“Optimization of LNA/DNA co-oligoers as EGS inducers of mRRNA degradation by RNAse P: an alternative strategy to deal with antibiotic resistance”

SOLER BISTUE, A J C; DAVIES SALA, C.; CARPIO DE; ZORREGUIETA, A; TOLMASKY, M E

San Diego

Congreso; General Meeting of the ASM; 2010

American Society for Microbiology

Background: External Guide Sequence (EGS) technology has proven effective to silence resistance genes and achieve phenotypic conversion to susceptibility.  It is based on the utilization of endogenous RNase P and an EGS, a short oligomer that elicits RNase P-mediated cleavage of a target RNA molecule. This general strategy is viable only if efficient nuclease-resistant oligonucleotide analogs that induce RNAse P-mediated degradation of the target mRNA are developed.  We have recently shown that hybrid locked nucleic acids (LNA)/DNA oligomers are good prospective EGSs.  Here we present an optimization of LNA/DNA oligomers that induce degradation of the aminoglycoside 6´-N-acetyltransferase type Ib (aac(6’)-Ib) mRNA. Methods: EGS-mediated digestion of 5´-end-labeled aac(6´)-Ib mRNA was assayed by incubating radiolabeled aac(6´)-Ib mRNA with the appropriate EGS followed by addition of the protein and RNA components of RNase P.  The products were analyzed by 5% denaturing GTG-PAGE. Results: After identification of an aac(6´)-Ib mRNA region available for interaction with a 17-residues EGS, LNA/DNA co-oligomers with varying configurations were assayed.  The configurations that mediated the highest rate of mRNA cleavage contained 5 and 4 LNA residues at the 5’ and the 3’ ends, respectively. An increase in the number of LNA residues resulted in a reduction in the ability to elicit mRNA cleavage.  Addition of 2 EGSs targeting different regions in the mRNA resulted in additive but not a synergic effect. Conclusion:  A balance between the increased binding ability conferred by the LNA residues and the structural properties conferred by the DNA residues must be found to generate a nuclease-resistant EGS that can be active in vivo.