Near-Infrared Optogenetic Module for Conditional Protein Splicing

Abstract

Optogenetics has emerged as a powerful tool for spatiotemporal control of biological processes. Near-infrared (NIR) light, with its low phototoxicity and deep tissue penetration, holds particular promise. However, the optogenetic control of polypeptide bond formation has not yet been developed. In this study, we introduce a NIR optogenetic module for conditional protein splicing (CPS) based on the gp41-1 intein. We optimized the module to minimize background signals in the darkness and to maximize the contrast between light and dark conditions. Next, we engineered a NIR CPS gene expression system based on the protein ligation of a transcription factor. We applied the NIR CPS for light-triggered protein cleavage to activate gasdermin D, a pore-forming protein that induces pyroptotic cell death. Our NIR CPS optogenetic module represents a promising tool for controlling molecular processes through covalent protein linkage and cleavage.

Keywords: DrBphP; MagRed; pyroptosis; split intein; tTA.

Figure 1.. Engineering and optimization of the NIR CPS module with split mCherry reporter.

(a) (top) The mechanism of NIR light-induced splicing of split mCherry based on split intein reconstitution. Upon 660 nm light DrBphP changes its conformation from Pr to Pfr state in which it binds Aff bringing N- (Int^N) and C-terminal (Int^C) fragments of split intein into close proximity. The assembled intein performs splicing of N- and C-terminal exteins that represent split mCherry, which then folds and becomes fluorescent after maturation. (bottom) Schematic representation of the module arrangement in the designed protein fusion construct pairs #1 (mCherry^N-Int^N-DrBphP and Aff-Int^C-mCherry^C) and #2 mCherry^N-Int^N-Aff and DrBphP-Int^C-mCherry^C). (b) Screening of gp41-1 split site variants using split mCherry splicing. Respective Int^N and Int^C variants were cloned into pair #1. Transiently transfected HEK293T cells were kept in darkness or illuminated with 660 nm light for 6h with subsequent 14h of darkness to allow maturation of spliced mCherry. Then, cells were analyzed with flow cytometry. Mean mCherry fluorescence (left) was measured for EGFP-positive cells (EGFP is coexpressed from one of the vectors being placed after mCherry^C through “self-cleaving” T2A peptide conjunction). Statistical analysis of respective sample pairs was performed using an unpaired t-test. Significance levels are represented as ****P < 0.0001, ***P < 0.0001, while "ns" indicates non-significance. Background autofluorescence in the mCherry channel measured in a respective control is subtracted from mean values. Calculated light-to-dark ratios are presented on the right bar graph. (c) Performance comparison for construct pairs #1 and #2 that differ in DrBphP and Aff arrangement. Transiently transfected HEK293T cells were kept in darkness or illuminated at 660 nm for 26 h and analyzed with flow cytometry. Mean mCherry fluorescence is measured for EGFP-positive cellsMean values for individual samples and light-to-dark ratios ±S.E.M. are given (n = 3). (d) Representative flow cytometry plots corresponding to (c).

Figure 2.. Characterization of optimized NIR CPS module with split mCherry reporter.

(a) The illumination protocol scheme. HEK293T cells transiently transfected with vectors carrying optimized NIR CPS module with split mCherry reporter (N23+C2 variant (Supplementary Figure 2)) were illuminated with 660 nm for the indicated times, and then incubated in darkness for 6 h allowing maturation of spliced mCherry. Finally, samples were analyzed using flow cytometry. (b) (left) The dynamics of NIR light-triggered split mCherry splicing. Mean mCherry fluorescence is measured for DrBphP and EGFP double-positive cells (EGFP is coexpressed from C2 vector through “self-cleaving” T2A peptide conjunction). (right) The corresponding light-to-dark ratio for each time point. Mean values for individual samples ±S.E.M. are given (n = 3). Statistical significance was determined using an unpaired t-test. ****P < 0.0001 (c) An overlay of representative flow cytometry histograms corresponding to (b).

Figure 3.. Performance of nanobodies LDB-3 and LDB-14 in the NIR CPS module with split mCherry.

(a) Schematic representation of the module arrangement in the designed constructs. Optimized NIR CPS module relying on DrBhpP-Aff interaction corresponds to N23+C2 variant (Supplementary Figure 2) (b) Transiently transfected HEK293T cells were kept in darkness or illuminated with 660 nm for 6 h with subsequent 13 h of darkness to allow spliced mCherry to mature. Samples were analyzed with flow cytometry. Mean mCherry fluorescence was measured for EGFP-positive cells (n = 3; error bars are S.E.M.). Statistical analysis of sample means was conducted using a two-way ANOVA with multiple comparisons of each sample mean to the non-fluorescent Ctrl sample. All comparisons were found to be non-significant, except for the indicated ones. ****p < 0.0001, **P < 0.01, *P < 0.05.

Figure 4.. The NIR light-activated intein-based gene expression system (NIR-IGES).

(a) (top) Suggested mechanism corresponding to the optimized NIR-IGES-NES-NLS that involves tTA (TetR-vp16) splicing. In the darkness, the Aff-Int^C-mCherry^C-NLS is sequestered in the nucleus due to fused NLS with a slight amount diffusing to the cytoplasm. Under illumination, it relocalizes to the cytoplasm through DrBphP-Aff interaction with the TetR-Int^N-DrBphP-NES that is retained in the cytoplasm due to strong NES. At the same time, split intein associates and perform splicing of tTA that starts the transcription of the Gaussia luciferase (Gluc) reporter. (bottom) Schematic representation of the designed constructs for NIR-IGES and the MagRed-GES. NIR-IGES components are modified with respective NES (PKI), NLS (MycA1), or both signals according to the scheme. (b,c) Performance comparison for combinations of constructs displayed on (a) with secreted Gluc reporter. Transiently transfected HeLa cells were kept in darkness or illuminated with 660 nm for 48 h. Raw signals (b) corresponding to the time course of illumination and calculated light-to-dark ratios (c) are shown (n = 3; error bars are S.E.M.).

Figure 5.. NIR light-activated GSDMD (NIR-AG).

(a) (top) The suggested mechanism of NIR light-triggered pyroptosis via optogenetic activation of GSDMD using NIR CPS. Upon 660 nm illumination, DrBphP binds Aff resulting in gp41-1-mediated splicing of autoinhibiting GSDMD^C domain to a short extein effectively releasing it from the GSDMD complex. Free GSDMD^N oligomerizes upon insertion into the plasma membrane forming pores and ultimately leading to pyroptotic cell death. (bottom) Schematic representation of the designed NIR-AG vectors. (b) Dynamics of NIR light-induced cell death with NIR-AG packed in two vectors and optimized transfection. HEK293T cells were transfected with NIR-AG vectors and illuminated with 660 nm for 8 and 25 h. Then cells were harvested, stained with SYTOX Dead Cell Stain Kit and analyzed by flow cytometry. Percentages of stained cells are shown for the EGFP-positive cell population. (c) An overlay of respective flow cytometry histograms for mock, dark, and 25h samples. (d) Dynamics of NIR light-induced cell death triggered with NIR-AG packed in a single vector with attenuated expression of GSDMD-containing component. Cells were illuminated for indicated times or kept in darkness and processed as described in (b). Percentages of stained cells are shown for the DrBphP-positive cell population. (e) An overlay of respective flow cytometry histograms corresponding to (d). Mean values for individual samples ±S.E.M. are given (n = 3). Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons test. ****P < 0.0001. BioRender clip arts (https://biorender.com/) were used to create (a).