Efficient opto- and chemogenetic control in a single molecule driven by FRET-modified bioluminescence

Abstract

. Significance Bioluminescent optogenetics (BL-OG) offers a unique and powerful approach to manipulate neural activity both opto- and chemogenetically using a single actuator molecule (a LuMinOpsin, LMO). Aim To further enhance the utility of BL-OG by improving the efficacy of chemogenetic (bioluminescence-driven) LMO activation. Approach We developed novel luciferases optimized for Förster resonance energy transfer when fused to the fluorescent protein mNeonGreen, generating bright bioluminescent (BL) emitters spectrally tuned to Volvox Channelrhodopsin 1 (VChR1). Results A new LMO generated from this approach (LMO7) showed significantly stronger BL-driven opsin activation compared to previous and other new variants. We extensively benchmarked LMO7 against LMO3 (current standard) and found significantly stronger neuronal activity modulation ex vivo and in vivo , and efficient modulation of behavior. Conclusions We report a robust new option for achieving multiple modes of control in a single actuator and a promising engineering strategy for continued improvement of BL-OG.

Engineering luminopsins with improved coupling efficiencies

“…We previously found that radiance decreased for opsin-tethered light emitters, consistent with a loss of photon emission to direct energy transfer to the nearby opsin chromophore. 21 We further observed a larger decrease for LMO7 (NCS2-VChR1) versus LMO3 (sbGLuc-VChR1). This is consistent with FRET from the light emitter to opsin being more efficient for NCS2 to VChR1 (LMO7) than for sbGLuc to VChR1 (LMO3).…”

“…Interestingly, when paired with VChR1 in an LMO, the coupling efficiency of NCS2 to VChR1 (LMO7) proved to be significantly higher than for NCS1 (LMO6), suggesting that the orientation of luciferase and fluorescent protein relative to the optogenetic channel may also be critical (i.e., mNeonGreen-eKL9h-VChR1 is superior to sbALuc1-mNeonGreen-VChR1). 21 We applied this principle successfully with LMOs 10 and 11 (mNeonGreen-SSLuc and GeNL_SS fused to VChR1 and ChRmine, respectively; this study).…”

“…5 This trend continued, though not as dramatically, when we replaced sbGLuc in LMO3 with a luciferasefluorescent protein Förster resonance energy transfer (FRET) probe (NCS2, a fusion of eKL9h and mNeonGreen), resulting in LMO7. 21…”

“…5,6,[12][13][14][15][16][17][18][19] We and others have since continued to combine more potent light emitters with more light sensitive opsins to generate LMOs with still higher efficacies. These efforts include use of novel luciferase mutants, such as GLucM23, 20 molecularly evolved luciferase-fluorescent protein fusions, such as NCS2 (eKL9h-mNeonGreen), 21 and opsins with higher light sensitivities, such as step function opsins. 22 These further improvements in LMO efficacy enable more effective neuron activation with less luciferin or via easier application routes (for example, intraperitoneal instead of intravenous).…”