eting 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 technology is licensed to several companies. For example, BioSearch Technologies sells molecular beacons starting at $95 for 10 nanomoles of a short oligonucleotide hairpin sequence labeled at both ends.
TRACKING AU NATUREL
Hands off that RNA
Both of the previous techniques require modifying the noncoding region of the RNA you’d like to track. If you’d like to understand the behavior of completely natural, unmodified RNA molecules, you need a different system.
Philip Santangelo and his team at Georgia Institute of Technology solved this problem using multiply labeled tetravalent RNA imaging probes (MTRIPs). These probes use oligonucleotides that are each labeled with two or three fluorophores of a single color and then covalently attached to biotin, which binds strongly to streptavidin, clumping the oligonucleotides together to produce clusters of about 10 fluorophores. Two or three MTRIPs targeted to different sequences on the native RNA can achieve sufficient signal for single molecules to stand out from background with up to three different dye colors (Meth Enzymol, 505:383-99, 2012).
Pros & Cons
You’ll have to find a way to deliver the MTRIPs into cells, Santangelo says. His group typically uses streptolysin O, a bacterial toxin that reversibly opens pores in the cell membrane, though it’s also possible to microinject them as Tyagi does with his probes.
Availability and cost
Although these probes are not commercially available, the components are easy to obtain and are not difficult to assemble, Santangelo says. His group estimates that it costs $0.15 per well to make a 30 nM concentration of MTRIPs using a 24-well plate (Nat Meth, 6:347-49, 2009).
STRIPPING DOWN GFP
A new technique on the block may circumvent some of the limitations of other RNA tracking approaches.
Samie Jaffrey and his colleagues at Weill Cornell Medical College in New York stripped GFP’s fluorescence down to its essence—the fluorophore that glows as the protein folds around it in a rigid structure. But instead of stabilizing the fluorophore