“Inhibition of amikacin resistance using an RNAse P based strategy to silence aac(6’)-Ib

SOLER BISTUE, A J C; HA, H; ZORREGUIETA A; TOLMASKY, M E

Mar del Plata

Congreso; 43th Annual Meeting SAIB; 2007

SAIB

Spread of aac(6’)-Ib among pathogenic bacteria is a growing concern as it generates resistance to the clinically important aminoglycoside amikacin (Ak). A possible strategy to overcome this problem is to silence aac(6’)-Ib. Several 17-nucleotide RNA molecules complementary to the five single stranded regions of aac(6’)-Ib mRNA, carrying the consensus sequence for RNAseP ACCA in it’s 3’-end were designed. These External Guided Sequences (EGS) were assessed in vitro for their capacity to bind to aac(6’)-Ib mRNA and their ability to direct RNAseP digestion of the messenger. These results led to the selection of five candidates to perform in vivo experiments. In vivo expression of EGS demonstrated that EGSA2 and EGSC3 were able to reduce Ak resistance, being the latter one the most effective. In this strain the mRNA level showed a 50% reduction. Degradation of EGS is a problem to further develop this technology. To face this we designed antisense compounds with the EGSC3 sequence using several non-hydrolysable nucleic acid analogs: phosphorothioates, 2’-O-Methyl and Locked Nucleic Acids (LNA) derivatives. Their binding to the mRNA and their capacity to direct RNAseP-mediated cleavage of mRNA was studied. Our results suggest that LNA derivatives are able to induce RNAse P cleavage in vitro. The use of LNA-EGS might be a viable strategy to deal with antibiotic resistance.