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 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.