Now Cluzel and his associates, using a technique called fluorescence correlation spectroscopy (FCS), have shown how to monitor the link between RNA synthesis and promoters-small pieces of DNA that turn genes on and off-in a living cell.
"Because RNA molecules are intrinsically unstable, it has been extremely challenging to measure and characterize the mode of action of RNA molecules within a living cell," Cluzel said.
The techniques previously used to study RNA required killing and breaking up a cell in order to extract an RNA molecule. With those techniques, "one cannot detect in real time the underlying dynamics of RNA synthesis associated with cellular activities," he said. But the scientists in his laboratory have found a way around this with a technique called fluorescence correlation spectroscopy (FCS).
First, they use an RNA molecule that will bind to a protein called MS2. This MS2 protein also is fused to a green fluorescent protein that a laser can detect within a microscopically small volume.
In the absence of RNA, the MS2 fused to the fluorescent protein moves rapidly. But when bound to RNA, the fused proteins move more slowly. From the speed of motion, "we can infer the concentration of RNA present inside the detection volume and a single living cell," said Thuc Le, a graduate student in Biochemistry & Molecular Biology and the lead author of the PNAS paper.
This new technique will now facilitate research that may reveal how RNA molecules, like proteins, turn genes on and off, Cluzel said.