Coumarin-Based Photolabile Solid Support Facilitates Nonchromatographic Purification of RNA Oligonucleotides
Ian McClain, Hilal Dagci, Bhoomika Pandit, Maksim Royzen
TL;DR
This paper introduces a new coumarin-based method for purifying RNA without using chromatography, using visible light instead of UV.
Contribution
A visible-light-responsive coumarin anchor for RNA purification that avoids UV incompatibility with phosphorothioate backbones.
Findings
The coumarin anchor was successfully used to purify a 20-nt poly-U RNA model.
The method works for both structured and unstructured RNA with phosphodiester and phosphorothioate backbones.
A 103-nt sgRNA was purified and shown to function correctly in CRISPR experiments.
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.
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Taxonomy
TopicsPhotochromic and Fluorescence Chemistry · DNA and Nucleic Acid Chemistry · Radical Photochemical Reactions