August 11, 2022

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A new mRNA labeling technology not affect the activity of mRNA molecules

A new type of mRNA labeling technology that does not affect the activity of mRNA molecules

 

 

A new mRNA labeling technology not affect the activity of mRNA molecules.  A method of labeling mRNA molecules has been developed, relying on fluorescent analogs of cytosine.

Messenger RNA molecules combined with cytosine analogues not only maintain their natural properties and activity, but can also be tracked in real time through a microscope. This method is of great significance for promoting the development of new rna drugs.


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Although there are several methods for labeling and tracking RNA in living cells, they severely tend to be an annoying trade-off. In an extreme case, some tags will send out strong signals and disrupt the activity of messenger RNA. At the other extreme, some tags can maintain the natural activity of messenger RNA and send out weak signals. A new method developed by researchers at Chalmers University of Technology may solve this problem.

The new method can be used to generate mRNA transcripts containing tCO triphosphate, which is a fluorescent tricyclic cytosine analog. These transcripts are easy to visualize and track. Moreover, they behave like ordinary mRNA transcripts, because fluorescent cytosines are very similar to ordinary cytosines. Fluorescent cytosine, 1,3-diazo-2-oxazine, can be incorporated into RNA in large quantities by end labeling reaction and free transcriptase.

In a recent study, the Chalmers team found that fluorescent cytosine can replace all natural cytosines in 1.2 kb long mRNA, which encodes histone H2B fused to green fluorescent protein (H2B:GFP).

Researchers report that this messenger RNA retains the ability to translate in vitro and in human cells. In addition, the researchers proved that their labeled mRNA has enough fluorescence to be directly observed in living human cells using a confocal microscope. Therefore, the researchers suggest that their method can be used to study mRNA delivery and protein translation in the context of drug delivery.

The detailed research results were published in an article titled “Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery” in the Journal of the American Chemical Society.

“Analysis of transcripts shows that tCO is actually as effectively integrated into RNA as natural CTP, and therefore constitutes a true fluorescence modification that mimics nature in this respect,” the author of the article wrote. “Surprisingly, we also found that both in vitro and in living cells, tco-labeled mRNA is translated into correctly folded and positioned protein products.”

They added: “We showed the first example of nucleic acid labeled with fluorescent base analogues, which can be directly visualized in living cells, indicating that the brightness of tCO and the absorption at 405 nm are sufficient to overcome the biological application of FBA probes. “In addition, we showed how it is convenient to realize the spatiotemporal monitoring of uptake, transport and organelle co-localization of chemically transfected mRNA in a living cell model, while detecting its translation into H2B:GFP protein. “

 

A new mRNA labeling technology not affect the activity of mRNA molecules
A new method of labeling mRNA molecules has been developed, which can then be observed under a microscope and tracked in real time. This new technology does not affect the properties of mRNA molecules or subsequent activity, and may be of great significance in promoting the development of new RNA drugs. [Chalmers University of Technology]


The authors of this article emphasize that their method can facilitate the development of new and improved delivery strategies for a new generation of nucleic acid drugs, as well as the further development of recent successful mrna vaccines.

 

RNA-based treatment methods provide a series of new opportunities for the prevention, treatment and potential cure of diseases. But at present, the efficiency of RNA therapy into cells is very low.

Dr. Marcus Wilhelmsson of Chalmers said: “Because our method can help solve one of the biggest problems in drug discovery and development, we see that this research can facilitate a paradigm shift from traditional medicine to RNA-based treatment.”

One of the challenges of working with messenger RNA is that these molecules are very large and carry an electric charge, but at the same time they are very fragile. They cannot enter the cell directly, so they must be packaged. The most successful method proved so far is to use very small droplets called lipid nanoparticles to encapsulate mRNA. There is still a great need to develop new and more efficient lipid nanoparticles-Chalmers researchers are also working on this. To do this, it is necessary to understand how mRNA is taken up by cells. Therefore, the ability to monitor how lipid nanoparticles and mRNA are distributed in cells in real time is an important tool.

Dr. Elin Esbjörner of Chalmers, associate professor, second lead author of the article.

Wilhelmsson added that so far, it has not been possible to measure the natural rate and efficiency of RNA acting in cells. This means that when you try to develop a new drug, you will get the wrong answer. For example, if you want to know the rate at which a process occurs, and your method gives an answer of one-fifth the correct rate, then drug discovery becomes difficult. “

In order to ensure the commercialization of the method, the researchers have submitted a patent application and plan to establish a business with the support of business incubators Chalmers Ventures and Chalmers Innovation Office. Spin off the company.

“We believe,” the author of the article concludes, “the development reported here will benefit the pharmaceutical industry, clinical laboratories, and academic partners, aiming to further understand absorption and endocytic escape mechanisms and allow them to take important steps to New and improved delivery strategies.”

 

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