A variety of genetically encoded calcium indicators are currently available for visualization of calcium dynamics in cultured cells and in vivo. Only one of them, called NIR-GECO1, exhibits fluorescence in the near-infrared region of the spectrum. NIR-GECO1 is engineered based on the near-infrared fluorescent protein mIFP derived from bacterial phytochromes. However, NIR-GECO1 has an inverted response to calcium ions and its excitation spectrum is not optimal for the commonly used 640 nm lasers. Using small near-infrared bacterial phytochrome GAF-FP and calmodulin/M13-peptide pair, we developed a near-infrared calcium indicator called GAF-CaMP2. In vitro, GAF-CaMP2 showed a positive response of 78% and high affinity (K_d of 466 nM) to the calcium ions. It had excitation and emission maxima at 642 and 674 nm, respectively. GAF-CaMP2 had a 2.0-fold lower brightness, 5.5-fold faster maturation and lower pH stability compared to GAF-FP in vitro. GAF-CaMP2 showed 2.9-fold higher photostability than smURFP protein. The GAF-CaMP2 fusion with sfGFP demonstrated a ratiometric response with a dynamic range of 169% when expressed in the cytosol of mammalian cells in culture. Finally, we successfully applied the ratiometric version of GAF-CaMP2 for the simultaneous visualization of calcium transients in three organelles of mammalian cells using four-color fluorescence microscopy.

Hydrogen peroxide (H₂O₂) plays an important role in modulating cell signaling and homeostasis in live organisms. The HyPer family of genetically encoded indicators allows the visualization of H₂O₂ dynamics in live cells within a limited field of view. The visualization of H₂O₂ within a whole organism with a single cell resolution would benefit from a slowly reducible fluorescent indicator that integrates the H₂O₂ concentration over desired time scales. This would enable post hoc optical readouts in chemically fixed samples. Herein, we report the development and characterization of NeonOxIrr, a genetically encoded green fluorescent indicator, which rapidly increases fluorescence brightness upon reaction with H₂O₂, but has a low reduction rate. NeonOxIrr is composed of circularly permutated mNeonGreen fluorescent protein fused to the truncated OxyR transcription factor isolated from E. coli. When compared in vitro to a standard in the field, HyPer3 indicator, NeonOxIrr showed 5.9-fold higher brightness, 15-fold faster oxidation rate, 5.9-fold faster chromophore maturation, similar intensiometric contrast (2.8-fold), 2-fold lower photostability, and significantly higher pH stability both in reduced (pK_a of 5.9 vs. ≥7.6) and oxidized states (pK_a of 5.9 vs.≥ 7.9). When expressed in the cytosol of HEK293T cells, NeonOxIrr demonstrated a 2.3-fold dynamic range in response to H₂O₂ and a 44 min reduction half-time, which were 1.4-fold lower and 7.6-fold longer than those for HyPer3. We also demonstrated and characterized the NeonOxIrr response to H₂O₂ when the sensor was targeted to the matrix and intermembrane space of the mitochondria, nucleus, cell membranes, peroxisomes, Golgi complex, and endoplasmic reticulum of HEK293T cells. NeonOxIrr could reveal endogenous reactive oxygen species (ROS) production in HeLa cells induced with staurosporine but not with thapsigargin or epidermal growth factor. In contrast to HyPer3, NeonOxIrr could visualize optogenetically produced ROS in HEK293T cells. In neuronal cultures, NeonOxIrr preserved its high 3.2-fold dynamic range to H₂O₂ and slow 198 min reduction half-time. We also demonstrated in HeLa cells that NeonOxIrr preserves a 1.7-fold ex vivo dynamic range to H₂O₂ upon alkylation with N-ethylmaleimide followed by paraformaldehyde fixation. The same alkylation-fixation procedure in the presence of NP-40 detergent allowed ex vivo detection of H₂O₂ with 1.5-fold contrast in neuronal cultures and in the cortex of the mouse brain. The slowly reducible H₂O₂ indicator NeonOxIrr can be used for both the in vivo and ex vivo visualization of ROS. Expanding the family of fixable indicators may be a promising strategy to visualize biological processes at a single cell resolution within an entire organism.

