Targeting GI-SINE (Growth inducing B2-SINE) RNA in DRG neurons by Antisense Oligonucleotides (ASO)

We identified 2 specific motifs that are enriched in GI-SINE RNA expressed in sciatic-nerve injured DRG neurons compared to non-regulated B2-SINE RNAs. We designed a mix of 5 ASOs (21nt phosphorothioate DNA with LNA-flanks) that target these motifs and transfected those into cultured dorsal root ganglia (DRG) neurons. Control ASO was based on a 21nt non-targetting sequence from shControl backbone (addgene plasmid #85741). Primary DRG neurons were transfected using Dharmafect-4 with ASO at 50nM final concentration 1 hour after plating. Cultures were processed for RNA extraction 48h after transfection for RNA sequencing to identify changes in B2-SINE expression. Overall design: Each replicate (1-4) is based on indepdendent total DRG culture preparation, from 1 mouse in each repeat that was distributed between experimental conditions. Experimental conditions are: ASO control 50nM, ASO mix (GI-SINE ASOs) and untransfected cultures.

我们在坐骨神经损伤背根神经节（Dorsal Root Ganglion, DRG）神经元表达的GI-SINE RNA中，鉴定出相较于非调控型B2-SINE RNA（B2短散在核RNA）显著富集的2个特定基序（motif）。我们设计了由5条靶向上述基序的反义寡核苷酸（Antisense Oligonucleotide, ASO）组成的混合体系，该反义寡核苷酸为带有锁核酸（Locked Nucleic Acid, LNA）侧翼的21nt硫代磷酸DNA，并将其转染至体外培养的背根神经节神经元中。对照反义寡核苷酸的序列取自shControl骨架的21nt非靶向序列，对应Addgene质粒#85741。原代背根神经节神经元在铺板1小时后，使用Dharmafect-4转染试剂，以终浓度50nM的剂量完成反义寡核苷酸转染。转染48小时后，对培养细胞进行RNA提取并开展RNA测序，以鉴定B2-SINE RNA的表达变化。整体实验设计：每一组重复样本（1-4号）均基于独立的背根神经节细胞培养体系，每次重复使用1只小鼠的组织，样本被分配至各实验分组中。实验分组包括：50nM对照反义寡核苷酸组、GI-SINE靶向反义寡核苷酸混合体系组，以及未转染细胞培养组。