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Published on: December 5, 2020
A dual fluorescence-based reporter assay for real-time determination of siRNA- and antisense oligonucleotide-mediated
Felix Heß1,2, Margarete Odenthal3, Elena Wasserburger-Zichel1,2
1Institute for Translational Epigenetics, Faculty of medicine and University Hospital Cologne, 50931 Cologne, Germany.
Abstract:
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.
Insights
Researchers developed FluoroDetect, a sensitive assay for testing nucleic acid (NA) therapeutics like small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs) in vivo. This tool aids in screening drug candidates and optimizing NA delivery for targeted therapies.
Area of Science:
- Biotechnology
- Molecular Biology
- Drug Discovery
Background:
- Nucleic acid (NA)-based therapeutics offer targeted treatment for diseases.
- Assessing the in vivo functionality of NA therapeutics is a critical developmental challenge.
Purpose of the Study:
- To develop a sensitive assay for evaluating NA therapeutics, including small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs).
- To provide a tool for screening NA drug candidates and optimizing NA delivery in vivo.
Main Methods:
- Development of a dual fluorescence-based assay named FluoroDetect.
- Incorporation of a target RNA (HSat3) into the 3' untranslated region (3'UTR) of a fluorescent protein (mCherry).
- Adaptability of the assay for transient or long-term experiments using In-Fusion cloning and lentiviral reporters.
Main Results:
- The FluoroDetect assay demonstrated sensitivity in testing siRNAs, ASOs, and RNA-binding compounds.
- Readout is achievable via fluorescence microscopy, plate reading, or flow cytometry.
- The assay enables measurement of cellular distribution for NA therapeutics.
Conclusions:
- FluoroDetect is a versatile tool for screening NA drug candidates.
- Facilitates optimization and quantification of NA delivery in NA-based therapies.
- Supports the advancement of targeted cancer and rare disease treatments.
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