The recently developed genetically encoded calcium indicator (GECI), called NTnC, has a novel design with reduced size due to utilization of the troponin C (TnC) as a Ca2+-binding moiety inserted into the mNeonGreen fluorescent protein. NTnC binds two times less Ca2+ ions while maintaining a higher fluorescence brightness at the basal level of Ca2+ in neurons as compared with the calmodulin-based GECIs, such as GCaMPs. In spite of NTnC’s high brightness, pH-stability, and high sensitivity to single action potentials, it has a limited fluorescence contrast (F-Ca2+/F+Ca2+) and slow Ca2+ dissociation kinetics. Herein, we developed a new NTnC-like GECI with enhanced fluorescence contrast and kinetics by replacing the mNeonGreen fluorescent subunit of the NTnC indicator with EYFP. Similar to NTnC, the developed indicator, named iYTnC2, has an inverted fluorescence response to Ca2+ (i.e. becoming dimmer with an increase of Ca2+ concentration). In the presence of Mg2+ ions, iYTnC2 demonstrated a 2.8-fold improved fluorescence contrast in vitro as compared with NTnC. The iYTnC2 indicator has lower brightness and pH-stability, but similar photostability as compared with NTnC in vitro. Stopped-flow fluorimetry studies revealed that iYTnC2 has 5-fold faster Ca2+ dissociation kinetics than NTnC. When compared with GCaMP6f GECI, iYTnC2 has up to 5.6-fold faster Ca2+ association kinetics and 1.7-fold slower dissociation kinetics. During calcium transients in cultured mammalian cells, iYTnC2 demonstrated a 2.7-fold higher fluorescence contrast as compared with that for the NTnC. iYTnC2 demonstrated a 4-fold larger response to Ca2+ transients in neuronal cultures than responses of NTnC. iYTnC2 response in neurons was additionally characterized using whole-cell patch clamp. Finally, we demonstrated that iYTnC2 can visualize neuronal activity in vivo in the hippocampus of freely moving mice using a nVista miniscope. We demonstrate that expanding the family of NTnC-like calcium indicators is a promising strategy for the development of the next generation of GECIs with smaller molecule size and lower Ca2+ ions buffering capacity as compared with commonly used GECIs.

The biologically most significant genotoxic metabolite of the environmental pollutant benzo[a]pyrene (B[a]P), (+)‐7R,8S‐diol 9S,10R‐epoxide, reacts chemically with guanine in DNA, resulting in the predominant formation of (+)‐trans‐B[a]P‐N²‐dG and, to a lesser extent, (+)‐cis‐B[a]P‐N²‐dG adducts. Here, we compare the effects of the adduct stereochemistry and conformation on the methylation of cytosine catalyzed by two purified prokaryotic DNA methyltransferases (MTases), SssI and HhaI, with the lesions positioned within or adjacent to their CG and GCGC recognition sites, respectively. The fluorescence properties of the pyrenyl residues of the (+)‐cis‐B[a]P‐N²‐dG and (+)‐trans‐B[a]P‐N²‐dG adducts in complexes with MTases are enhanced, but to different extents, indicating that aromatic B[a]P residues are positioned in different microenvironments in the DNA–protein complexes. We have previously shown that the (+)‐trans‐isomeric adduct inhibits both the binding and methylating efficiencies (k_cat) of both MTases [Subach OM, Baskunov VB, Darii MV, Maltseva DV, Alexandrov DA, Kirsanova OV, Kolbanovskiy A, Kolbanovskiy M, Johnson F, Bonala R, et al. (2006) Biochemistry45, 6142–6159]. Here we show that the stereoisomeric (+)‐cis‐B[a]P‐N²‐dG lesion has only a minimal effect on the binding of these MTases and on k_cat. The minor‐groove (+)‐trans adduct interferes with the formation of the normal DNA minor‐groove contacts with the catalytic loop of the MTases. However, the intercalated base‐displaced (+)‐cis adduct does not interfere with the minor‐groove DNA–catalytic loop contacts, allowing near‐normal binding of the MTases and undiminished k_cat values.

The mRubyFT is a monomeric genetically encoded fluorescent timer based on the mRuby2 fluorescent protein, which is characterized by the complete maturation of the blue form with the subsequent conversion to the red one. It has higher brightness in mammalian cells and higher photostability compared with other fluorescent timers. A high-resolution structure is a known characteristic of the mRubyFT with the red form chromophore, but structural details of its blue form remain obscure. In order to obtain insight into this, we obtained an S148I variant of the mRubyFT (mRubyFT^S148I) with the blocked over time blue form of the chromophore. X-ray data at a 1.8 Å resolution allowed us to propose a chromophore conformation and its interactions with the neighboring residues. The imidazolidinone moiety of the chromophore is completely matured, being a conjugated π-system. The methine bridge is not oxidized in the blue form bringing flexibility to the phenolic moiety that manifests itself in poor electron density. Integration of these data with the results of molecular dynamic simulation disclosed that the OH group of the phenolic moiety forms a hydrogen bond with the side chain of the T163 residue. A detailed comparison of mRubyFT^S148I with other available structures of the blue form of fluorescent proteins, Blue102 and mTagBFP, revealed a number of characteristic differences. Molecular dynamic simulations with the combined quantum mechanic/molecular mechanic potentials demonstrated that the blue form exists in two protonation states, anion and zwitterion, both sharing enolate tautomeric forms of the C=C–O⁻ fragment. These two forms have similar excitation energies, as evaluated by calculations. Finally, excited state molecular dynamic simulations showed that excitation of the chromophore in both protonation states leads to the same anionic fluorescent state. The data obtained shed light on the structural features and spectral properties of the blue form of the mRubyFT timer.