An orthogonal strategy for dual-location RNA labeling with APEX2 and singlet oxygen mediated methods in live cells

Abstract

<p>Emerging transcriptomic studies uncover that the precise RNA localization at a specific time is essential to perform their biological functions.^[1] Though there are various biochemical strategies to dissect RNA localization in live cells,^[2] they typically focus on one subcellular location at a time and thus can’t fully capture RNA dynamics. Herein, we adopted mechanistically orthogonal APEX2 and singlet oxygen (¹O₂) mediated methods to perform dual location labeling in live cells. APEX2, an enzyme catalyzing the hydrogen peroxide (H₂O₂)-dependent formation of phenoxyl/aniline radicals, has been applied in diverse subcellular locales.^[2] In addition, the short-lived ¹O₂ with high reactivity has been utilized to map RNA subcellular localization. The ¹O₂ source can be either photosensitizers with specific targeting motifs or MiniSOG, a ¹O₂ generation protein.^[2] Since APEX2 mediated labeling requires the expression of exogenous fusion protein (such as mito-APEX2 cells stably expressing APEX2 in mitochondria matrix), we selected small molecule-based ¹O₂ generation for dual mode labeling to avoid expressing another protein. DRAQ5, a commercially available DNA-intercalating photosensitizer ^[3] can be employed readily in mito-APEX2 cells for the nucleus-restricted labeling with ¹O₂.</p><p>Tagging RNA with orthogonally clickable handles at different subcellular locations sequentially can effectively describe the translocation process on the transcriptome-wide level. To achieve the overarching aim, the phenol derivative, Ph_N₃ was designed for APEX2 mediated labeling in mitochondrial matrix, and propargyl amine (PA) was adopted for ¹O₂ mediated labeling in nucleus. As a proof of concept, we characterized two sequential labeling procedures to illustrate the orthogonality between ¹O₂ and APEX2 mediated proximity labeling in mito-APEX2 cells. At the imaging level, the APEX2 and ¹O₂-induced labeled sites were visualized by strain-promoted alkyne-azide cycloaddition with DBCO conjugated fluorophore and copper(I)-catalyzed alkyne-azide cycloaddition with azide-fluorophore, respectively. In terms of transcriptomic labeling, we confirmed that Ph_N₃ rarely showed reactivity to RNA with ¹O₂, while PA failed to efficiently tag RNA with APEX2 and H₂O₂ in live cells. Overall, the dual location labeling was successfully achieved in mito-APEX2 cells via the two mechanistically orthogonal methods. We envision our report will inspire more studies to unveil complex RNA translocation in different biological systems.</p>