title: The translation start codon region is sensitive to antisense PNA inhibition in Escherichia coli.
abstract: Antisense peptide nucleic acids (PNA) can inhibit bacterial gene expression with gene and sequence specificity. Using attached carrier peptides that aid cell permeation, the antisense effects when targeting essential genes are sufficient to prevent growth and even kill bacteria. However, many design uncertainties remain, including the difficult question of target sequence selection. In this study, we synthesized 90 antisense peptide-PNAs to target sequences in a head to tail manner across the entire length of the mRNA encoding beta-lactamase. The results from this scan pointed to the start codon region as most sensitive to inhibition. To confirm and refine the result, a higher-resolution scan was conducted over the start codon region of the beta-lactamase gene and the essential Escherichia coli acpP gene. For both genes, the start codon region, including the Shine-Dalgarno motif, was sensitive, whereas antisense agents targeted outside of this region were largely ineffective. These results are in accord with natural antisense mechanisms, which typically hinder the start codon region, and the sensitivity of this region should hold true for most bacterial genes as well as for other RNase H-independent antisense agents that rely on a steric blocking mechanism. Therefore, although other design parameters are also important, the start codon region in E. coli mRNA is the most reliable target site for antisense PNAs.
authors.affiliation: Center for Genomics and Bioinformatics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Rikard
authors.initials: R
authors.lastname: Dryselius
authors.forename: Satish Kumar
authors.initials: SK
authors.lastname: Aswasti
authors.forename: Gunaratna K
authors.initials: GK
authors.lastname: Rajarao
authors.forename: Peter E
authors.initials: PE
authors.lastname: Nielsen
authors.forename: Liam
authors.initials: L
authors.lastname: Good
mesh_terms: D000213:Acyl Carrier Protein / Q000235:genetics / Q000378:metabolism; D001059:Apoproteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D018387:Codon, Initiator / Q000037:antagonists & inhibitors* / Q000235:genetics; D015536:Down-Regulation; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D054889:Fatty Acid Synthase, Type II; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology*; D010442:Peptide Chain Initiation, Translational; D020135:Peptide Nucleic Acids / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D044366:Transition Temperature; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
chemical_list: D000213:Acyl Carrier Protein; D001059:Apoproteins; D018387:Codon, Initiator; D029968:Escherichia coli Proteins; D016376:Oligonucleotides, Antisense; D020135:Peptide Nucleic Acids; D010455:Peptides; D012333:RNA, Messenger; C040157:acpP protein, E coli; D001618:beta-Lactamases; D054889:Fatty Acid Synthase, Type II