Genetically Encoded FRET-Based Nanosensor for Insect Juvenile Hormone

High Resolution Image Download MS PowerPoint Slide Juvenile hormone (JH) plays a central role in insect development and reproductive maturation. However, the molecular transport mechanism remains unclear because JH is difficult to detect nondestructively and in real-time. A new genetically encoded fluorescent biosensor based on Förster resonance energy transfer (FRET) is composed of a JH-binding protein (JHBP) derived from Bombyx mori, which is fused with two fluorescent protein pairs (mTFP1 and mVenus) and exhibits a ratiometric readout. The mTFP1-JHBP-mVenus proteins, flanked by the two fluorescent proteins at the N- and C-termini, respectively, exhibited no discernible FRET as a primary FRET JH sensor. This suggests that inducible FRET by JH binding may have been compromised. Optimizing the insertion of mTFP1 into JHBP enabled inducible FRET in the presence of JH. The resulting construct, the FRET JH Indicator Agent (FREJIA), is the first ratiometric, genetically encoded, and fluorescent biosensor for JH detection. FREJIA interacts with the binding protein and emits a resonance energy transfer signal in response to binding to JH I, II, and III and methoprene at nanomolar ranges. FREJIA enables ratiometric imaging of JH III in live mammalian cells. Our results showed that FRET can be applied to the selective and ratiometric monitoring of JH levels.