Near-infrared light–controlled systems for gene transcription regulation, protein targeting and spectral multiplexing

Abstract

Near-infrared (NIR, 740-780 nm) optogenetic systems are well-suited to spectral multiplexing with blue-light-controlled tools. Here, we present two protocols, one for regulation of gene transcription and another for control of protein localization, that use a NIR-responsive bacterial phytochrome BphP1-QPAS1 optogenetic pair. In the first protocol, cells are transfected with the optogenetic constructs for independently controlling gene transcription by NIR (BphP1-QPAS1) and blue (LightOn) light. The NIR and blue-light-controlled gene transcription systems show minimal spectral crosstalk and induce a 35- to 40-fold increase in reporter gene expression. In the second protocol, the BphP1-QPAS1 pair is combined with a light-oxygen-voltage-sensing (LOV) domain-based construct into a single optogenetic tool, termed iRIS. This dual-light-controllable protein localization tool allows tridirectional protein translocation among the cytoplasm, nucleus and plasma membrane. Both procedures can be performed within 3-5 d. Use of NIR light-controlled optogenetic systems should advance basic and biomedical research.

Optogenetic regulation of endogenous proteins

“…Combining optogenetic tools controlled by light of different wavelengths enabled tridirectional protein targeting 27 , which may be used to control multifunctional proteins or to increase dynamic range of bidirectional localizers, as discussed 31 . A NIR-blue-light inducible shuttle (iRIS) construct was shown to be effective for dualwavelength-controlled exogenous protein relocalization 16,19 . In this system, the QPAS1 protein is fused to a blue-light-sensing AsLOV2-based nuclear localization controller.…”

“…Third, although the bacterial phytochromebased optogenetic tools benefit from the abundance of biliverdin in mammalian cells, they may depend on its concentration in a particular tissue. This technical obstacle can be overcome by using stable expression of the phytochrome part of the iBincorporating optogenetic tools 16 .…”

“…Among these, bacterial phytochromes, sensitive to nearinfrared (NIR) light, cryptochromes, and LOV domains, sensitive to blue light, do not need supply of exogenous cofactors to efficiently function in mammalian cells. The distinct spectral sensitivity of these photoreceptors allows their effective spectral multiplexing 16 . The light-dependent structural reorganization, homo-and heterodimerization of these non-opsin photoreceptors were successfully applied to gene expression, regulation of its epigenetic state, control of cell signaling, cell cycle progression, and apoptosis.…”

Light‐Controlled Mammalian Cells and Their Therapeutic Applications in Synthetic Biology

“…[68] Upon exposure to red light, PhyB changes conformation and binds to its partner phytochrome interacting factor (PIF). [69][70][71] BphP1 is a bacterial phytochrome and uses biliverdin, which is abundant in eukaryotic cells, as a chromophore. [68] A recently developed light-switchable transgene system based on BphP1-PpsR2 or BphP1-QPAS1 interaction can be activated and inactivated by light in the near-infrared region (740-780 nm) and red light (660 nm), respectively.…”

The Impact of Optogenetics on Regenerative Medicine

“…Advances in tissue engineering and regenerative medicine (TERM) have shed light on important scenarios in medicine, with a specific interest in tissue repairing. Optogenetics is pretty useful in the study of mechanisms supporting cell development and regeneration, in order to better understand the physiological processes of tissues and organs replacement [57].…”