ASO Synthesis and Purification

LNA-modified gapmers were designed with fully modified phosphorothioate backbones and were synthesized on a MerMade 192X synthesizer (Bioautomation, TX, USA) following standard phosphoramidite protocols. The final 5′-dimethoxytrityl (DMT) group was left on the oligonucleotide. After synthesis, the oligonucleotides were cleaved from the solid support using aqueous ammonia and subsequently deprotected at 65°C for 5 h. The oligonucleotides were purified by solid-phase extraction in TOP DNA cartridges (Agilent, Glostrup, Denmark) using the lipophilic DMT group as a chromatographic retention probe. After eluting impurities, the DMT group was removed by treatment with dichloroacetic acid. As the last step in the purification process, the oligonucleotides were eluted from the cartridge and the eluate was evaporated to dryness. The oligonucleotides were dissolved in phosphate-buffered saline (PBS) and the oligonucleotide concentration in solution determined using Beer-Lambert’s law by calculating the extinction coefficient and measuring UV absorbance. Oligonucleotide identity and purity were determined by reversed-phase ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS).