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.