Comparison of Configurations and Chemistries of External Guide Sequences to Inhibit Gene Expression with Antisense Technologie

The use of antibiotics to treat bacterial infections is increasingly ineffective worldwide. Genes disseminated by bacteria such as aac(6')-Ib, render aminoglycoside antibiotics ineffective, requiring discovery of new antibiotics or alternatives strategies for decreasing this genetic expression. Antisense technology with complementary external guide sequences (EGS) can target mRNA to elicit ribozyme RNase P degradation and reduce gene expression. EGS analogs composed of both locked nucleic acid residues and deoxyribonucleic acid residues have shown potential to induce RNase P activity. A newer generation of bridged nucleic acids was recently synthesized that displayed positive binding affinity to RNA. In this study, EGS analogs of various configurations of LNA/DNA and BNANC/DNA with the same nucleotide sequence to target two regions of aac(6')-IbmRNA. mRNA degradation was measured to determine which configuration of hybrid analogs efficiently recruited RNase P cleavage. LNA/DNA gapmer analogs with five 5'-end and four LNA 3'-end residue substitutions displayed equivalent or better mRNA degradation compared to natural RNA EGSs. BNANC/DNA EGSs did not induce substantial RNase P cleavage activity. Chemistry differences between the two generations of bridged nucleic acids impacted the structural nature of the EGS-mRNA complex in which RNase P activity was not induced effectively. Configurations that best induced cleavage in these two regions should be tested in alternate genes to confirm universality.