Because PNAs specifically block amplification of DNA by Taq DNA polymerase in vitro, we postulated that PNAs might block DNA replication, in vivo. To explore this possibility, we allowed a PNA, directed against segments of the Ampr sequence, to bind to PUC19 prior to electroporation into E.coli. Colonies produced by this maneuver not only remained sensitive to ampicillin but were also incapable of color production on x-gal-containing media, thus demonstrating true blockade of PUC19 replication, rather than antisense activity. Exclusion of the plasmid from the bacteria was eliminated as the cause of these findings. Moreover, replication blockade, in vivo, could be shown to follow a dose-response curve that closely approximated the inhibition of Taq polymerase, in vitro, using the same PNA and PUC19. Similarly, the addition of lysines to the PNA produced an increase in inhibitory capacity, in vitro, that was closely mirrored, in vivo, when the same PNA was used for replication blockade. This suggests that such an in vitro system might be used to model appropriate sequence modifications and dose ranges for subsequent in vivo experiments. Finally, when a PNA complementary to a portion of the lacZ gene was directly electroporated into the bacteria, the number of colonies was diminished 2.5 fold compared to electroporation with a control PNA (p<0.001). This is the first demonstration of in vivo replication blocade by PNA and opens a wide vista of possibilities for new forms of specific antibiosis in both prokaryotic and eukaryotic cells.
|Original language||English (US)|
|State||Published - Mar 20 1998|
ASJC Scopus subject areas
- Molecular Biology