipulate DNA duplexes in a virtually sequence-unrestricted manner.