JAC Advance Access originally published online on December 22, 2007
Journal of Antimicrobial Chemotherapy 2008 61(2):262-272; doi:10.1093/jac/dkm482
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Original research |
Anti-gene padlocks eliminate Escherichia coli based on their genotype
1 Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA 2 Department of Comparative Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA 3 Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA 4 Department of Microbiology and Molecular Genetics, The University of Vermont, 224 Stafford Hall, Burlington, VT 05405, USA
Received 23 August 2007; returned 1 October 2007; revised 16 November 2007; accepted 20 November 2007
* Corresponding author. Tel: +1-410-955-3511; Fax: +1-410-614-0671; E-mail: jeshlema{at}jhmi.edu
Objectives: Several therapeutic strategies that target nucleic acids exist; however, most approaches target messenger RNA, rather than genomic DNA. We describe a novel oligonucleotide-based strategy, called anti-gene padlocks (AGPs), which eliminate Escherichia coli based on their genotype.
Methods: The strategy employs an oligonucleotide with a double hairpin structure where both strands of the AGP are complementary to both strands of a target gene. We tested AGPs for in vitro binding and inhibition of DNA polymerization. AGPs were electroporated into bacterial cells with and without gene targets along with an ampicillin resistance plasmid, and cell survival was measured.
Results: In vitro, AGPs bound the DNA target in a sequence-dependent fashion and inhibited DNA synthesis. When transformed into bacterial cells containing 10, 20 or 30 bp lacZ or 20 bp proA DNA targets in their genomes, AGPs selectively killed or otherwise inhibited growth of these cells, while those lacking the target demonstrated little, if any, toxicity. A single transformation resulted in 
30% to 40% loss of target-bearing cells. Structure–function experiments were performed to define essential AGP requirements.
Conclusions: These results suggest that AGPs may be a useful tool to eliminate specific cell populations.
Keywords: novel therapeutics , oligonucleotide , gene targeting , antimicrobial agents , antibiotics , padlock probes
These authors have contributed equally.
Present address. Department of Oncology, National University Hospital, Singapore, 5 Lower Kent Ridge Road, Singapore 119074.