Bacterial gene expression inhibition with antisense peptide nucleic acids

Sammanfattning: Regulated gene expression is widely useful for gene function studies and as a means to control genes important in disease. There are many methods for regulating the expression of genes, and one approach is to use antisense oligonucleotides that bind complementary mRNAs to inhibit translation. A number of nucleic acid analogues and mimics have been developed to improve antisense efficiency. Peptide nucleic acid (PNA) is a DNA mimic with many interesting properties. PNA has proven effective in a variety of applications and mRNA has been a frequent target with the aim to silence gene expression in a sequence specific manner. However, mRNA targeting antisense PNA has almost exclusively been used on eukaryotic cells and relatively little is known about inhibition of bacterial gene expression. In this thesis antisense PNA is used to target bacterial genes. The focus is on design features such as antisense length and uptake and also on selection of appropriate target sites and target genes. Moreover, practical applications for antisense PNAs are examined including their use as inhibitors of essential bacterial genes. In terms of design features, the results show that antisense effects are much improved when a cell penetrating peptide is conjugated to the PNA. Also, relatively short molecules show higher efficiency. It is further demonstrated that the ribosome binding site is most likely to be susceptible to antisense inhibition and that antisense PNA directed to genes within operons may strongly affect the expression of cotranscribed genes. In terms of practical applications it was observed that an antisense PNA directed to an essential bacterial gene inhibited growth and was bactericidal. Furthermore, the PNA was able to rescue a human cell culture from infection and select for PNA resistant bacteria in a mixed population, indicating that efficient killing effects can be both cell type and strain specific. Also, growth inhibitory properties of antisense PNAs were examined in combination with conventional antimicrobial drugs. Combinations of mRNA specific antisense PNAs and protein specific drugs with shared genetic targets commonly displayed antimicrobial synergy, suggesting that antisense agents can be used to potentiate conventional antimicrobial drugs. In summary, the results described here provide guidelines for the design of sequence specific antisense PNAs and demonstrate practical uses for antisense agents as inhibitors of bacterial genes.