Inhibition of gene expression in Escherichia coli by antisense phosphorodiamidate morpholino oligomers.
Antisense phosphorodiamidate morpholino oligomers (PMOs) were tested for the ability to inhibit gene expression in Escherichia coli. PMOs targeted to either a myc-luciferase reporter gene product or 16S rRNA did not inhibit luciferase expression or growth. However, in a strain with defective lipopolysaccharide (lpxA mutant), which has a leaky outer membrane, PMOs targeted to the myc-luciferase or acyl carrier protein (acpP) mRNA significantly inhibited their targets in a dose-dependent response. A significant improvement was made by covalently joining the peptide (KFF)(3)KC to the end of PMOs. In strains with an intact outer membrane, (KFF)(3)KC-myc PMO inhibited luciferase expression by 63%. A second (KFF)(3)KC-PMO conjugate targeted to lacI mRNA induced beta-galactosidase in a dose-dependent response. The end of the PMO to which (KFF)(3)KC is attached affected the efficiency of target inhibition but in various ways depending on the PMO. Another peptide-lacI PMO conjugate was synthesized with the cationic peptide CRRRQRRKKR and was found not to induce beta-galactosidase. We conclude that the outer membrane of E. coli inhibits entry of PMOs and that (KFF)(3)KC-PMO conjugates are transported across both membranes and specifically inhibit expression of their genetic targets.
D000213:Acyl Carrier Protein / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002463:Cell Membrane Permeability; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter / Q000187:drug effects / Q000235:genetics; D016259:Genes, myc / Q000187:drug effects / Q000235:genetics; D056968:Lac Repressors; D008156:Luciferases / Q000037:antagonists & inhibitors / Q000235:genetics; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D009025:Morpholines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D060172:Morpholinos; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D012336:RNA, Ribosomal, 16S / Q000187:drug effects / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D001616:beta-Galactosidase / Q000378:metabolism
D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D026901:Membrane Transport Proteins; D009025:Morpholines; D060172:Morpholinos; D016376:Oligonucleotides, Antisense; D012336:RNA, Ribosomal, 16S; D012097:Repressor Proteins; D008156:Luciferases; D001616:beta-Galactosidase