title: Inhibition of gene expression in Escherichia coli by antisense phosphorodiamidate morpholino oligomers.
abstract: 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.
mesh_terms: 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
chemical_list: 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