Genetically encoded FRET-based nanosensor for insect juvenile hormone

Abstract

Juvenile hormone (JH) plays a pivotal role in regulating insect development and reproductive maturation. Disruption of JH biosynthesis, transport, or degradation results in aberrant or incomplete development, a phenomenon that has been exploited in pest control through the use of insect growth regulators (IGRs) that interfere with hormonal signaling. However, the identification of novel IGR candidates has been hindered by the absence of a rapid and sensitive method for quantifying JH, limiting progress in this area. To address this limitation, we developed a genetically encoded fluorescent biosensor for JH detection, based on Förster resonance energy transfer (FRET). The sensor is constructed from the JH-binding protein (JHBP) of Bombyx mori, flanked by two fluorescent proteins—mTFP1 and mVenus—at the N- and C-termini, respectively. The initial construct, mTFP1-JHBP-mVenus, showed no observable FRET in the absence of ligand, suggesting that conformational changes necessary for FRET induction upon JH binding were not adequately achieved. Through structural optimization, including the insertion of mTFP1 into a flexible region of JHBP, a FRET signal became inducible upon JH binding. This optimized construct, termed FRET JH Indicator Agent (FREJIA), represents the first ratiometric, genetically encoded fluorescent biosensor specific to JH. FREJIA responds to JH I, II, III, as well as the synthetic JH analog methoprene, by emitting a FRET signal upon ligand binding. The sensor exhibits a detection threshold below 1.0 µM for JH III, enabling sensitive and quantitative ratiometric imaging of JH in living mammalian cells.

Our findings demonstrate that FREJIA enables real-time, selective detection of juvenile hormones and provides a powerful tool for screening and evaluating novel IGR candidates. By facilitating high-throughput, non-invasive monitoring of JH activity in live-cell systems, FREJIA offers a promising platform for advancing the discovery and development of next-generation insect growth regulators.

Keywords: JH, juvenile hormone, JHBP, juvenile hormone binding protein, FRET, Förster resonance energy transfer, IGR, insect growth regulator, FREJIA FREt Jh Indicator Agent, HTS, high-throughput screening, HEK293T, Human Embryonic