Antisense-Inhibition der bakteriellen RNase P

In the present work the in vitro and in vivo inhibition of bacterial RNase P by antisense oligonucleotides (AS-ON) had been studied. Ribonuclease P is a ubiquitous and essential ribonucleoprotein enzyme which generates the mature 5´ ends of tRNAs. For inhibition experiments RNase P RNAs of the two existing classes had been chosen, with the RNase P RNA of Escherichia coli representing the A- and the RNase P RNA of Mycoplasma hyopneumoniae representing the B type. In vitro, the RNA subunit of bacterial RNase P is catalytically active in the absence of the protein subunit. In most bacteria, as a prerequisite for efficient substrate cleavage, the loop 15/16 region of the RNase P RNA active site associates with the tRNA CCA 3' end via Watson-Crick base pairing interaction. This CCA binding site appears to be a good target for an inhibitory antisense RNA strategy because of its importance for the cleavage reaction, its exposed position within the RNase P molecule and because eucaryotic RNase P enzymes do not possess a similar CCA binding site. The sequences around the CCA-binding site are not highly conserved among bacteria, which allows the design of AS-ON specific for a target organism. This opens up new perspectives to combat bacterial pathogens by inactivation of RNase P. The strategy we initially employed to design AS-ONs was adapted from a naturally occurring antisense mechanism in E. coli, thus it was possible to inhibit E. coli RNase P RNA by RNA hairpin oligonucleotides but not the RNA subunit of M. hyopneumoniae RNase P. Experiments to gain insight in the inhibition mechanism revealed, that singlestranded AS-ON derived of that part of the hairpin oligonucleotide that was complementary to RNase P RNA, was a much better inhibitor than the hairpin oligonucleotide itself. Therefore the singlestranded AS-ON (called 15mer) had been used for further optimization regarding oligonucleotide length which results in the most efficient so-called 14mer with a Ki of 2,2 nM. Since we liked to use the AS-ONs in in vivo experiments the inhibition potential of more nuclease resistant analogues like LNA, PNA and DNA versions of the best performing RNA inhibitor were tested. The Ki-value for the RNA and LNA 14mer were in the same range, while the PNA 14mer had a reduced efficacy (Ki = 12.5 nM) and the inhibition potential of the DNA 14mer was even lower. Specificity of the 14mers were tested in in vitro assays with RNase P RNAs from bacteria showing increased sequence variation around the CCA-binding site compared with the RNase P RNA of E. coli. The PNA 14mer had clearly better discrimination properties than the RNA analog, while the LNA 14mer showed the lowest specificity. In a next step the velocity of association was determined, with the PNA analog owing the fastest association followed by the LNA- and RNA 14mer and the DNA 14mer. The antisense mechanism was verified by lead cleavage experiments. All 14mer analogs protect RNase P RNA from lead cleavage over their entire length. These data together with data of other authors suggest that inhibitors not only block ptRNA cleavage but also fix RNase P in an inactive conformation. Because of its high specificity and fast association rate the PNA-oligomer was tested in E. coli cell cultures. Limited cellular uptake is a general problem with antisense agents, in particular for bacteria. Another advantage of PNA-oligonucleotides is that they can be easily connected with invasive peptides derived from the innate immune system. By their amphipatic nature invasive peptides preferentially interact with bacterial membranes and enhance permeability of bacterial cell envelopes. The PNA 14mer was connected with two different linkers (the new glycine-linker and the commonly used AEEA-linker) with the invasive peptide KFFKFFKFFK. First the conjugates were tested in the in vitro inhibition assay and the results suggest that neither the linker nor the peptide disturb helix formation. In vivo experiments were performed in 10 % LB-Medium and inhibition measured by a plating assay. At an assay concentration of 10 µM PNA-G-peptide no surviving E. coli wild type cells, K12 as well as AS19 (LPS defect) cells, had been detected, while the PNA-AEEA-peptide show just partial inhibition. In general inhibition patterns of PNA-peptide constructs was very similar for both with a stronger inhibition of the LPS defect strain AS19. This results suggest that still the cell envelops retard the inhibitors from cell entry, but differences in inhibition efficacy of the PNA-peptide conjugates and the PNA 14mer alone indicate also that cell entry is strongly enhanced by the attached invasive peptide. Also the uptake seamed to be very efficient as 10 minutes after addition of the PNA-G-peptide almost no surviving cells could be ascertained.