Locked nucleic acid-modified antisense oligonucleotides (ASOs) can achieve strongly different degrees of target knockdown despite having similar biophysical properties and 100% homology with their target. The determinants for this observation remain largely unknown. We used multi-specific ASOs that have 100% sequence complementarity with a common target (IDO1) and a different number of diverse targets and investigated their effect on gene expression in a cell line by RNA-sequencing. We observed a significant higher chance for downregulation of long genes compared to short genes, of genes with high compared to lower expression, and of genes that have more than one binding site for the respective ASO. By investigating the expression of genes that have binding sites for more than one ASO we identified the individual binding site being an important determinant for activity. Under the selected experimental conditions we have not seen indications that availability of RNase H is a limiting factor as the number of degraded target RNA molecules correlated significantly with the number of predicted target RNA molecules. Taken together, by using multi-specific ASOs as tool compounds we identified determinants for ASO activity that can be taken into consideration to improve the selection process of highly potent and selective ASOs in the future.

中文翻译：

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通过 RNA 测序研究针对多个基因的反义寡核苷酸的活性

锁定核酸修饰的反义寡核苷酸 (ASO) 尽管具有相似的生物物理特性并且与其靶标具有 100% 的同源性，但可以实现不同程度的靶标敲除。这一观察结果的决定因素在很大程度上仍然未知。我们使用与一个共同目标（ IDO1）具有 100% 序列互补性的多特异性 ASO) 和不同数量的不同靶点，并通过 RNA 测序研究了它们对细胞系中基因表达的影响。我们观察到与短基因相比，长基因下调的机会显着更高，与低表达相比，高表达的基因，以及对各自的 ASO 具有多个结合位点的基因。通过研究具有多个 ASO 结合位点的基因的表达，我们确定了单个结合位点是活性的重要决定因素。在选定的实验条件下，我们没有看到迹象表明 RNase H 的可用性是一个限制因素，因为降解的靶 RNA 分子的数量与预测的靶 RNA 分子的数量显着相关。综合起来，