Development of a genetically encodable FRET system using fluorescent RNA aptamers

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.

Aptamers: Uptake mechanisms and intracellular applications

“…To obtain FRET, the Spinach and Mango were placed in close proximity on the scaffolds and genetically encoded in the plasmid. The apta-FRET constructs expressed in E. coli showed the tolerable FRET output [37], suggesting that the apta-FRET system might be used as real-time imaging in living cells and biosensors.…”

“…For the molecular interactions in living cells, two fluorescent RNA aptamers, Spinach and Mango, were engineered using RNA origami scaffolds for detection of conformational changes, called aptamersbased Förster resonance energy transfer (apta-FRET) [37]. To obtain FRET, the Spinach and Mango were placed in close proximity on the scaffolds and genetically encoded in the plasmid.…”

Nucleic‐Acid Structures as Intracellular Probes for Live Cells

“…These probes gave higher sensitivity compared to Spinach . Jepsen et al developed a genetically encoded FRET‐based strategy for imaging intracellular analytes by incorporating Spinach and Mango aptamers in close proximity in single‐stranded RNA origami scaffolds . The validity of this system was demonstrated by imaging SAM in E. coli cells.…”

An Enzymatic Active Site Embedded in a DNA Nanostructure

“…The functionalisation of nanodevices presents a serious challenge in the field of nanotechnology and is often inspired by the existing solutions found in nature. To achieve its function, a state of the art nanodevice employs binding of enzymes [1,2,3], fluorophores [3,4,5,6], antibodies [7,8], aptamers [5,8,9] and other active elements [12,38], while nanostructures itself act as a scaffold for the organisational purposes. Biological solutions however, are not easily implemented.…”