A platform for live-cell metabolic incorporation of fluorescent nucleosides for real-time imaging of RNA dynamics

Background

RNA plays a central role in cellular biology, governing processes such as gene expression, protein synthesis, and the response to environmental stimuli. Understanding the dynamics of RNA—how it is synthesized, trafficked, and degraded within living cells—is crucial for unraveling the complexities of cellular function and disease mechanisms. Live-cell imaging of RNA provides a powerful means to study these processes in real time, offering insights into RNA behavior during physiological events and under stress conditions. However, the field has long faced challenges in developing tools that allow for the direct, transcriptome-wide visualization of RNA in living cells, as most available techniques are limited in scope or require disruptive sample preparation.

Current approaches to live-cell RNA imaging, such as MS2 tagging, RNA aptamers, CRISPR-Cas-based systems, and chemoenzymatic labeling, suffer from significant limitations. These methods are often restricted to monitoring individual RNA transcripts and typically require the introduction of non-native sequences or proteins, which can perturb normal cellular function. Additionally, metabolic labeling strategies using modified nucleosides (e.g., with alkyne or azide groups) generally necessitate cell fixation due to the incompatibility of bioorthogonal labeling chemistries with live-cell conditions. As a result, these approaches cannot provide real-time, transcriptome-wide information in living cells. Direct metabolic incorporation of fluorescent nucleosides could, in principle, overcome these barriers, but most such nucleosides have only been used in vitro due to poor cellular uptake, inefficient enzymatic processing, or inadequate fluorescence properties in the cellular environment.

Technology

This technology enables real-time, live-cell imaging of RNA dynamics by metabolically incorporating fluorescent cytidine analogues—specifically pyrroloC and tC—into cellular RNA. The process is facilitated by engineered overexpression of uridine-cytidine kinase 2 (UCK2), which phosphorylates these nucleosides, allowing them to enter the nucleotide salvage pathway and be integrated into newly synthesized RNA. Once incorporated, the intrinsic fluorescence of these analogues permits direct visualization of RNA synthesis, trafficking, and degradation in living cells using confocal or epifluorescence microscopy. The system achieves single-cell resolution without requiring cell fixation or non-native tagging sequences, making it possible to monitor RNA behavior during normal physiological processes as well as under conditions of oxidative stress. The method is rapid, with detectable labeling within 30 minutes, and is compatible with a range of experimental conditions.

This technology is differentiated by its operational simplicity, versatility, and ability to provide transcriptome-wide labeling of RNA in living cells. Unlike previous RNA imaging methods—which often require complex genetic tagging, fixation, or bioorthogonal chemistries incompatible with live-cell studies—this approach leverages the cell’s own salvage pathways and engineered kinase expression to achieve efficient, direct labeling. The use of fluorescent nucleosides that base pair canonically ensures minimal perturbation to RNA structure and function, while the specificity of UCK2 for these analogues ensures robust incorporation. The platform not only enables real-time tracking of RNA synthesis and turnover but also facilitates the discovery of novel RNA-protein granules and insights into RNA metabolism under stress. Its adaptability to other nucleoside analogs and cell types further broadens its utility for fundamental research and biomedical applications.

Benefits

• Enables direct, real-time visualization of RNA synthesis, trafficking, and degradation in living cells at single-cell resolution.
• Overcomes limitations of previous RNA imaging methods by allowing transcriptome-wide labeling without cell fixation or non-native tagging sequences.
• Utilizes metabolically incorporated fluorescent cytidine analogues (pyrroloC and tC) compatible with the nucleotide salvage pathway via engineered UCK2 overexpression.
• Facilitates rapid RNA labeling detectable within 30 minutes, suitable for dynamic studies including responses to oxidative stress.
• Allows discovery and study of novel RNA-protein granules and RNA metabolism alterations under physiological and stress conditions.
• Operationally simple and adaptable to various cell types and nucleoside analogues, supporting broad biomedical and fundamental RNA research applications.

Applications

• Live-cell RNA synthesis imaging
• High-throughput RNA drug screening
• Real-time RNA stress response analysis
• Single-cell RNA turnover studies
• Visualization of RNA-protein interactions

Additional info

This technology utilizes engineered overexpression of uridine-cytidine kinase 2 (UCK2) to metabolically incorporate fluorescent cytidine analogues, such as pyrroloC and tC, into cellular RNA. These substrates enter the nucleotide salvage pathway, allowing real-time, single-cell resolution imaging of RNA synthesis, trafficking, and turnover. This approach enables direct visualization of transcriptome dynamics in living cells via microscopy without requiring fixation or non-native tagging sequences.