title: 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.
abstract: 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.
authors: Dept of Neurogenetics AMC, 1105 AZ Amsterdam, The Netherlands. k.fluiter@amc.uva.nl
authors: Kees
authors: K
authors: Fluiter
authors: Olaf R F
authors: OR
authors: Mook
authors: Jeroen
authors: J
authors: Vreijling
authors: Niels
authors: N
authors: Langkjaer
authors: Torben
authors: T
authors: Højland
authors: Jesper
authors: J
authors: Wengel
authors: Frank
authors: F
authors: Baas
mesh_terms: D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry*; D055785:Gene Knockdown Techniques / Q000379:methods; D006801:Humans; D009841:Oligonucleotides / Q000737:chemistry*; D016376:Oligonucleotides, Antisense / Q000737:chemistry*; D016914:Ribonuclease H / Q000737:chemistry*
chemical_list: D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; C477371:locked nucleic acid; D004247:DNA; D016914:Ribonuclease H