Abstract
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.
Highlights
Fluorescent RNA aptamers are useful as markers for tracking RNA molecules inside cells and for creating biosensor devices
We set out to construct a Förster resonance energy transfer (FRET) system based on fluorescent RNA aptamers and examined the spectral properties of fluorophores bound by Spinach[6] (Fig. 1a) and Mango[8, 26] (Fig. 1b)
The modified fluorophore difluoro-4-hydroxybenzylidene imidazolinone (DFHBI)-1T displays brighter fluorescence compared to DFHBI and is slightly red-shifted to better fit with standard Green fluorescent protein (GFP) filters[27]
Summary
Fluorescent RNA aptamers are useful as markers for tracking RNA molecules inside cells and for creating biosensor devices. Similar sensors based on riboswitches have been produced for cyclic di-GMP13, cyclic di-AMP14, and thiamine 5′pyrophosphate[15] The folding of such riboswitches has been studied using FRET, where regions of interest on the RNA were chemically labeled with small-molecule fluorophores making it possible to follow changes in conformation due to ligandbinding[16,17,18], such labeled riboswitches are not expressible inside cells. Gene expression under different promotors were quantified by expressing two different RNA aptamers that bound externally added Cy3- or Cy5-modified ligands, providing an expressible system for real-time assessment of promotor activity[19] This system was genetically encodable, it lacked structural control of the transcribed aptamers needed to make a system that could change conformation upon binding of a molecule of interest. We demonstrate that the apta-FRET constructs are genetically encodable by observing FRET when expressing the constructs in E. coli
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
