To identify chemistries and strategies to improve the potency of MOE second generation ASOs, we have evaluated gapmer antisense oligonucleotides containing BNAs having N−O bonds. These modifications include N-MeO-amino BNA, N-Me-aminooxy BNA, 2′,4′-BNA^NC[NMe], and 2′,4′-BNA^NC bridged nucleoside analogues. These modifications provided increased thermal stability and improved in vitro activity compared to the corresponding ASO containing the MOE modification. Additionally, ASOs containing N-MeO-amino BNA, N-Me-aminooxy BNA, and 2′,4′-BNA^NC[NMe] modifications showed improved in vivo activity (>5-fold) compared to MOE ASO. Importantly, toxicity parameters, such as AST, ALT, liver, kidney, and body weights, were found to be normal for N-MeO-amino BNA, N-Me-aminooxy BNA, and 2′,4′-BNA^NC[NMe] ASO treated animals. The data generated in these experiments suggest that N-MeO-amino BNA, N-Me-aminooxy BNA, and 2′,4′-BNA^NC[NMe] are useful modifications for applications in both antisense and other oligonucleotide based drug discovery efforts.