Chemists at the University of Massachusetts Amherst have developed a fluorescent imaging approach that allows multiple messenger RNA molecules to be visualized simultaneously inside living mammalian cells. The method enables real-time observation of RNA dynamics using three distinct colors and is described in Nature Methods.
RNA plays a central role in cellular regulation, yet its behavior inside cells has been difficult to study directly. “There are many diseases that result from something going wrong with RNA,” said Daisy Pham, a graduate student in chemistry at UMass Amherst and lead author of the study. “And RNA plays a crucial role inside our cells: it’s the messenger that tells the cell how to make the proteins for which DNA is the master blueprint, they can turn on and off specific genes, they can organize and shape cellular structures, and other functions that need to happen perfectly for our cells to remain healthy.”
To better study RNA, the research team developed a method built upon established RNA hairpin labeling strategies that rely on fluorescent protein tags. According to senior author Jiahui Wu, assistant professor of chemistry, the challenge lies in direct observation. “We’re very curious about all of RNA’s many functions,” Wu said. “The best answer is to actually observe them inside a living cell, but they’re tiny.”
The team improved the approach by engineering fluorescent proteins that bind specific RNA motifs and emit light only when attached to their target. This conditional activation reduces background fluorescence that can obscure the signal in conventional methods. In addition, the researchers created three spectrally distinct protein tags that fluoresce green, red, or far red, allowing different RNA populations to be tracked at the same time within a single cell.
“We can now watch as different kinds of RNA in action inside a living cell,” Pham said, “and we can better understand how they do what they do.”
The authors have made the method publicly available, providing a new experimental option for studying RNA localization and dynamics in live-cell systems.
Publication Details
Pham, T.G., Ajayi, O., He, J. et al. Orthogonal RNA-regulated destabilization domains for three-color RNA imaging with minimal RNA perturbation. Nat Methods (2025). https://doi.org/10.1038/s41592-025-02905-x
RNA plays a central role in cellular regulation, yet its behavior inside cells has been difficult to study directly. “There are many diseases that result from something going wrong with RNA,” said Daisy Pham, a graduate student in chemistry at UMass Amherst and lead author of the study. “And RNA plays a crucial role inside our cells: it’s the messenger that tells the cell how to make the proteins for which DNA is the master blueprint, they can turn on and off specific genes, they can organize and shape cellular structures, and other functions that need to happen perfectly for our cells to remain healthy.”
To better study RNA, the research team developed a method built upon established RNA hairpin labeling strategies that rely on fluorescent protein tags. According to senior author Jiahui Wu, assistant professor of chemistry, the challenge lies in direct observation. “We’re very curious about all of RNA’s many functions,” Wu said. “The best answer is to actually observe them inside a living cell, but they’re tiny.”
The team improved the approach by engineering fluorescent proteins that bind specific RNA motifs and emit light only when attached to their target. This conditional activation reduces background fluorescence that can obscure the signal in conventional methods. In addition, the researchers created three spectrally distinct protein tags that fluoresce green, red, or far red, allowing different RNA populations to be tracked at the same time within a single cell.
“We can now watch as different kinds of RNA in action inside a living cell,” Pham said, “and we can better understand how they do what they do.”
The authors have made the method publicly available, providing a new experimental option for studying RNA localization and dynamics in live-cell systems.
Publication Details
Pham, T.G., Ajayi, O., He, J. et al. Orthogonal RNA-regulated destabilization domains for three-color RNA imaging with minimal RNA perturbation. Nat Methods (2025). https://doi.org/10.1038/s41592-025-02905-x