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.
Gruegelsiepe
Heike
H
Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, D-35037 Marburg, Germany.
Brandt
Ole
O
Hartmann
Roland K
RK
D001412:Bacillus subtilis / Q000378:metabolism; D001483:Base Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010455:Peptides / Q000737:chemistry; D017433:Protein Structure, Secondary; D012313:RNA / Q000737:chemistry; D016337:RNA, Catalytic / Q000737:chemistry; D043262:Ribonuclease P / Q000037:antagonists & inhibitors* / Q000235:genetics*
D010455:Peptides; D016337:RNA, Catalytic; D012313:RNA; D043262:Ribonuclease P; D008274:Magnesium