Inteins are a unique class of proteins that are capable of self-excision from larger polypeptides by ligation of flanking exteins (external proteins) through a native peptide bond formation in a process called protein splicing.1, 2 While some inteins are contiguous, i.e., composed of a single protein fragment, others are naturally split into two fragments. In the case of split inteins, the process is called protein trans-splicing (PTS). It involves an additional step of association and folding of intein fragments to form a splicing-competent complex.3, 4 This complex undergoes a series of nucleophilic attacks accompanied by conformational shifts in intein structure and results in N- and C-terminal exteins linked together with a native peptide bond5 (Supplementary Figure 1a).

The introduction of external control to the process of protein splicing, known as conditional protein splicing (CPS), provides a powerful approach to studying biological processes and regulating protein activity via covalent protein modification.6 Since splicing of all natural inteins, with rare exceptions, occurs spontaneously, several engineering approaches have been employed to achieve CPS. These include chemical modification of reactive groups and fusion of additional domains that can be regulated with small molecules,7, 8, 9 proteolytic cleavage,10, 11 temperature12, 13 or pH14 change.

Among all the methods of external control, light offers the best spatiotemporal precision for regulating molecular processes. Light-dependent CPS can be achieved through the photocaging of reaction groups15, 11 or by fusing inteins to genetically encoded protein photoreceptors.16, 17, 18 Chemical photocaging methods typically involve ex vivo peptide synthesis or the introduction of unnatural amino acids in bacterial systems, which is complicated by the limitations of methods for delivering modified proteins into mammalian cells. Optogenetic CPS has been achieved thus far using a blue light-sensing AsLOV2 from Avena sativa in bacteria16 and mammalian cells16, 18 and near-infrared (NIR) light-regulated plant phytochrome heterodimerization pair PhyB-PIF3 from Arabidopsis thaliana in yeast.17 The last system was used for splicing of non-functional model exteins, namely affinity and solubility tags, and requires the addition of exogenous chromophore PCB, which is absent in both yeast and mammalian cells. Overall, the above systems are characterized by substantial non-specific activity.

The high-energy wavelengths of UV and blue light can cause phototoxic damage to cells and tissues,19, 20 as well as alter cell signaling21, 22 and morphology.23 In contrast, the NIR range of light (600–800 nm) does not cause phototoxic effects. And as NIR light penetrates deeply into the tissue,24, 25 NIR optogenetic tools (OTs) hold great potential for in vivo applications.

Among protein photoreceptors sensing NIR light, bacterial phytochromes are particularly promising for use in mammalian cells because they utilize biliverdin (BV), a product of heme catabolism that is abundant in mammalian tissue, as their chromophore.26, 27 Therefore, there is no need to supply additional chemicals, as is the case with plant phytochromes such as PhyB. Bacterial phytochromes undergo a conformational change between so-called Pr and Pfr states upon illumination with 640–700 nm, and between Pfr and Pr states with 740–790 nm light. One of these forms is a stable inactive state, to which they spontaneously revert in the darkness through thermal relaxation.28 Until recently, BphP1 from Rhodopseudomonas palustris with its binding partner PpsR229 or its smaller engineered analog QPAS130 was the only available bacterial phytochrome hetero-dimerizer, which has been applied to control and study cellular function with 740–780 nm light in vitro29, 31, 32, 33 as well as in vivo.29, 34 Recently, several binders specific to the active (Pfr) form of another bacterial phytochrome, DrBphP from Deinococcus radiodurans, have been developed, including the affibody Aff6_V18FN (hereafter referred to as Aff) in the MagRed system35 and a pair of nanobodies LDB-3 and LDB-14 in nanoReD systems.36 DrBphP, which is activated with 660 nm light, demonstrates low background binding to its partners.36, 35

In this study, we developed a NIR CPS optogenetic module by using NIR light-controlled DrBphP-Aff interaction (Supplementary Figure 1b) to bring together parts of the gp41-1, one of the smallest and fastest split inteins known to date.37, 38, 39, 40 Thanks to gp41-1 characteristics, it has been utilized in multiple synthetic biology applications, from the splicing of antibiotic resistance markers for the selection of bacterial and mammalian cells containing two plasmids41, 42, 43 to the engineering of complex logic circuits in bacteria.44 The native gp41-1 has a split site located at 88/89 position (hereafter numbering of gp41-1 amino acid residues follows that in PDB ID: 6QAZ37). It is characterized by efficient spontaneous splicing,39 which makes this site unsuitable for CPS engineering.

First, we identified the optimal split site in gp41-1 that mediates the weakest self-association of intein fragments and optimized our NIR CPS module applied to a reconstitution of split mCherry. Next, we demonstrated that the NIR CPS module can be used for transcriptional activation by splicing a DNA-binding protein with a transactivator. Additionally, we were able to cleave the protein at a designated site analogous to protease action by using a short extein sequence. To illustrate this feature, we engineered NIR light-triggered cleavage of Gasdermin D (GSDMD) to induce pyroptotic cell death.