POE Immunoassay: Plate-based oligonucleotide electro-chemiluminescent immunoassay for the quantification of nucleic acids in biological matrices
Abstract Oligonucleotide therapeutics use short interfering RNA (siRNA) or antisense oligonucleotide (ASO) molecules to exploit endogenous systems—neutralizing target RNA to prevent subsequent protein translation. While the potential clinical application is vast, delivery efficiency and extrahepatic...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2020-06-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-020-66829-6 |
| _version_ | 1818851477169373184 |
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| author | Mai B. Thayer Sara C. Humphreys Kyu S. Chung Julie M. Lade Kevin D. Cook Brooke M. Rock |
| author_facet | Mai B. Thayer Sara C. Humphreys Kyu S. Chung Julie M. Lade Kevin D. Cook Brooke M. Rock |
| author_sort | Mai B. Thayer |
| collection | DOAJ |
| description | Abstract Oligonucleotide therapeutics use short interfering RNA (siRNA) or antisense oligonucleotide (ASO) molecules to exploit endogenous systems—neutralizing target RNA to prevent subsequent protein translation. While the potential clinical application is vast, delivery efficiency and extrahepatic targeting is challenging. Bioanalytical assays are important in building understanding of these complex relationships. The literature currently lacks description of robust and sensitive methods to measure siRNA and ASOs in complex biological matrices. Described herein is a non-enzymatic hybridization-based immunoassay that enables quantification of individual siRNA strands (antisense or sense) in serum, urine, bile, and liver and kidney homogenates. Assay utility is also demonstrated in ASOs. The assay improves upon previous works by abolishing enzymatic steps and further incorporating Locked Nucleic Acid (LNA) nucleotide modifications to increase analyte hybridization affinity and improve sensitivity, specificity, and robustness. We report an assay with an ultrasensitive dynamic range of 0.3 to 16,700 pM for siRNA in serum. The assay was submitted to full qualification for accuracy and precision in both serum and tissue matrices and assay performance was assessed with single and mixed analytes. The reliable LNA-hybridization-based approach removes the need for matrix sample extraction, enrichment or amplification steps which may be impeded by more advanced chemical modifications. |
| first_indexed | 2024-12-19T07:05:39Z |
| format | Article |
| id | doaj.art-22cff2a3388640828a7f309b40b4cccd |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-19T07:05:39Z |
| publishDate | 2020-06-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-22cff2a3388640828a7f309b40b4cccd2022-12-21T20:31:17ZengNature PortfolioScientific Reports2045-23222020-06-011011910.1038/s41598-020-66829-6POE Immunoassay: Plate-based oligonucleotide electro-chemiluminescent immunoassay for the quantification of nucleic acids in biological matricesMai B. Thayer0Sara C. Humphreys1Kyu S. Chung2Julie M. Lade3Kevin D. Cook4Brooke M. Rock5Amgen Research, Pharmacokinetics & Drug Metabolism, Amgen Inc.Amgen Research, Pharmacokinetics & Drug Metabolism, Amgen Inc.Amgen Research, Pharmacokinetics & Drug Metabolism, Amgen Inc.Amgen Research, Pharmacokinetics & Drug Metabolism, Amgen Inc.Amgen Research, Pharmacokinetics & Drug Metabolism, Amgen Inc.Amgen Research, Pharmacokinetics & Drug Metabolism, Amgen Inc.Abstract Oligonucleotide therapeutics use short interfering RNA (siRNA) or antisense oligonucleotide (ASO) molecules to exploit endogenous systems—neutralizing target RNA to prevent subsequent protein translation. While the potential clinical application is vast, delivery efficiency and extrahepatic targeting is challenging. Bioanalytical assays are important in building understanding of these complex relationships. The literature currently lacks description of robust and sensitive methods to measure siRNA and ASOs in complex biological matrices. Described herein is a non-enzymatic hybridization-based immunoassay that enables quantification of individual siRNA strands (antisense or sense) in serum, urine, bile, and liver and kidney homogenates. Assay utility is also demonstrated in ASOs. The assay improves upon previous works by abolishing enzymatic steps and further incorporating Locked Nucleic Acid (LNA) nucleotide modifications to increase analyte hybridization affinity and improve sensitivity, specificity, and robustness. We report an assay with an ultrasensitive dynamic range of 0.3 to 16,700 pM for siRNA in serum. The assay was submitted to full qualification for accuracy and precision in both serum and tissue matrices and assay performance was assessed with single and mixed analytes. The reliable LNA-hybridization-based approach removes the need for matrix sample extraction, enrichment or amplification steps which may be impeded by more advanced chemical modifications.https://doi.org/10.1038/s41598-020-66829-6 |
| spellingShingle | Mai B. Thayer Sara C. Humphreys Kyu S. Chung Julie M. Lade Kevin D. Cook Brooke M. Rock POE Immunoassay: Plate-based oligonucleotide electro-chemiluminescent immunoassay for the quantification of nucleic acids in biological matrices Scientific Reports |
| title | POE Immunoassay: Plate-based oligonucleotide electro-chemiluminescent immunoassay for the quantification of nucleic acids in biological matrices |
| title_full | POE Immunoassay: Plate-based oligonucleotide electro-chemiluminescent immunoassay for the quantification of nucleic acids in biological matrices |
| title_fullStr | POE Immunoassay: Plate-based oligonucleotide electro-chemiluminescent immunoassay for the quantification of nucleic acids in biological matrices |
| title_full_unstemmed | POE Immunoassay: Plate-based oligonucleotide electro-chemiluminescent immunoassay for the quantification of nucleic acids in biological matrices |
| title_short | POE Immunoassay: Plate-based oligonucleotide electro-chemiluminescent immunoassay for the quantification of nucleic acids in biological matrices |
| title_sort | poe immunoassay plate based oligonucleotide electro chemiluminescent immunoassay for the quantification of nucleic acids in biological matrices |
| url | https://doi.org/10.1038/s41598-020-66829-6 |
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