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Fluorescent RNA aptamers are useful as markers for tracking RNA molecules inside cells and for creating biosensor devices. Förster resonance energy transfer (FRET) based on fluorescent proteins has been used to detect conformational changes, however, such FRET devices have not yet been produced using fluorescent RNA aptamers. Here we develop an RNA aptamer-based FRET (apta-FRET) system using single-stranded RNA origami scaffolds. To obtain FRET, the fluorescent aptamers Spinach and Mango are placed in close proximity on the RNA scaffolds and a new fluorophore is synthesized to increase spectral overlap. RNA devices that respond to conformational changes are developed, and finally, apta-FRET constructs are expressed in E. coli where FRET is observed, demonstrating that the apta-FRET system is genetically encodable and that the RNA nanostructures fold correctly in bacteria. We anticipate that the RNA apta-FRET system could have applications as ratiometric sensors for real-time studies in cell and synthetic biology. FRET has been used to study protein conformational changes but has never been applied to RNA aptamers. Here the authors develop a genetically encodable RNA aptamer-based FRET system on single-stranded RNA origami scaffolds, and demonstrate it can be used to study RNA conformational changes.

In the original version of this Article the last section of the Methods describing Fluorescence microscopy was inadvertently omitted during the production process. This has now been corrected in the PDF and HTML versions of the Article.