Abstract
Near-infrared (NIR) optogenetic systems for transcription regulation are in high demand because NIR light exhibits low phototoxicity, low scattering, and allows combining with probes of visible range. However, available NIR optogenetic systems consist of several protein components of large size and multidomain structure. Here, we engineer single-component NIR systems consisting of evolved photosensory core module of Idiomarina sp. bacterial phytochrome, named iLight, which are smaller and packable in adeno-associated virus. We characterize iLight in vitro and in gene transcription repression in bacterial and gene transcription activation in mammalian cells. Bacterial iLight system shows 115-fold repression of protein production. Comparing to multi-component NIR systems, mammalian iLight system exhibits higher activation of 65-fold in cells and faster 6-fold activation in deep tissues of mice. Neurons transduced with viral-encoded iLight system exhibit 50-fold induction of fluorescent reporter. NIR light-induced neuronal expression of green-light-activatable CheRiff channelrhodopsin causes 20-fold increase of photocurrent and demonstrates efficient spectral multiplexing.
Highlights
Near-infrared (NIR) optogenetic systems for transcription regulation are in high demand because NIR light exhibits low phototoxicity, low scattering, and allows combining with probes of visible range
The IsPadC-photosensory core module (PCM) was fused to a C terminus of the DNA-binding domain (DBD: amino acid residues 1–87)[20] of LexA408-mutated repressor of the Escherichia coli SOS regulon, which binds mutated operator and does not interfere with endogenous wildtype LexA protein and operator regions in bacterial SOS signaling pathway[21]
The second plasmid encoded a lightsensitive repressor LexA408DBD-IsPadC-PCM-monomeric superfolder green fluorescent protein (msfGFP) under control of a weak constitutive promoter J23116 and the mCherry reporter under the control of a constitutive promoter ColE with the LexA408 operator located after the promoter
Summary
Near-infrared (NIR) optogenetic systems for transcription regulation are in high demand because NIR light exhibits low phototoxicity, low scattering, and allows combining with probes of visible range. Red-light-triggered heterodimerization of a plant phytochrome B (PhyB) and a phytochrome-interacting factor 6 (PIF6) from Arabidopsis is successfully applied to transcriptional control[9], cell signaling[10], and protein localization[11]. The substantial drawback of the currently available NIR optogenetic systems is the requirement to co-express two large protein components (i.e., PhyB phytochrome and PIF6 partner or RpBphP1 phytochrome and RpPpsR2 partner), meaning the need to cotransfect two plasmids or to co-transduce with two adeno-associated viruses (AAVs)[18]. Another drawback of the RpBphP1–RpPpsR2 system is its rather high background in darkness
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