Although there are several methods for labeling and tracking RNA inside living cells, they lean heavily toward one side or another of a vexing tradeoff. At one extreme, there are labels that emit a strong signal and perturb mRNA activity. At the other extreme, there are labels that preserve natural mRNA activity and emit a weak signal. This dilemma may be resolved by a new method that has been developed by researchers at Chalmers University of Technology.
The new method can be used to generate mRNA transcripts that incorporate the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. These transcripts are readily visualized and easy to track. Also, they behave just like ordinary mRNA transcripts because the fluorescent cytosine is very much like ordinary cytosine. The fluorescent cytosine, 1,3-diaza-2-oxophenoxazine, can be enzymatically incorporated in high numbers into RNA via end-labeling reactions as well as cell-free transcription.
In a recent study, the Chalmers team showed that fluorescent cytosines can replace all the natural cytosines in a 1.2-kb-long mRNA encoding for the histone H2B fused to green fluorescent protein (H2B:GFP). This mRNA, the researchers reported, retains translation competence both in vitro and in human cells. Moreover, the researchers demonstrated that their labeled mRNA is sufficiently fluorescent to be directly visualized by confocal microscopy in a living human cell. Accordingly, the researchers suggested that their method could be used to study mRNA delivery and protein translation in a drug delivery context.
Detailed findings appeared in the Journal of the American Chemical Society, in an article titled, “Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery.”
“Analysis of the transcription products demonstrated that tCO is incorporated into RNA virtually as efficiently as native CTP and therefore constitutes a true nature-mimicking fluorescent modification in this respect,” the article’s authors wrote. “Astoundingly, we also found that tCO-labeled mRNA is translated into its correctly folded and localized protein product, both in vitro and in live cells.”
“We present the first example of a fluorescent nucleobase analogue–labeled nucleic acid that can be directly visualized in live cells, showing that tCO’s brightness and absorption at 405 nm are sufficient to overcome previous limitations with FBA probes in biological applications,” they added. “Moreover, we demonstrate how this conveniently allows for spatiotemporal monitoring of uptake, trafficking, and organelle colocalization of chemically transfected mRNA in a live cell model with simultaneous detection of its translation into H2B:GFP protein.”
The article’s authors emphasized that their method could facilitate the development of new and improved delivery strategies for next-generation nucleic acid-based drugs as well as further development of the recently successful mRNA-based vaccines.
RNA-based therapeutics offer a range of new opportunities to prevent, treat, and potentially cure diseases. But currently, the delivery of RNA therapeutics into the cell is inefficient.
“Since our method can help solve one of the biggest problems for drug discovery and development, we see that this research can facilitate a paradigm shift from traditional drugs to RNA-based therapeutics,” said Chalmers’ Marcus Wilhelmsson, one of the main authors of the article.
A challenge when working with mRNA is that the molecules are very large and charged, but at the same time fragile. They cannot get into cells directly and must therefore be packaged. The method that has proven most successful to date uses very small droplets known as lipid nanoparticles to encapsulate the mRNA. There is still a great need to develop new and more efficient lipid nanoparticles—something which the Chalmers researchers are also working on. To be able to do that, it is necessary to understand how mRNA is taken up into cells. The ability to monitor, in real time, how the lipid nanoparticles and mRNA are distributed through the cell is therefore an important tool.
“The great benefit of this method is that we can now easily see where in the cell the delivered mRNA goes, and in which cells the protein is formed, without losing RNA’s natural protein-translating ability,” noted Chalmers’ Elin Esbjörner, the second lead author of the article.
“Until now, it has not been possible to measure the natural rate and efficiency with which RNA acts in the cell, Wilhelmsson added. “This means that you get the wrong answers to the questions you ask when trying to develop a new drug. For example, if you want an answer to what rate a process takes place at, and your method gives you an answer that is a fifth of the correct, drug discovery becomes difficult.”
To ensure useful commercialization of the method, the researchers have submitted a patent application and are planning for a spin-off company, with the support of the business incubator Chalmers Ventures and the Chalmers Innovation Office.
“We believe,” the article’s authors concluded, “that the development reported here will benefit pharmaceutical industry, clinical laboratories, and academic partners aiming at furthering their understanding of uptake and endosomal escape mechanisms and allow them to take vital steps toward new and improved delivery strategies.”