Small-molecule based Proximity Labeling for RNA Localization Studies

Abstract

<p>Cellular activities rely on the coordination among various biomolecules. Over the years, proximity labeling methods have becoming more and more important and popular thanks to the informative insights revealed by this type of methodology. Such molecular tools can combine the spatial information with the identity information available from a different technology, such as proteomics and RNA sequencing, thus providing unprecedented molecular interaction information. For many years, proximity labeling has been focused on protein-protein interactions and it was not until 2017 that the concept has been achieved with RNAs, later further supported with high throughput transcriptomic analysis. Currently available approaches relying on fusion proteins to achieve proximity labeling with high spatiotemporal resolution is limited by the inevitable introduction of exogenous proteins inside cells being studied. In lieu of extending such powerful molecular interaction analysis tools into complex biological systems, such as clinical samples, it is important to enrich the chemical toolbox for proximity labeling with functional small molecules that can be introduced into complex biological systems readily with limited perturbation to the system of interest.</p><p>Here, we report two types of molecular design to achieve RNA proximity labeling in live cells. The first approach focuses on labeling chromatin-associated RNAs with green light irradiation, where a photosensitizer (dibromofluorescein, DBF) is conjugated to a Hoechst motif, a stain commonly used in fluorescence confocal imaging for nucleus indication. The Hoechst motif and the photosensitizer was linked with an ethylene glycol linker, allowing Hoechst to effectively prevent DBF from generating singlet oxygen. Due to the interaction between dsDNA and Hoechst motif, DBF will be guided to chromatin in the nucleus and then activated since Hoechst motif is no longer available to quench DBF and occupied by the minor groove of dsDNA. The other molecular design relies on selenium containing Nile Blue derivatives (SeNB), which can generate enhanced yield of singlet oxygen upon near-infrared light irradiation at the presence of lipid membranes. Conjugating with different tail groups, SeNB derivatives can target organelle membranes selectively, thus allowing RNA proximity labeling respectively at different membrane locations. This method can be readily applied in more complex biological systems due to its wash-free and deep penetration features.</p>