nal technique for tracking RNA inside living cells, developed about 15 years ago, uses GFP as the fluorescent reporter molecule. Previously, researchers had used the coat protein from the MS2 bacteriophage, which binds to a defined 19-nucleotide sequence of RNA, as a way to capture RNA and RNA-binding proteins. By fusing GFP to this MS2 protein, Robert Singer and his colleagues at Albert Einstein College of Medicine in Bronx, New York, first demonstrated that they could follow RNA as it migrated within cells (Molecular Cell, 2:437-45, 1998).
The first step is adding copies of MS2’s RNA-binding sequence, which forms a characteristic stem loop structure, to the noncoding region of the gene of interest. In addition, the cells need to be transfected with a plasmid so that they can express the MS2-GFP fusion protein. There’s also an inherent problem to surmount—the background fluorescence of the fusion protein before it binds to the target RNA. Singer and his colleagues realized that they’d need to insert many copies—24, and sometimes even 48—of the MS2 stem loop within the RNA of interest to overcome this “haze of GFP” and get sufficient signal to track a single molecule moving within the cell in real time.
Researchers have used the technique in many different cell types, including yeast, Drosophila, and various mammalian cells. Singer and his colleagues have even developed MS2-GFP transgenic mice that track mRNA generated by the ß-actin gene (Nat Methods, 8:165-70, 2011). They are currently developing a second transgenic mouse, and others could use the technique to develop their own mouse models.
Pros & Cons
On the plus side, because the probes are expressed within the cell, you don’t have to worry about delivering large, highly charged reagents across the cell membrane. One potential problem, however, is that the MS2 protein includes a nuclear trafficking signal, prompting questions about whether that signal could modify the localization behavior of the RNA-protein complex. Right now, because there is only one targeting sequence, you can only track one RNA sequence at a time. The Singer lab is working on a two-color system.
Equipment and cost
Singer has contributed the plasmids encoding the MS2-GFP fusion protein and 24 copies of the MS2 stem loops to the nonprofit repository Addgene, which sells them for $65 each. Addgene receives nearly 300 requests per month for these reagents, Singer says.
When MS-2 GFP fusion isn’t enough
Let’s say you want to track multiple RNAs at the same time, or you’re concerned about the effects of a bulky MS2-GFP fusion protein on the mobility of your RNA. A newer approach addresses these needs by employing smaller probes that recognize their RNA targets through complementary Watson-Crick base pairing.
Sanjay Tyagi and his colleagues at the University of Medicine and Dentistry of New Jersey in Newark developed molecular beacons designed to recognize specific sequences engineered into the noncoding region of a gene of interest. The molecular beacons are hairpin sequences of RNA with a fluorescent dye at one end and a quencher molecule at the other. When these free structures move around a cell in a closed conformation, the quencher blocks fluorescence. As the hairpin binds to the complementary RNA target, it opens and the dye lights up.
Pros & Cons
Because some RNAs are relatively rare within a cell, with an expression rate of just 20 to 50 copies, getting a reliable signal for a single molecule of RNA can still require some tweaking, says Tyagi. You’ll need multiple probes, typically 32, or multiple copies of the same probe. On the plus side, molecular beacons can be designed to recognize different sequences and glow with different colors, so you can monitor more than one RNA molecule at the same time. Tyagi and his team recently used molecular beacons labeled with two different dyes to follow splicing patterns of genes (Cell, 147:1054-65, 2011). To deliver these synthetic probes across the cell membrane, Tyagi’s team microinjects the probes directly into the cells they are studying.
Tyagi’s te