Detection of Antisense Oligonucleotides from Biological Samples by Ligase Detection Reaction Using T4 RNA Ligase 2

This study developed a detection method for antisense oligonucleotides (ASOs) by ligase detection reaction (LDR) using T4 RNA ligase 2 (T4Rnl2). The LDR was applied to detect a nusinersen-based ASO, an 18-mer 2'-O-methoxyethyl-modifiedoligonucleotide with phosphorothioate linkages. The method enabled detection of the ASO at concentrations of 0.08-10 nM in aqueous samples. In addition, when combined with mixed-mode solid phase extraction for ASO spiked samples, the method detected 0.8-100 nM in serum and 0.04-25 nmol/g tissue in spinal tissue. The method was further applied to determine ASO levels in plasma and spinal tissue following intrathecal injection in mice. These results demonstrated that LDR using T4Rnl2 was a simple and sensitive approach for detecting ASOs in biological samples.