Antisense inhibition of RNase P: mechanistic aspects and application to live bacteria.

We explored bacterial RNase P as a drug target using antisense oligomers against the P15 loop region of Escherichia coli RNase P RNA. An RNA 14-mer, or locked nucleic acid (LNA) and peptide nucleic acid (PNA) versions thereof, disrupted local secondary structure in the catalytic core, forming hybrid duplexes over their entire length. Binding of the PNA and LNA 14-mers to RNase P RNA in vitro was essentially irreversible and even resisted denaturing PAGE. Association rates for the RNA, LNA, and PNA 14-mers were approximately 10(5) m(-1) s(-1) with a rate advantage for PNA and were thus rather fast despite the need to disrupt local structure. Conjugates in which the PNA 14-mer was coupled to an invasive peptide via a novel monoglycine linker showed RNase P RNA-specific growth inhibition of E. coli cells. Cell growth could be rescued when expressing a second bacterial RNase P RNA with an unrelated sequence in the target region. We report here for the first time specific and growth-inhibitory drug targeting of RNase P in live bacteria. This is also the first example of a duplex-forming oligomer that invades a structured catalytic RNA and inactivates the RNA by (i) trapping it in a state in which the catalytic core is partially unfolded, (ii) sterically interfering with substrate binding, and (iii) perturbing the coordination of catalytically relevant Mg2+ ions.

Evaluation of bacterial RNase P RNA as a drug target.

RNA has gained increasing importance as a therapeutic target. However, so far mRNAs rather than stable cellular RNAs have been considered in such studies. In bacteria, the tRNA-processing enzyme RNase P has a catalytic RNA subunit. Fundamental differences in structure and function between bacterial and eukaryotic RNase P, and its indispensability for cell viability make the bacterial enzyme an attractive drug target candidate. Herein we describe two approaches utilized to evaluate whether the catalytic RNA subunit of bacterial RNase P is amenable to inactivation by antisense-based strategies. In the first approach, we rationally designed RNA hairpin oligonucleotides targeted at the tRNA 3'-CCA binding site (P15 loop region) of bacterial RNase P RNA by attempting to include principles derived from the natural CopA-CopT antisense system. Substantial inactivation of RNase P RNA was observed for Type A RNase P RNA (such as that in Escherichia coli) but not for Type B (as in Mycoplasma hyopneumoniae). Moreover, only an RNA oligonucleotide (Eco 3') complementary to the CCA binding site and its 3' flanking sequences was shown to be an efficient inhibitor. Mutation of Eco 3' and analysis of other natural RNase P RNAs with sequence deviations in the P15 loop region showed that inhibition is due to interaction of Eco 3' with this region and occurs in a highly sequence-specific manner. A DNA version of Eco 3' was a less potent inhibitor. The potential of Eco 3' to form an initial kissing complex with the P15 loop did not prove advantageous. In a second approach, we tested a set of oligonucleotides against E. coli RNase P RNA which were designed by algorithms developed for the selection of suitable mRNA targets. This approach identified the P10/11-J11/12 region of bacterial RNase P RNA as another accessible region. In conclusion, both the P15 loop and P10/11-J11/12 regions of Type A RNase P RNAs seem to be promising antisense target sites since they are easily accessible and sufficiently interspersed with nonhelical sequence elements, and oligonucleotide binding directly interferes with substrate docking to these two regions.

Antisense inhibition of Escherichia coli RNase P RNA: mechanistic aspects.

The ribonucleoprotein enzyme RNase P catalyzes endonucleolytic 5'-maturation of tRNA primary transcripts in all domains of life. The indispensability of RNase P for bacterial cell growth and the large differences in structure and function between bacterial and eukaryotic RNase P enzymes comply with the basic requirements for a bacterial enzyme to be suitable as a potential novel drug target. We have identified RNA oligonucleotides that start to show an inhibitory effect on bacterial RNase P RNAs of the structural type A (for example, the Escherichia coli or Klebsiella pneumoniae enzymes) at subnanomolar concentrations in our in vitro precursor tRNA (ptRNA) processing assay. These oligonucleotides are directed against the so-called P15 loop region of RNase P RNA known to interact with the 3'-CCA portion of ptRNA substrates. Lead probing experiments demonstrate that a complementary RNA or DNA 14-mer fully invades the P15 loop region and thereby disrupts local structure in the catalytic core of RNase P RNA. Binding of the RNA 14-mer is essentially irreversible because of a very low dissociation rate. The association rate of this oligonucleotide is on the order of 10(4) M(-1) s(-1) and is thus comparable to those of many other artificial antisense oligonucleotides. The remarkable inhibition efficacy is attributable to the dual effect of direct interference with substrate binding to the RNase P RNA active site and induction of misfolding of the catalytic core of RNase P RNA. Based on our findings, the P15 loop region of bacterial RNase P RNAs of the structural type A can be considered the "Achilles' heel" of the ribozyme and therefore represents a promising target for combatting multiresistant bacterial pathogens.