Genetically encoded photosensitizers are increasingly used as optogenetic tools to control cell fate or trigger intracellular processes. A monomeric red fluorescent protein called SuperNova has been recently developed, however, it demonstrates suboptimal characteristics in most phototoxicity-based applications. Here, we applied directed evolution to this protein and identified SuperNova2, a protein with S10R substitution that results in enhanced brightness, chromophore maturation and phototoxicity in bacterial and mammalian cell cultures.

Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on Förster resonance energy transfer (FRET). The WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module. The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with the WWE domain of RNF146 E3 ubiquitin ligase protein. This bipartite sensor named sPARroW (sensor for PAR relying on WWE) enabled monitoring of PAR accumulation and depletion in live mammalian cells in response to different stimuli, namely hydrogen peroxide treatment, UV irradiation and hyperthermia.

Epigenetic modifications (methylation, acetylation, etc.) of core histones play a key role in regulation of gene expression. Thus, the epigenome changes strongly during various biological processes such as cell differentiation and dedifferentiation. Classical methods of analysis of epigenetic modifications such as mass-spectrometry and chromatin immuno-precipitation, work with fixed cells only. Here we present a genetically encoded fluorescent probe, MPP8-Green, for detecting H3K9me3, a histone modification associated with inactive chromatin. This probe, based on the chromodomain of MPP8, allows for visualization of H3K9me3 epigenetic landscapes in single living cells. We used this probe to track changes in H3K9me3 landscapes during the differentiation of induced pluripotent stem cells (iPSCs) into induced neurons. Our findings revealed two major waves of global H3K9me3 reorganization during 4-day differentiation, namely on the first and third days, whereas nearly no changes occurred on the second and fourth days. The proposed method LiveMIEL (Live-cell Microscopic Imaging of Epigenetic Landscapes), which combines genetically encoded epigenetic probes and machine learning approaches, enables classification of multiparametric epigenetic signatures of single cells during stem cell differentiation and potentially in other biological models.

Genetically encoded photosensitizers are widely used in fundamental research and translational medicine due to their ability to generate reactive oxygen species (ROS) after photosensitizing. Previously, it was shown in mice that red dimeric fluorescent protein KillerRed is a potential photosensitizer that can be used for photodynamic therapy of cancer. In addition, it was demonstrated that HeLa cells expressing KillerRed fused to histone H2B cease proliferation upon illumination. A DNA repair protein, X-ray repair cross-complementing protein 1 (XRCC1), redistributed in the cell nuclei, indicating that the mechanism of phototoxic action of the construct involved DNA breaks generation. Here, we have constructed and tested a new genetically encoded photosensitizer molecule which introduces DNA breaks and activates the repair system in cancer-derived and embryonic cell lines more efficiently than previously described. The molecule consists of two parts: a SuperNova2 (monomeric mutant of KillerRed with enhanced phototoxicity) and methyl-CpG binding protein MECP2. The complex activates XRCC1 redistribution after illumination with lower power compared to the previously used construct. We suppose it can be explained by the tighter contact between photosensitizer and DNA. In addition, we hypothesize that the system should be error-prone for the expressed genes as it is targeted to the DNA which is silenced by methylation. Taking everything into consideration, the new genetically encoded construct has shown the improved ability to generate DNA breaks in the cancer cell lines.