Imaging and Tracking RNA in Live Mammalian Cells via Fluorogenic Photoaffinity Labeling

• DOI: 10.1021/acschembio.4c00848
• Publication Date: 2025-02-15T13:58:36Z
• Authors: Alexandria L. Qui...; Diane B. Karloff; Tewoderos M. Ayele; Tatiana F. Flores; Gerry Chen; Zachary T. McEachin; Arielle N. Valdez...; Jennifer M. Heemstra
• Keywords: Photoaffinity labeling; Live cell imaging; Affinity label; Fluorescent labelling

Cellular RNA labeling using light-up aptamers that bind to and activate fluorogenic molecules has gained interest in recent years as an alternative protein-based approaches. Aptamer-based systems are genetically encodable cover the entire visible spectrum. However, inherently temporary nature of noncovalent aptamer–fluorogen interaction limits utility these imaging does not withstand dye washout, dissociation can compromise tracking. We propose limitations be averted through covalent labeling. Here, we describe a photoaffinity approach which aptamer ligand is functionalized with photoactivatable diazirine reactive group such irradiation UV light results attachment interest. In addition robustness linkage, this benefits from ability achieve spatiotemporal control over To demonstrate approach, incorporated linker into malachite green fused single copy Cajal body-associated small nuclear well cytoplasmic mRNA. observed improved sensitivity for live cell target upon demonstrated visualization dynamics time scale minutes. The uniquely enables time-resolved experiments, whereas approaches, molecule transferred between different molecules, compromising envision future applications method wide range investigations cellular localization, dynamics, protein-binding properties RNAs.