Non-hydrolyzable antisense oligonucleotides direct aac(6?)-Ib mRNA cleavage by RNAse P

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

Rosario

Congreso; V Congreso Argentino de Microbiología General SAMIGE; 2008

SAMIGE

Resistance to aminoglycosides (Ag) is mostly due to a wide variety of modifying enzymes. Spread of aac(6?)-Ib among pathogenic bacteria is a growing concern as it generates resistance to the clinically important Ag amikacin (Ak). A possible strategy to overcome this problem is to silence aac(6?)-Ib. RNA molecules complementary to single stranded regions of aac(6?)-Ib mRNA, carrying the consensus sequence for RNAse P ACCA in it?s 3?-end were designed. These External Guided Sequences (EGS) were assessed in vitro and in vivo for their ability to direct RNAseP digestion of the messenger. Two of them, EGSA2 and EGSC3, were able to reduce Ak resistance. Degradation of EGS is a problem to further develop this technology. To face this we designed antisense compounds with the EGSC3 sequence using non-hydrolysable nucleic acid analogs. Phosphorotioates, 2?-O-Methyl RNA and Locked Nucleic Acids (LNA) were assayed. Among them, only oligonucleotides composed of LNA derivatives and deoxinucleotides (DNT), LNA/DNT EGS, were able to direct RNAse P-mediated precise cleavage of aac(6?)-Ib mRNA. Time course experiments showed that mRNA cleavage mediated by LNA/DNT EGS occurred at a similar rate to RNA EGS. Deoxyoligonucleotides with LNA substitutions in different positions of the EGS were designed and assayed for binding to the messenger.and mRNA cleavage in the presence of RNAse P .  Results showed that configuration of substitutions were very important for both inducing RNAse P mediated cleavage of the messenger and for binding of these EGS to acc(6?)-Ib mRNA. Stability of LNA/DNT EGS and RNA EGS to degradation in pure bacterial cultures was assessed. Importantly, LNA/DNA EGS were more resistant than RNA EGS to degradation if exposed to pure E.coli cultures. This is the first report of nucleic acid EGS analogs being able to direct mRNA degradation by RNase P. Our results suggest that LNA/DNA EGS might be an effective tool for aac(6?)-Ib silencing by an RNAse P mechanism, however in vivo evidence is needed to further support this idea.