Evaluation of Gene Expression Knock‐Down by Chemically and Structurally Modified Gapmer Antisense Oligonucleotides

Abstract

We analyzed the effect of modified nucleotides within gapmer antisense oligonucleotides on RNase H mediated gene silencing. Additionally, short hairpins were introduced into antisense oligonucleotides as structural motifs, and their influence on biological and physicochemical properties of pre-structured gapmers was investigated for the first time. The results indicate that two LNA residues in specified positions of the gap flanking regions are sufficient and favorable for efficient knock-down of the β-actin gene. Furthermore, the introduction of other modified nucleotides, i. e. glycyl-amino-LNAÀ T, 2'-O-propagyluridine, polyamine functionalized uridine, and UNA, in specified positions, also increases the inhibition of β-actin expression. Importantly, the presence of hairpins within the gapmers improves their silencing properties.

Investigation of Sequence‐Penetration Relationships of Antisense Oligonucleotides

“…In a recent study that used PS-modified gapmers flanked with LNA-modified residues, the addition of a small (3 bp) or medium (5 bp) double-stranded stem to the 3'-end of the gapmer resulted in a more active oligonucleotide, while the addition of a 7 bp stem did not improve activity. [53] When the same length stems were added on the 5'-end the ASO with the 5 bp stem was the least active, which implies that the local context of the secondary structure also matters. The authors hypothesized that the increasing efficiency of the oligos with the hairpin structure was a result of higher stability against exonucleases, which is especially relevant for gapmers which have unmodified residues.…”

“…There is some prior work that suggested that secondary structure formation can affect ASO activity. In a recent study that used PS‐modified gapmers flanked with LNA‐modified residues, the addition of a small (3 bp) or medium (5 bp) double‐stranded stem to the 3′‐end of the gapmer resulted in a more active oligonucleotide, while the addition of a 7 bp stem did not improve activity [53] . When the same length stems were added on the 5′‐end the ASO with the 5 bp stem was the least active, which implies that the local context of the secondary structure also matters.…”