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 approximately 70 nM). In contrast, the efficiency of a 2'-O-methyl-modified oligonucleotide (IC50 approximately 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.