Comparison of different antisense strategies in mammalian cells using locked nucleic acids, 2′‐O‐methyl RNA, phosphorothioates and small interfering RNA

Locked nucleic acids (LNAs) and double‐stranded small interfering RNAs (siRNAs) are rather new promising antisense molecules for cell culture and in vivo applications. Here, we compare LNA–DNA–LNA gapmer oligonucleotides and siRNAs with a phosphorothioate and a chimeric 2′‐O‐methyl RNA–DNA gapmer with respect to their capacities to knock down the expression of the vanilloid receptor subtype 1 (VR1). LNA–DNA–LNA gapmers with four or five LNAs on either side and a central stretch of 10 or 8 DNA monomers in the center were found to be active gapmers that inhibit gene expression. A comparative co‐transfection study showed that siRNA is the most potent inhibitor of VR1–green fluorescent protein (GFP) expression. A specific inhibition was observed with an estimated IC50 of 0.06 nM. An LNA gapmer was found to be the most efficient single‐stranded antisense oligonucleotide, with an IC50 of 0.4 nM being 175‐fold lower than that of commonly used phosphorothioates (IC50 ∼70 nM). In contrast, the efficiency of a 2′‐O‐methyl‐modified oligonucleotide (IC50 ∼220 nM) was 3‐fold lower compared with the phosphorothioate. The high potency of siRNAs and chimeric LNA–DNA oligonucleotides make them valuable candidates for cell culture and in vivo applications targeting the VR1 mRNA.