Methods for the quantification of antisense oligonucleotides (AONs) provide insightful information on biodistribution and intracellular trafficking. However, the established methods have not provided information on the absolute number of molecules in subcellular compartments or about how many AONs are needed for target gene reduction for unconjugated AONs. We have developed a new method for nuclear AON quantification that enables us to determine the absolute number of AONs per nucleus without relying on AON conjugates such as fluorophores that may alter AON distribution. This study describes an alternative and label-free method using subcellular fractionation, nucleus counting, and locked nucleic acid (LNA) sandwich enzyme-linked immunosorbent assay to quantify absolute numbers of oligonucleotides in nuclei. Our findings show compound variability (diversity) by which 247,000-693,000 LNAs/nuclei results in similar target reduction for different compounds. This method can be applied to any antisense drug discovery platform providing information on specific and clinically relevant AONs. Finally, this method can directly compare nuclear entry of AON with target gene knockdown for any compound design and nucleobase sequence, gene target, and phosphorothioate stereochemistry.

中文翻译：

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未结合的，无标记的锁定核酸寡核苷酸的核和细胞质定量。

定量反义寡核苷酸（AONs）的方法提供了有关生物分布和细胞内运输的有见地的信息。但是，已建立的方法尚未提供有关亚细胞区室中分子的绝对数目或未结合的AON的靶基因还原需要多少个AON的信息。我们已经开发了一种用于核AON定量的新方法，该方法使我们能够确定每个原子核AON的绝对数量，而无需依赖于可能会改变AON分布的AON共轭物（例如荧光团）。这项研究描述了一种使用亚细胞分级分离，核计数和锁定核酸（LNA）夹心酶联免疫吸附测定法来定量核中寡核苷酸绝对数量的替代方法和无标记方法。我们的研究结果显示了247,000-693,000 LNA /细胞核的化合物变异性（多样性），导致不同化合物的目标减少相似。该方法可应用于提供有关特定和临床相关AON信息的任何反义药物发现平台。最后，该方法可以直接比较AON的核进入与靶基因敲低的任何化合物设计和核碱基序列，基因靶以及硫代磷酸酯立体化学。