 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 that chemists could synthesize the fluorophore relatively easily. The fluorophore is also commercially available: Lucerna Technologies sells 5 mg for $299.99.