Abstract
We describe the synthesis and application of a coumarin-based photocleavable anchor for solid-phase synthesis and nonchromatographic purification of RNA oligonucleotides. In contrast to standard nitroaryl anchors, which are cleaved with ultraviolet (UV) light, the described construct requires visible light (λ = 456 nm) for photocleavage. This is especially important for RNAs that contain a phosphorothioate backbone, which is not compatible with UV light. We optimized the synthesis and purification process using a model 20-nt poly-U RNA and subsequently applied it to structured and unstructured RNA oligonucleotides that contain both phosphodiester and phosphorothioate backbones. Lastly, we purified 103-nt sgRNA and checked its functional fidelity by CRISPR experiments.