Creating Red Light-Controlled Protein Dimerization Systems as Genetically Encoded Actuators with High Specificity

Protein dimerization systems that can be controlled by red light with increased tissue penetration depth are a highly needed optogenetic tool for clinical applications such as cell and gene therapies. However, existing red light-induced dimerization systems are all based on phytochrome photoreceptors and naturally occurring binding partners with complex structures and suboptimal in vivo performance, limiting mammalian applications. Here, we introduce an efficient, generalizable method (COMBINES-LID) for creating highly specific light-induced dimerization systems. Proof-of-principle was provided by creating nanobody-based, red light-induced dimerization (nanoReD) systems comprising a truncated bacterial phytochrome sensory module using a mammalian endogenous chromophore, biliverdin, and light-form specific nanobodies. Selected nanoReD systems were biochemically characterized and exhibited low dark activity and high induction specificity for in vivo activation of gene expression. Overall, COMBINES-LID opens new opportunities for creating genetically encoded actuators for the optical manipulation of biological processes.