A New Highly Efficient Molecule for Both Optogenetic and Chemogenetic Control Driven by FRET Amplification of BioLuminescence

Abstract

The ability to manipulate neuronal activity both opto- and chemogenetically with a single actuator molecule presents unique and flexible means to study neural circuit function. We previously developed methodology to enable such bimodal control using fusion molecules called luminopsins (LMOs), where a channelrhodopsin actuator can be activated using either physical (LED driven) or biological (bioluminescent) light. While activation of LMOs using bioluminescence has previously allowed manipulation of circuits and behavior in mice, further improvement would advance the utility of this technique. Thus, we here aimed to increase the efficiency of bioluminescent activation of channelrhodopsins by development of novel FRET-probes with bright and spectrally matched emission tailored to Volvox channelrhodopsin 1 (VChR1). We find that pairing of a molecularly evolved Oplophorus luciferase variant with mNeonGreen significantly improves the efficacy of bioluminescent activation when tethered to VChR1 (construct named LMO7) as compared to previous and other newly generated LMO variants. We proceed to extensively benchmark LMO7 against previous LMO standard (LMO3) and find that LMO7 outperforms LMO3 in the ability to drive bioluminescent activation of VChR1 both in vitro and in vivo, and efficiently modulates animal behavior following intraperitonial injection of fluorofurimazine. In conclusion, we demonstrate a rationale for improving bioluminescent activation of optogenetic actuators using a tailored molecular engineering approach and provide a new tool to bimodally manipulate neuronal activity with increased bioluminescence-driven efficacy.

Strength of Activation and Temporal Dynamics of BioLuminescent-Optogenetics in Response to Systemic Injections of the Luciferin

“…The luminopsin-7 (LMO7) molecule comprises a fused fluorescent and bioluminescent protein, known as NCS2 (mNeonGreen-eKL9h, a modified Oplophorus -based luciferase), that is tethered to the N-terminus of Volvox Channelrhodopsin 1 (VChR1; see Figure 1A ) (Björefeldt et al, 2023). LMO7 was encoded within an adeno-associated viral 9 (AAV-9) construct under the human synapsin (h-Syn) promotor.…”