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

SOLER BISTUE A; HA H; ZORREGUIETA A; SOLER BISTUÉ AJ, MARTÍN FA, PETRONI A, FACCONE D, GALAS M, TOLMASKY ME, ZORREGUIETA A.

Mar del Plata

Congreso; Reunión Anual de SAIB; 2007

Resistance to aminoglycosides(Ag) is mostly due to a wide variety of modifying enzymes. Spread of aac(6')-Ib among pathogenic bacteria isa growing concern as it generates resistance to the clinically important Agamikacin (Ak). A possible strategy to overcome this problem is to silence aac(6')-Ib. Several 17 nucleotide RNAmolecules complementary to the five single stranded regions of aac(6')-Ib mRNA, carrying the consensussequence for RNAseP ACCA in it's 3'-end were designed. These External GuidedSequences (EGS) were assessed in vitro for their capacity to bind to aac(6')-Ib mRNA and their ability todirect RNAseP digestion of the messenger. These results led to the selection offive candidates to perform in vivo experiments. In vivo expression of EGSdemonstrated that EGSA2 and EGSC3 were able to reduce Ak resistance, being thelatter one the most effective as seen in growth curves. In this strain the mRNAlevel showed a 50% reduction. Degradation of EGS is a problem to further developthis technology. To face this we designed antisense compounds with the EGSC3sequence using several non-hydrolysable nucleic acid analogs:phosphorothioates, 2'-O-Methyl and Locked Nucleic Acids (LNA) derivatives. Their binding to the mRNA and theircapacity direct RNAseP-mediated cleavage of mRNA was studied. Ourresults suggest that LNA derivatives are able to induce RNAse P cleavage.