Enhancement of the Inhibitory Activity of oatp Antisense Oligonucleotides by Incorporation of 2‘-O,4‘-C-Ethylene-Bridged Nucleic Acids (ENA) without a Loss of Subtype Selectivity

Abstract

Antisense oligonucleotides (AONs) that specifically target the genes of rat organic anion transporting polypeptide (oatp) subtypes were selected by using antisense in vitro selection (AIVS) and a conventional gene alignment program (GAP). When we incorporated several of our original 2'-O,4'-C-ethylene-bridged nucleic acid (ENA) residues into AONs, which were designed as gapmers containing a series of 2'-deoxynucleotides in the center, at both the 3' and 5' ends, the inhibitory activity of these oatp AONs was enhanced and their inhibition was mediated by RNase H cleavage. Moreover, these ENA AONs did not lose their oatp selectivity. These strategies of using AIVS and GAP to select AONs followed by incorporation of ENA residues were effective for synthesizing oatp subtype-specific AONs.

Inhibition of VEGF mRNA by 2′-O,4′−C−Ethylene-Bridgednucleicacids (ENA®) Antisense Oligonucleotides and Their Influence on Off-Target Gene Expressions

“…We have confirmed the intracellular antisense activity of ENA oligonucleotides against organic anion transporting polypeptide (oatp). [10] The ENA oligonucleotides also exhibit excellent triplex formation with dsDNA. [11] They could be used for exon skipping to bind dystrophin pre-mRNA and inhibit its splicing reaction.…”

Antisense applications for biological control

“…It has been demonstrated that fully modified ASOs (i.e., with 2 0 ribose modifications at all nucleotide positions) act predominantly by inhibiting mRNA translation [Crooke, 1999;Fulford et al, 2000]. However, modified ASOs with six to eight DNA bases in the center lacking ribose modification at the 2 0 position (known as ''GAPmers'') efficiently activate RNase H to degrade the target mRNA Zamaratski et al, 2001;ten Asbroek et al, 2002]. Like unmodified and fully modified ASOs, GAPmers form stable duplexes with target mRNA, although the stability is decreased with increasing length of the alkyl chain and with an increasing number of modified nucleotides in a sequence.…”

2'-O,4'-C-Ethylene-Bridged Nucleic Acids (ENA) as Next-Generation Antisense and Antigene Agents

“…17,19,20) ENA-DNA-ENA gapmer-designed oligonucleotides that can undergo RNase H-mediated degradation are used as AONs. When ENA-modified AONs against vascular endothelial growth factor (VEGF) mRNA were introduced into human cancer A549 cells in the presence of a cationic polymer, more than 90% inhibition of VEGF mRNA production was observed after RT-PCR analysis.…”

“…19,20) When several ENA residues were incorporated into AONs, the inhibitory activity of these oatp AONs was enhanced. Moreover, these ENA AONs did not lose their oatp-subtype selectivity.…”