A new microscopic technique allows the real-time study of RNA G-quadruplexes in living cells, which has implications for the fight against amyotrophic lateral sclerosis.
Amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig’s disease and Stephen Hawking’s disease, is a neurodegenerative disease that causes us to gradually lose control over the body’s muscles. It is currently incurable and the cause of the disease is unknown in more than 90% of all cases – although genetic and environmental factors are believed to be involved.
The research groups of Dr. Akira Kitamura at the Department of Advanced Life Science, Hokkaido University, and Prof. Jerker Widengren from the KTH Royal Institute of Technology, Sweden, has developed a new method that can detect the structure of RNA in real time in living cells. This method, based on fluorescence-microscopic spectroscopy, was published in the journal Nucleic Acids Research.
One of the genetic factors believed to be involved in the development of ALS is a specific sequence of RNA that forms a four-stranded structure, called a G-quadruplex. Normally, these structures control gene expression. However, a mutation on chromosome 9 in humans results in the formation of G-quadruplexes that may play a role in neurodegenerative diseases including ALS.”
Dr. Akira Kitamura, first author of the study
One of the major obstacles to understanding the precise role of G-quadruplexes in disease has been the limitations of studying their structure and location in living cells in real time. Kitamura and Widengren’s teams have succeeded in creating a simple, powerful and widely used method that solves the problems at hand.
This method uses a cyanine dye called Alexa Fluor 647 (AF647). When labeled with RNA, the fluorescence intensity of the dye is changed by the formation of RNA G-quadruplexes. The teams analyzed the RNA labeled AF647 using a microscopy technique called TRAST (TRAnsient State) to detect this fluorescence blink in real time.
“Visually, time-resolved changes in fluorescence intensity appear to blink”, said Kitamura, explaining the method. “In TRAST, we expose the cells to a specific method of changing the light intensity and measure the average fluorescence intensity from the dye combined with the RNA in the cells over a certain period of time. RNA inside the cell.”
The team tested their experiments under laboratory conditions, determining exactly how much fluorescence blinking corresponds to RNA G-quadruplexes. From this information, they were able to locate RNA G-quadruplexes in living cells using TRAST.
This work proves that cyanine dyes can give clear data to read the states of RNA G-quadruplexes in living cells, even in single cells. This, in turn, allows the possibility to study RNA G-quadruplexes in real time at the intra-cellular level. It can also be used to study the folding and unfolding of proteins in cells.
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Journal reference:
Kitamura, A., and al. (2023) Trans-cis isomerization kinetics of cyanine dyes reports on the folding states of abnormal RNA G-quadruplexes in living cells. Nucleic Acids Research. doi.org/10.1093/nar/gkac1255.
