FAP-MG Fluorogenic Photosensitizer for Non-wash RNA Proximity Labeling upon Near-infrared (NIR) Light Activation

Abstract

<p>The survival of cells relies on the intricate cooperation of biomolecules involved. Over the years, increasing evidence has suggested that well-regulated RNA translocation inside live cells is ubiquitous and important for cell survival. Various strategies have been developed for RNA proximity labeling to study RNA localization, including utilization of enzymes, small functional molecules, or construction of protein-ligand systems. Comparing to enzyme-based approaches whose labeling capacity could be sensitive to specific subcellular environment, chemical photosensitizers are more robust and the labeling on-off can be precisely controlled by light irradiation.</p><p>More photosensitizers in near infrared (NIR) regions are needed to versatile the proximity labeling toolbox, allowing deep penetration in tissue samples as well as integrative studies on biomolecule translocation in live cells. The previous reported photosensitizers for proximity labeling mainly relies on irradiation in the visible region (< 550 nm). For example, miniSOG enzyme utilizes a naturally occurring photosensitizer flavin to achieve singlet oxygen (¹O₂) generation with 365 nm light irradiation over a long period of time for RNA proximity labeling and the chemical photosensitizer dibromofluorescein-HaloTag can be spatiotemporally activated with light of 530 nm to produce ¹O₂with higher efficiency and durability.</p><p>The complex self-assembled by fluorogen activating protein (FAP) and malachite green (MG) is a well-established protein-ligand system for biomolecular tracking as well as <em>in situ</em> oxidation perturbation. Meanwhile, the MG derivatives only functional-on after binding to FAP through rotation restriction, which results in high spatial resolution without any washing steps. Together with the ability to generate ¹O₂ with long wavelength of light, FAP-MG affords a platform to achieve RNA proximity labeling upon NIR light irradiation. Through systematic characterization of different halogen-substituted MG derivatives, we revamped the FAP-MG system by introducing monohalogen-subsituted MG. This new FAP-MG system keeps a satisfactory <em>Φ</em>_FL with a relatively high yield of ¹O₂ than the corresponding dihalogen-substituted derivatives. Herein, the MG-HI with high ¹O₂generation efficiency were applied for RNA proximity labeling via 660-nm light irradiation in live cells, where the spatial specificity was achieved by expressing FAP in a particular subcellular location. Two cell lines stably expressing FAP in nucleus and endoplasmic reticulum lumen were utilized for proof-of-concept studies. The fast cellular uptake and spatial-restricted activation of MG probes were validated by live cell confocal microscope imaging. Benefited from the high binding affinity between MG derivatives and FAP, MG in nanomole range can occupy almost all available FAP proteins expressed in both cell lines. The fluorescence images of in-situ labeled biomolecules, with RNA dot blot and RT-qPCR results indicated RNA proximity labeling was achieved. Such proximity labeling is also compatible to high throughput transcriptome wide analysis. We anticipate that this newly developed no-wash RNA proximity labeling method with NIR light irradiation will help elucidate RNA dynamics in more complex biological systems.</p>