Filling the gap in LNA antisense oligo gapmers: the effects of unlocked nucleic acid (UNA) and 4'-C-hydroxymethyl-DNA modifications on RNase H recruitment and efficacy of an LNA gapmer

• Published in: Molecular bioSystems 2009-01, Vol.5 (8), p.838-843
• Main Authors: Fluiter, Kees, Mook, Olaf R F, Vreijling, Jeroen, Langkjaer, Niels, Højland, Torben, Wengel, Jesper, Baas, Frank
• Format: Article
• Language: English
• Subjects: Cell Line, Tumor; DNA - chemistry; Gene Knockdown Techniques - methods; Humans; Oligonucleotides - chemistry; Oligonucleotides, Antisense - chemistry; Ribonuclease H - chemistry
• ISSN: 1742-206X; 1742-2051
• DOI: 10.1039/b903922h

Stability against nucleases, affinity for the targeted mRNA and the ability to recruit RNase H are prerequisites for antisense oligonucleotide (AON) applications where gene expression knockdown is required. Typically chimeric gapmer AON designs are used with a central continuous stretch of RNase H recruiting nucleotides (e.g. phosphorothioate DNA), flanked by affinity and stability-enhancing modified nucleotides. However, many types of nucleotide modifications in the central DNA gap can disturb RNase H function. Here we present studies into two different types of nucleotide modifications, a flexible acyclic RNA analog named unlocked nucleic acid (UNA) and 4'-C-hydroxymethyl-DNA in the gap of an LNA (locked nucleic acid) flanked gapmer. We compared the efficacy of mRNA degradation by the gap modified LNA antisense gapmers in cell-free assays and cultured cells. This study shows that both UNA and 4'-C-hydroxymethyl-DNA gap insertions are compatible with RNase H activity when used sparingly. However, multiple 4'-C-hydroxymethyl-DNA modifications are better tolerated by RNase H than multiple UNA modifications in the gap. Furthermore, this report shows that LNA gapmer AONs with multiple 4'-C-hydroxymethyl-DNA moieties in the gap can mediate target knockdown in vivo.