Development of RNA proximity labeling approaches mediated by fluorogenic photosensitizers

Abstract

RNA localization is crucial for maintaining cellular biological functions. Proximity labeling methods using enzymes, protein-ligand systems, or small molecules have been developed to study RNA localization transcriptome-wide. Among these methods, light-triggered proximity labeling mediated by photosensitizers provides high spatiotemporal resolution. A spatial-restricted functional turn-on of the photosensitizer in the designated location is essential for precise labeling. However, most synthetic photosensitizers remain active, regardless of their locations, increasing the risk of non-specific labeling. A fluorogenic photosensitizer that only initiates proximity labeling at the designated site can address this issue. We developed two chemical approaches for live cell RNA proximity labeling, whose labeling on-off is precisely controlled by the light-triggered photosensitization of designed fluorogenic photosensitizers. 1) The near-infrared (NIR)-activated fluorogen activating protein (FAP)-malachite green (MG) system achieves precise localization and functional turn-on of small molecule MG derivatives in the site of FAP expression. 2) The green light-activated self-localized Hoe-Ac2DFB affords proximity labeling in the chromatin region. The photophysical properties of halogenated MG derivatives with and without FAP binding were systematically investigated experimentally and computationally in Chapter 2. Next, in Chapter 3, monoiodo-substituted MG derivative (MG-HI), hypothesized to be more effective than the other MG derivatives, was synthesized and confirmed to possess the highest 1O2 generation ability and RNA labeling ability in vitro. MG-HI mediated RNA proximity labeling was further validated at a transcriptomic level in two cell lines expressing FAP in nucleus and endoplasmic reticulum, together with its protein proximity labeling ability. In Chapter 4, three self-localized Hoe-Ac2DBFs photosensitizers were designed and synthesized with the aim of achieving proximity labeling in the chromatin region without genetic manipulation. The linkers with different lengths and hydrophobicity were utilized to construct the Hoe-EG3-Ac2DBF, Hoe-EG1-Ac2DBF, and Hoe-C4-Ac2DBF. Their fluorogenic turn-on properties were compared both in vitro and in cellulo. In summary, two types of fluorogenic photosensitizers, NIR-activated FAP-MG-HI and self-localized Hoe-EG3-Ac2DBF, were developed and applied in RNA proximity labeling in live cells. We anticipate that these two approaches, with high spatiotemporal labeling resolution and convenient on-off control by light, will have broad applications in complex biological systems.