Targeting nucleic acids in bacteria with synthetic ligands

Abstract

There is a need for new antibacterial agents, and one attractive strategy is to develop nucleic acid ligands that inhibit pathogen genes selectively. Also, such ligands can be used as molecular biology probes to study gene function and nucleic acid structures. In this thesis, bacterial genes were selectively inhibited with antisense peptide nucleic acid (PNA) and the higher order structures formed by (GAA)n repeats were probed with the intercalator benzoquinoquinoxaline (BQQ).

A majority of bacterial genes belong to tight clusters and operons, and regulation within cotranscribed genes has been difficult to study. We examined the effects of antisense silencing of individual ORFs within a natural and synthetic operon in Escherichia coli. The results indicate that expression can be discoordinated within a synthetic operon but only partially discoordinated within a natural operon.

Bacteria use natural antisense mechanisms to regulate gene expression and an established model for sense/antisense RNA pairing is the hok/sok toxin antitoxin (TA) locus. We aimed to test this model in cells and also the idea that sequestration of Sok-RNA could provide a novel antimicrobial strategy using antisense agents. The results support the hok/sok sense/antisense interaction model and the idea that PNA can out-compete this interaction and provide potent killing activity.

Certain antisense agents are effective in bacteria, yet it is unclear how these relatively large molecules overcome stringent bacterial barriers. Here we determined the transit kinetics of peptide-PNAs and observed an accumulation of cell-associated PNA in E. coli and slow efflux. Consistent with cell accumulation and retention, the post-antibiotic effect (PAE) of a PNA that targets the growth essential fatty acid biosynthesis gene acpP was greater than seven hours.

At the DNA level, polypyrimidine/polypurine rich sequences have the potential to form intramolecular triple helix structures (H-DNA). Triplet (GAA)n repeats are pathological in Friedreich's ataxia (FA) disease, and may form H-DNA. Here we probed for triplex structures in (GAA)n sequences using the triple-helix specific stabilizing compound BQQ. The results showed that E. coli plasmid carrying a (GAA)n repeat sequence forms H-DNA, suggesting that these structures may play a role in the pathology of FA .