Inactivation of expression of several genes in a variety of bacterial species by EGS technology

The expression of gene products in bacteria can be inhibited by the use of RNA external guide sequences (EGSs) that hybridize to a target mRNA. Endogenous RNase P cleaves the mRNA in the complex, making it inactive. EGSs participate in this biochemical reaction as the data presented here show. They promote mRNA cleavage at the expected site and sometimes at other secondary sites. Higher-order structure must affect these reactions if the cleavage does not occur at the defined site, which has been determined by techniques based on their ability to find sites that are accessible to the EGS oligonucleotides. Sites defined by a random EGS technique occur as expected. Oligonucleotides made up primarily of defined or random nucleotides are extremely useful in inhibiting expression of the gyrA and rnpA genes from several different bacteria or the cat gene that determines resistance to chloramphenicol in Escherichia coli. An EGS made up of a peptide-phosphorodiamidate morpholino oligonucleotide (PPMO) does not cleave at the same site as an unmodified RNA EGS for reasons that are only partly understood. However, PPMO-EGSs are useful in inhibiting the expression of targeted genes from Gram-negative and Gram-positive organisms during ordinary growth in broth and may provide a basis for broad-spectrum antibiotics.