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Antimicrobial Agents and Chemotherapy, October 2003, p. 3233-3239, Vol. 47, No. 10
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.10.3233-3239.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Inhibition of Gene Expression in Escherichia coli by Antisense Phosphorodiamidate Morpholino Oligomers

B. L. Geller,1,2* J. D. Deere,2 D. A. Stein,2 A. D. Kroeker,2 H. M. Moulton,2 and P. L. Iversen2

Oregon State University,1 AVI Biopharma, Inc., Corvallis, Oregon2

Received 26 February 2003/ Returned for modification 24 April 2003/ Accepted 2 July 2003

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)3KC to the end of PMOs. In strains with an intact outer membrane, (KFF)3KC-myc PMO inhibited luciferase expression by 63%. A second (KFF)3KC-PMO conjugate targeted to lacI mRNA induced ß-galactosidase in a dose-dependent response. The end of the PMO to which (KFF)3KC 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 ß-galactosidase. We conclude that the outer membrane of E. coli inhibits entry of PMOs and that (KFF)3KC-PMO conjugates are transported across both membranes and specifically inhibit expression of their genetic targets.

* Corresponding author. Mailing address: Department of Microbiology, Nash Hall 220, Oregon State University, Corvallis, OR 97331-3804. Phone: (541) 737-1845. Fax: (541) 737-0496. E-mail: gellerb{at}orst.edu.

Antimicrobial Agents and Chemotherapy, October 2003, p. 3233-3239, Vol. 47, No. 10
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.10.3233-3239.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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