Shin, MinwookMeda Krishnamurthy, PranathiDevi, GitaliWatts, Jonathan K2023-04-252023-04-252021-12-17Shin M, Meda Krishnamurthy P, Devi G, Watts JK. Quantification of Antisense Oligonucleotides by Splint Ligation and Quantitative Polymerase Chain Reaction. Nucleic Acid Ther. 2022 Feb;32(1):66-73. doi: 10.1089/nat.2021.0040. Epub 2021 Dec 17. PMID: 34928745; PMCID: PMC8817697.2159-334510.1089/nat.2021.004034928745https://hdl.handle.net/20.500.14038/51988Reliable detection and quantification of antisense oligonucleotides (ASOs) in experimental and clinical specimens are essential to understand the biological function of novel oligonucleotide-based therapeutics. In this study, we describe a method to detect and quantify ASOs in biological samples, whereby the ASO acts as a splint to direct the ligation of complementary probes and quantitative real-time PCR was used to monitor ligation products. Low levels of 2'-O-methoxyethyl (2'-O-MOE) gapmer ASO in serum, liver, kidney, lung, heart, muscle, and brain tissues can be detected over a 6-log linear range for detection using this method. This method allows quantification of various types of chemically modified ASOs, including phosphorothioate linkage, 2'-O-methyl, 2'-O-MOE, and locked nucleic acid, as well as siRNAs. This method does not require probe modifications, and can be performed using standard laboratory equipment; making it a fast, sensitive, and reliable technique that can be widely applied. This detection method may find potential applications in detection of therapeutic oligonucleotides in biological samples.enThis Open Access article is distributed under the terms of the Creative Commons License [CC-BY] (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Attribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/SplintR ligaseantisense oligonucleotidesqPCRquantificationJournal ArticleNucleic acid therapeutics