Chemically modified antisense oligonucleotides with phosphorothioate linkages (PS-ASOs) mediate site-specific cleavage of RNA by RNase H1 and are broadly used as research and therapeutic tools. PS-ASOs can enter cells via endocytic pathways and escape from membrane-enclosed endocytic organelles to reach target RNAs. We recently found that lysobisphosphatidic acid is required for release of PS-ASOs from late endosomes. Here, we evaluated the effects of other lipids on PS-ASO intracellular trafficking and activities. We show that free fatty acids, ceramide, and cholesterol increase PS-ASO activities. Free fatty acids induced formation of lipid droplets without changing the intracellular localization of PS-ASOs in early or late endosomes. Ceramide and cholesterol did not obviously induce the formation of lipid droplets, but cholesterol caused enlargement of endosome size and volume. Although none of those lipids appeared to influence PS-ASO internalization or intracellular trafficking processes, all led to an increase in leakiness of late endosomes. Thus, the membrane destabilization induced by these lipids likely contributes to PS-ASO release from late endosomes, which, in turn, increases PS-ASO activity.

具有硫代磷酸酯键（PS-ASO）的化学修饰的反义寡核苷酸可通过RNase H1介导RNA的位点特异性切割，被广泛用作研究和治疗工具。PS-ASO可以通过内吞途径进入细胞，并从膜封闭的内吞细胞器中逃逸，从而到达靶RNA。我们最近发现，溶血双磷脂酸是从晚期内体释放PS-ASO所必需的。在这里，我们评估了其他脂质对PS-ASO细胞内运输和活性的影响。我们表明，游离脂肪酸，神经酰胺和胆固醇会增加PS-ASO的活性。游离脂肪酸诱导脂质小滴的形成，而不改变早期或晚期内体中PS-ASO的细胞内定位。神经酰胺和胆固醇没有明显诱导脂质液滴的形成，但是胆固醇会引起内体体积和体积的增大。尽管这些脂质似乎均未影响PS-ASO的内在化或细胞内运输过程，但均导致晚期内体的渗漏增加。因此，由这些脂质引起的膜失稳可能有助于晚期内体释放PS-ASO，进而增加PS-ASO活性。