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

SOLER BISTUE A; HA H; CARPIO DE; JOAQUIN J

Rosario

Congreso; Reunión Anual de SAMIGE; 2008

Samige

Resistance to aminoglycosides (Ag) is mostly due to awide 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 moleculescomplementary to single stranded regions of aac(6')-Ib mRNA, carrying the consensus sequence for RNAse P ACCA in its 3'-end were designed. These External Guided Sequences (EGS) were assessed in vitro and invivo for their ability to direct RNAse P digestion of themessenger. 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 usingnon-hydrolysable nucleic acid analogs. Phosphorotioates, 2'-O-MethylRNA 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 substitutionswere very important for both inducing RNAse P mediated cleavageof the messenger and for binding of these EGS to acc(6')-IbmRNA. Stability of LNA/DNT EGS and RNA EGS to degradation inpure bacterial cultures was assessed. Importantly, LNA/DNAEGS were more resistant than RNA EGS to degradation if exposed to pure Escherichia coli cultures. This is the firstreport EGS of nucleic acid analogs being able to direct mRNA degradation by RNase P. Our results suggest that LNA/DNA EGS might bean effective tool for aac(6')-Ib silencing by an RNAse P mechanism.