Magana et al. (2025) tested and evaluated gapmers composed of a short oligodeoxynucleotide flanked by BNA residues in a BNA5-DNA8-BNA4 configuration, using available BNA variants. The variants studied were 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 BNANC (2′-O,4′-aminomethylene bridge). The research group tested the gapmers in vitro for their ability to directly cleave aac(6')-Ib mRNA. The aac(6)-Ib gene encodes the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] enzyme, responsible for the inactivation of amikacin in most amikacin-resistant Gram-negative bacteria. The inhibition of aac(6)-Ib expression would allow the use of amikacin and other aminoglycosides to treat severe multidrug-resistant infections, which would restore susceptibility to clinically important aminoglycosides.
Magana et al. showed that gapmers composed of a stretch of LNA residues flanking an oligodeoxynucleotide are most efficient at eliciting degradation by both RNases and can also reduce gene expression by steric hindrance, depending on the location of the complementary region in the target mRNA.
Bridge nucleic acid (BNA)-DNA-BNA gapmers are robust gene silencers that combine DNA-like (RNase H) and RNA-like (RNase P/steric hindrance) inhibition.
BNA gapmers use a central DNA "gap" for nuclease recruitment, such as RNase H, and chemically locked BNA wings, including BNANC, LNA, or cET, for stability and target affinity.
In a gapmer, the central DNA stretch when hybridizing to target mRNA, form a DNA/RNA duplex that recruits RNase H, which then cleaves and degrades the mRNA, preventing protein synthesis.
BNA containing gapmers can mimic RNA to act as external guide sequences (EGSs), recruiting RNase P to cleave the target RNA.
If targeted to specific regions, such as the ribosome-binding site (RBS), the gapmer physically blocks the ribosome or the splicing machinery, stopping protein production without degrading the mRNA. A central segment of deoxynucleotides, for example, 7-10 bases, binds the target RNA and serves as a substrate for RNase H. Locked Nucleic Acid (LNA) or other BNA analogs, like BNANC, cET, cMOE, on either side provide stability, increase binding affinity and increasing the melting temperature of the complement, and also improve nuclease resistance, ensuring the gapmer survives long enough in cell tissues.
Gapmers containing BNAs trigger multiple silencing pathways including RNase H and RNase P. Well designed gapmers can also create a steric block, maximizing their efficacy, resulting in enhanced binding and stability.
Reference
Magaña AJ, Phan K, Lopez J, Ramirez MS, Tolmasky ME. Bridge nucleic acid/DNA gapmers as inhibitors of gene expression by multiple antisense mechanisms. Res Sq [Preprint]. 2025 Sep 3:rs.3.rs-7390173. [PMC]
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