Readout of RNA structures in real time

Readout of RNA structures in real time

The fluorescent blinking of the cyanine dye bound to RNA (Alexa Fluor 647, pink star) changes depending on the structure of the RNA. When the RNA is folded like a hairpin, the fluorescent blink is fast, and when the RNA switches to a G-quadruplex, the blink is slow. Credit: Akira Kitamura

Amyotrophic Lateral Sclerosis (ALS), commonly known as Lou Gehrig’s disease and Stephen Hawking’s disease, is a neurodegenerative disease that results in the gradual loss of control over muscles in the body. It is currently incurable and the cause of the disease is unknown in over 90% of all cases – although both genetic and environmental factors are thought to play a role.

The research groups of Dr. Akira Kitamura at the Faculty of Advanced Life Science, Hokkaido University, and Prof. Jerker Widengren at the KTH Royal Institute of Technology, Sweden, have developed a novel technique capable of recognizing a characteristic structure of RNA in real time in living cells . The technique, which is based on fluorescence microscopic spectroscopy, was published in the journal Nucleic Acid Research.

“One of the genetic factors thought to be involved in the development of ALS is a specific RNA sequence that forms a four-stranded structure called a G-quadruplex,” explains Kitamura, first author of the study. “Normally, these structures regulate the expression of genes. However, a mutation on chromosome 9 in humans leads to the formation of G-quadruplexes that may play a role in neurodegenerative diseases, including ALS.”

One of the major hurdles in understanding the precise role of G-quadruplexes in disease has been the limitations of studying their formation and localization in living cells in real time. The Kitamura and Widengren groups succeeded in developing a simple, robust, and widely applicable technique that solves existing problems.

The technique follows a cyanine dye called Alexa Fluor 647 (AF647). When labeled with RNA, the fluorescent blinking state of the dye is altered as the RNA G-quadruplexes form. The groups analyzed the AF647-tagged RNA using a microscopy technique called TRAST (TRAnsient STate) monitoring to detect this fluorescent blink in real time.

“Visually, the time-resolved intensity changes of the fluorescence appear as blinking,” Kitamura describes the technique. “In TRAST, we expose cells to a specific pattern of changing light intensities and measure the average intensity of the fluorescence emitted by the RNA-bound dye in the cells over specific time intervals. By measuring changes in blinking properties, we can distinguish the structures of RNA in the cell.”

The team calibrated their experiment under laboratory conditions and determined exactly which fluorescent blinks corresponded to RNA G-quadruplexes. From this data, they were able to use TRAST to determine the position of RNA G-quadruplexes in living cells.

This work proves that cyanine dyes can provide sensitive reading parameters for the folding states of RNA G-quadruplexes in living cells and even for single cells. This in turn opens up the possibility of studying the RNA G-quadruplexes in disease in real time at the intracellular level. It can also be applied to study the folding and misfolding of proteins in cells.

More information:
Akira Kitamura et al., Trans-cis isomerization kinetics of cyanine dyes reports on the folding states of exogenous RNA G-quadruplexes in living cells, Nucleic Acid Research (2023). DOI: 10.1093/nar/gkac1255

Provided by the University of Hokkaido

Citation: Real-Time Reading of RNA Structures (2023, February 2) Retrieved February 2, 2023 from

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