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Detection of the phosphorothioate oligonucleotide fomivirsen using a ligase detection reaction with polymerase chain reaction

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  • Novel Analytical Technologies Contributing to Clinical and Pharmaceutical Research Fields
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Abstract

This study aimed to develop a simple and sensitive detection method for fomivirsen, a 21-nucleotide phosphorothioate oligonucleotide used as a nucleic acid medicine, using a ligase detection reaction. A ligation probe was designed to hybridize with fomivirsen and polymerase chain reaction (PCR) primers, with a deoxyuridine part between the primer binding sites. The probe was ligated to a circular product by Taq DNA ligase, and the resulting product was converted to a linear form through the removal of the uracil base using uracil DNA glycosylase. The linear product was then quantified using real-time PCR. The developed method could detect 0.025–6.4 nM of fomivirsen in water and HeLa genomic DNA solutions and 0.6–160 nM of fomivirsen in mouse serum in combination with an extraction method based on alkalinization and neutralization. This method could be useful for not only detecting fomivirsen but also other functional oligonucleotides composed of phosphorothioate oligonucleotides. In summary, this study presents a practical and effective approach to the detection of the nucleic acid medicine fomivirsen.

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Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

This work was supported by JSPS KAKENHI Grant Number 20K06991.

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Authors and Affiliations

  1. School of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi, Chiba, 274-8555, Japan

    Naoki Harikai, Haruka Kakuda, Takumi Uchiyama, Tsubaki Yamamoto, Kazumasa Zaima & Kazufusa Shinomiya

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  1. Naoki Harikai
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  2. Haruka Kakuda
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  3. Takumi Uchiyama
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  4. Tsubaki Yamamoto
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  5. Kazumasa Zaima
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  6. Kazufusa Shinomiya
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Contributions

NH designed the study. NH, HK, TU, and TY performed the experiments and analyzed the data. NH wrote the manuscript. KZ and KS critically revised the manuscript. All authors revised and approved the final manuscript.

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Correspondence to Naoki Harikai.

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The authors declare that they have no conflict of interest.

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Harikai, N., Kakuda, H., Uchiyama, T. et al. Detection of the phosphorothioate oligonucleotide fomivirsen using a ligase detection reaction with polymerase chain reaction. ANAL. SCI. 40, 965–971 (2024). https://doi.org/10.1007/s44211-024-00539-1

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  • DOI: https://doi.org/10.1007/s44211-024-00539-1

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