chnology 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 with a protein scaffold, he prepared an RNA library and did selection experiments to pull out RNA sequences that stabilized a small synthetic version of the GFP fluorophore and allowed it to fluoresce (Science, 333:642-46, 2011).
The minimum sequence required to induce fluorescence with the small molecule is 80 nucleotides long, and Jaffrey has christened it “Spinach.” The vegetable nomenclature riffs on widely used fluorescent proteins—such as mCherry, developed by Roger Tsien’s lab at the University of California, San Diego—that are named for fruit. Jaffrey is also working on orange and red versions of Spinach he’s calling Carrot and Radish. (See “It’s Easy Being Green,” The Scientist, January 2012.)
Pros & Cons
The method removes a step from the design process, says Tyagi, who is testing the technique in his own lab. “Now we don’t have to engineer the GFP, all we have to do is engineer the RNA.” It also avoids the delivery problems in some of the hybridization methods: the RNA sequences that bind the fluorophore are incorporated into the noncoding regions of the gene of interest, while the small-molecule fluorophore is added to the cell medium and easily crosses the cell membrane. Another perk is that unlike GFP, which can take up to 20 minutes to fold and fluoresce, Spinach becomes maximally fluorescent almost immediately, which could be useful for monitoring short-lived processes within cells, Jaffrey says. Initially, Jaffrey tested Spinach with a high-abundance, noncoding RNA in the cell. As with the other techniques, imaging low-abundance RNAs such as mRNAs may require tweaking the system, either by using concatenated sequences of Spinach, employing other colors such as red (which have less natural background fluorescence in the cell), or both.
Availability and cost
According to Jaffrey, Cornell has already licensed the technology, which could soon lead to commercially available plasmids that are specialized for particular applications. “I give [the plasmid] away to anyone who asks,” Jaffrey says, adding 