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GEN News Highlights : Mar 9, 2012
RNA Spinach Sensor Tracks Metabolites Not Amenable to Fluorescent Protein Tags
Method enabled fluorescence markers against any metabolite for real-time monitoring in live cells.!--h2>
An RNA sensor technology for fluorescence-based imaging of cellular metabolites in real time could provide scientists with a more widely applicable and sensitive approach for monitoring cellular processes associated with disease or drug activity than current imaging techniques, according to researchers at Cornell University’s Weill Medical College. The technique developed by Samie R. Jaffrey, Ph.D., and colleagues is based on the team’s previously reported RNA aptamer-fluorophore complex Spinach, which they have now linked to a ligand-binding RNA aptamer. The development of Spinach and other RNA-based mimics of green fluorescent protein (GFP) and enhanced GFP was described in Science last year.
Dr. Jaffrey’s team claims the Spinach-based imaging technique overcomes the problems associated with designing fluorescent proteins against many cell metabolites and could be used to image virtually any molecule in a cell. They report their approach and initial tests in E. coli in a paper in Science titled “Fluorescence Imaging of Cellular Metabolites with RNA.”
Oft-used techniques for the real-time imaging of small molecules in living cells rely on genetically encoded sensors comprising fluorescent proteins flanking a ligand-binding domain. Ligand binding to the sensor protein effectively induces conformational changes in the protein, triggering fluorescence, which is then detected by changes in Förster resonance energy transfer (FRET). However, Dr. Jaffrey and colleagues point out, this approach can’t be applied to every ligand because proteins that undergo conformational changes upon binding a desired target molecule aren’t always available.
To address such issues The Weill Medical College team has designed a form of RNA sensor based on ligand-selective RNA aptamers, which can be isolated using in vitro selection against virtually any small molecule. The RNA sensor construct comprises the ligand-binding aptamer linked to an RNA-fluorophore complex composed of Spinach RNA and the fluorophore 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI). Essentially, ligand binding to the aptamer induces binding of Spinach to DFHBI, leading to fluorescence.
The aptamer attaches to its ligand via one of the three Spinach stem loops the team found was essential for fluorescence. “Many aptamers are unstructured until they bind their targets,” the authors explain. “If a small molecule-binding aptamer and Spinach share the critical stem required for Spinach fluorescence, small-molecule binding can fold the aptamer and stabilize the stem, resulting in fluorescence.”
For initial tests the researchers designed candidate aptamers for ADP, S-adenosylmethionine (SAM), guanine, or GTP. These are all ligands for which there are no FRET-based sensors, they point out. The most efficient of the sensors for each of the intracellular metabolites exhibited up to 32-fold increases in fluorescence when bound to their cognate ligands. “The fluorescence increases were linear in physiological concentration ranges, the authors add. “Most sensors detected the intended target, but not related metabolites, and exhibited rapid fluorescence activation and deactivation kinetics.”
They moved on to use the RNA sensors for monitoring SAM metabolite dynamics in live DFHBI-treated E. coli expressing the SAM sensor and to follow ADP levels in E. coli using the ADP sensor. “These sensors produce about a 20-fold increase in fluorescence upon metabolite binding, unlike FRET sensors, which typically exhibit 30 to 100% increases,” the authors remark. “Because RNA aptamers can be readily generated against any biomolecule, the strategies described here should enable the design of sensors to image essentially any molecule.”
The new approach will help scientists monitor real time changes in the levels of metabolites involved in disease progression, or drug response, in single cells, Dr. Jaffrey suggests. “You could see how these levels change dynamically in response to signaling pathways or genetic changes. The ability to see metabolites in action will offer us new and powerful clues into how they are altered in disease and help us to find treatments that can restore their levels to normal. The amazing thing about RNA is that you can make RNA sequences that bind to essentially any small molecule you want.”
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