Bridge nucleic acid/DNA gapmers as inhibitors of gene expression by multiple antisense mechanisms

Antisense interference with gene expression is usually achieved using nuclease-resistant oligonucleotide analogs that act by mRNA degradation, recruiting endogenous RNase H or RNase P, or steric hindrance of translation. Bridge nucleic acids (BNAs) are promising nucleotide analogs, and their chemical structure allows the development of new variants. Building on previous research, we evaluated gapmers composed of a short oligodeoxynucleotide flanked by BNA residues in a BNA₅-DNA₈-BNA₄ configuration, using available BNA variants: the original locked nucleic acid (LNA; 2'-O-4'-methylene locked nucleic acid), cET (2'-O,4'-ethyl bridge), cMOE (2'-O,4'-methoxyethyl bridge), and BNA^NC (2'-O,4'-aminomethylene bridge). These gapmers were tested in vitro for their ability to direct cleavage of the aac(6')-Ib mRNA, which would restore susceptibility to clinically important aminoglycosides. The assays were carried out using gapmers that target a region of the mRNA previously identified as suitable for interaction with antisense oligomers. While all gapmers showed variable RNase H-mediated activity, only the LNA-containing gapmer (LDAA) elicited RNase P-dependent degradation, demonstrating ability to mimic both RNA and DNA. Coupled in vitro transcription-translation reactions using a cell lysate or a reconstituted system confirmed inhibition of expression and ruled out steric hindrance as mechanism of action. Gapmers with the LDAA structure can act as external guide sequences (EGSs), molecules that elicit RNAse P cleavage, and as antisense compounds that work via RNase H degradation. In contrast, gapmers targeting the ribosome binding site failed to recruit endogenous RNases but strongly inhibited expression by steric hindrance. Taken together, the results show that LNA-containing gapmers with the tested configuration can act through multiple mechanisms. A single molecule can elicit both RNase H- and RNase P-mediated degradation, and, when directed to other regions such as the ribosome binding site, inhibit expression through steric hindrance, supporting the potential for synergistic inhibition of gene expression when used in combination.