Blockade of plasmid replication mediated by peptide nucleic acids.
Because peptide nucleic acids (PNAs) are capable of blocking amplification of deoxyribonucleic acid (DNA) by Taq DNA polymerase in vitro, we postulated that PNAs might be able to block replication in vivo. To explore this possibility, we assessed the ability of PNA to specifically block the replication of pUC19 plasmids by allowing a PNA, directed against segments of the Ampr sequence to bind to pUC19 prior to electroporation into Escherichia coli, strain DH10B. Colonies produced by this maneuver not only remained sensitive to ampicillin but were also incapable of blue color production on X-gal-containing media, thus demonstrating true blockade of pUC19 replication, rather than antisense activity. The ability of the PNA to prevent pUC19 replication in these experiments was shown to be dose related. Attempts to prevent the replication of E. coli using a PNA directed against a portion of the lac Z sequence found within the bacterial genome were not uniformly successful. Subsequent experiments showed that the electroporated PNA did not consistently enter a sufficient number of cells for an effect to be demonstrated in the assays used. Nonetheless, this is the first demonstration of in vivo complete replication blockade by a PNA and opens up the potential for new forms of specific antibiosis in both prokaryotic and eukaryotic cells.
Liebling
Michael R
MR
Division of Rheumatology, Box 470/ E2 South, LAC Harbor-UCLA Medical Center,1000 West Carson Street, Torrance, CA 90509, USA. mliebling@rei.edu
Jou
Nainn-Tsyr
NT
Fang
Wayne
W
Louie
James S
JS
D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics* / Q000502:physiology; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D020135:Peptide Nucleic Acids / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D010957:Plasmids; D019914:Taq Polymerase / Q000378:metabolism; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D009841:Oligonucleotides; D020135:Peptide Nucleic Acids; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase