Optimization of LNA/DNA co-oligomers as EGS inducers of mRNA degradation by RNase P: an alternative strategy to deal with antibiotic resistance

ALFONSO SOLER BISTUE; DAVIES-SALA C; CARPIO DE; ZORREGUIETA A

San Diego

Congreso; 110th American Society for Microbiology (ASM) General Meeting; 2010

American Society for Microbiology

Background: External Guide Sequence (EGS) technology has proveneffective to silence resistance genes and achieve phenotypic conversion tosusceptibility.  It is based on theutilization of endogenous RNase P and an EGS, a short oligomer that elicitsRNase P-mediated cleavage of a target RNA molecule.  This general strategy is viable only ifefficient nuclease-resistant oligonucleotide analogs that induce RNAseP-mediated degradation of the target mRNA are developed.  We have recently shown that hybrid locked nucleicacids (LNA)/DNA oligomers are good prospective EGSs.  Here we present an optimization analysis ofLNA/DNA oligomers that induce degradation of the aminoglycoside6´-N-acetyltransferase type Ib (aac(6?)-Ib)mRNA.  Furthermore we tested the effectof combining two LNA/DNA oligomers targeting different regions of the mRNA.  Methods:  EGS-mediated digestion of aac(6´)-IbmRNA was assayed by incubating radiolabeled mRNA with theappropriate EGS followed by addition of the components of RNase P.  The products were analyzed by 5%denaturing GTG-PAGE.  Results:  After identification of an aac(6´)-IbmRNA region available for interaction with a 17-residues EGS, LNA/DNAco-oligomers with varying configurations were assayed.  The configurations that mediated the highestrate of mRNA cleavage contained 5 and 4 LNA residues at the 5? and the 3? ends,respectively.  An increase in the numberof LNA residues resulted in a reduction in the ability to elicit mRNAcleavage.  Addition of the same mass ofan equimolar mix of 2 EGSs targeting different regions in the mRNA resulted inadditive but not a synergic effect.  Conclusions:  Small structural changes in EGS stronglyimproved their activity in vitro.  Abalance between the increased binding ability conferred by the LNA residues andthe structural properties conferred by the DNA residues must be found to generatea nuclease-resistant EGS.  The effect of2 EGSs was additive but not synergic.