Heß Felix, Odenthal Margarete, Wasserburger-Zichel Elena, Grimm Christina, Schweiger Michal R
Institute 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.
Mol Ther Nucleic Acids. 2025 Jul 17;36(3):102631. doi: 10.1016/j.omtn.2025.102631. eCollection 2025 Sep 9.
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 . 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.
近年来，已经开发出许多用于癌症或罕见病靶向治疗的工具，包括基于核酸（NA）的疗法。其中一个开发方面的注意事项是对其功能的评估。因此，我们开发了一种基于双荧光的灵敏的FluoroDetect检测方法，用于检测小干扰RNA（siRNA）、反义寡核苷酸（ASO）和RNA结合化合物。作为原理验证，我们将非编码HSat3 RNA作为主要靶标插入到mCherry荧光蛋白的3'非翻译区（3'UTR）。FluoroDetect检测方法设计为可通过In-Fusion克隆适应任何所需的靶位点。该检测方法可作为短期实验的快速瞬时模型，或作为长期实验的慢病毒报告基因。该检测方法可通过荧光显微镜、酶标仪读数或流式细胞术进行读数，并可用于测量NA疗法、siRNA和ASO的细胞分布。因此，FluoroDetect检测方法是一种筛选NA候选药物的工具，有助于基于NA的疗法中NA递送的优化和定量。
