A dual fluorescence-based reporter assay for real-time determination of siRNA- and antisense oligonucleotide-mediated knockdown
In recent years, many tools have been developed for targeted treatments of cancer or rare diseases including nucleic acid (NA)-based therapeutics. One of the developmental caveats is the assessment of their functionality in vivo. We therefore developed a sensitive dual fluorescence-based FluoroDetec...
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Elsevier
2025-09-01
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| Series: | Molecular Therapy: Nucleic Acids |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2162253125001854 |
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| author | Felix Heß Margarete Odenthal Elena Wasserburger-Zichel Christina Grimm Michal R. Schweiger |
| author_facet | Felix Heß Margarete Odenthal Elena Wasserburger-Zichel Christina Grimm Michal R. Schweiger |
| author_sort | Felix Heß |
| collection | DOAJ |
| description | In recent years, many tools have been developed for targeted treatments of cancer or rare diseases including nucleic acid (NA)-based therapeutics. One of the developmental caveats is the assessment of their functionality in vivo. We therefore developed a sensitive dual fluorescence-based FluoroDetect assay for the testing of small interfering RNAs (siRNAs), antisense oligonucleotides (ASOs), and RNA-binding compounds. For a proof of principle, we inserted the noncoding HSat3 RNA as a prime target into the 3′UTR of an mCherry fluorescence protein. The FluoroDetect assay was designed to be adapted by the In-Fusion cloning with any wished target site. The assay can be used as a quick transient model for short-term experiments or as a lentiviral reporter for long-term experiments. Readout of the assay is possible with fluorescence microscopy, plate reading, or flow cytometry, and can be used to measure the cellular distribution of NA therapeutics, siRNAs, and ASOs. Thus, the FluoroDetect assay is a tool to screen NA drug candidates and facilitates the optimization and quantification of NA delivery in NA-based therapies. |
| format | Article |
| id | doaj-art-aee005c0ebf34a0d9f429da3ccbee5ab |
| institution | Matheson Library |
| issn | 2162-2531 |
| language | English |
| publishDate | 2025-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj-art-aee005c0ebf34a0d9f429da3ccbee5ab2025-08-03T04:42:37ZengElsevierMolecular Therapy: Nucleic Acids2162-25312025-09-01363102631A dual fluorescence-based reporter assay for real-time determination of siRNA- and antisense oligonucleotide-mediated knockdownFelix Heß0Margarete Odenthal1Elena Wasserburger-Zichel2Christina Grimm3Michal R. Schweiger4Institute for Translational Epigenetics, Faculty of medicine and University Hospital Cologne, 50931 Cologne, Germany; Center for Molecular Medicine Cologne (CMMC), Faculty of medicine and University Hospital Cologne, University of Cologne, 50931 Cologne, GermanyInstitute of Pathology, Faculty of medicine and University Hospital Cologne, University of Cologne, 50937 Cologne, GermanyInstitute for Translational Epigenetics, Faculty of medicine and University Hospital Cologne, 50931 Cologne, Germany; Center for Molecular Medicine Cologne (CMMC), Faculty of medicine and University Hospital Cologne, University of Cologne, 50931 Cologne, GermanyInstitute for Translational Epigenetics, Faculty of medicine and University Hospital Cologne, 50931 Cologne, Germany; Center for Molecular Medicine Cologne (CMMC), Faculty of medicine and University Hospital Cologne, University of Cologne, 50931 Cologne, GermanyInstitute for Translational Epigenetics, Faculty of medicine and University Hospital Cologne, 50931 Cologne, Germany; Center for Molecular Medicine Cologne (CMMC), Faculty of medicine and University Hospital Cologne, University of Cologne, 50931 Cologne, Germany; Cologne Center for Genomics (CCG) and West German Genome Center (WGGC), University of Cologne, 50931 Cologne, Germany; Corresponding author: Michal R. Schweiger, Institute for Translational Epigenetics, Faculty of medicine and University Hospital Cologne, 50931 Cologne, Germany.In recent years, many tools have been developed for targeted treatments of cancer or rare diseases including nucleic acid (NA)-based therapeutics. One of the developmental caveats is the assessment of their functionality in vivo. We therefore developed a sensitive dual fluorescence-based FluoroDetect assay for the testing of small interfering RNAs (siRNAs), antisense oligonucleotides (ASOs), and RNA-binding compounds. For a proof of principle, we inserted the noncoding HSat3 RNA as a prime target into the 3′UTR of an mCherry fluorescence protein. The FluoroDetect assay was designed to be adapted by the In-Fusion cloning with any wished target site. The assay can be used as a quick transient model for short-term experiments or as a lentiviral reporter for long-term experiments. Readout of the assay is possible with fluorescence microscopy, plate reading, or flow cytometry, and can be used to measure the cellular distribution of NA therapeutics, siRNAs, and ASOs. Thus, the FluoroDetect assay is a tool to screen NA drug candidates and facilitates the optimization and quantification of NA delivery in NA-based therapies.http://www.sciencedirect.com/science/article/pii/S2162253125001854MT: Oligonucleotides: Diagnostics and Biosensorssmall interfering RNAantisense oligonucleotideHSat3fluorescence assayFluoroDetect |
| spellingShingle | Felix Heß Margarete Odenthal Elena Wasserburger-Zichel Christina Grimm Michal R. Schweiger A dual fluorescence-based reporter assay for real-time determination of siRNA- and antisense oligonucleotide-mediated knockdown Molecular Therapy: Nucleic Acids MT: Oligonucleotides: Diagnostics and Biosensors small interfering RNA antisense oligonucleotide HSat3 fluorescence assay FluoroDetect |
| title | A dual fluorescence-based reporter assay for real-time determination of siRNA- and antisense oligonucleotide-mediated knockdown |
| title_full | A dual fluorescence-based reporter assay for real-time determination of siRNA- and antisense oligonucleotide-mediated knockdown |
| title_fullStr | A dual fluorescence-based reporter assay for real-time determination of siRNA- and antisense oligonucleotide-mediated knockdown |
| title_full_unstemmed | A dual fluorescence-based reporter assay for real-time determination of siRNA- and antisense oligonucleotide-mediated knockdown |
| title_short | A dual fluorescence-based reporter assay for real-time determination of siRNA- and antisense oligonucleotide-mediated knockdown |
| title_sort | dual fluorescence based reporter assay for real time determination of sirna and antisense oligonucleotide mediated knockdown |
| topic | MT: Oligonucleotides: Diagnostics and Biosensors small interfering RNA antisense oligonucleotide HSat3 fluorescence assay FluoroDetect |
| url | http://www.sciencedirect.com/science/article/pii/S2162253125001854 |
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