【JACS】3000倍的荧光开启响应！上海交大朱麟勇团队开发通用型近红外分子马达荧光染料#

Figure 1. Design of novel molecular rotor-based NIR fluorogenic dyes. (a) Schematic illustration of the strategy for the design of molecular rotor-based NIR fluorogenic dyes. Introduction of a low-steric-hindrance molecular rotor at the meso position of the SiR scaffold to increase rotational freedom. Constraining the intramolecular motion of the dyes would enable them to produce bright fluorescence. (b) Molecular structures of the NIR fluorogenic dyes. (c) Potential energy surface (PES) scans of SiR-Tp in the first excited singlet state (S1) as a function of the rotational angle φ. Optimized molecular structures that correspond to the planar conformation (I) and the butterfly conformation (II) are shown. (d) Calculated chemical properties and absorbance maximum values of the SiR derivatives. φ represents the dihedral angle between the aromatic ring and the SiR scaffold in the ground state. E_a (eV) represents the rotational energy barrier, and Δ_E (eV) denotes the energy gap between the HOMO and LUMO. λabs represents the absorbance maximum wavelengths in glycerol. (e) Fluorescence intensity of the fluorogenic SiR derivatives in solution with distinct viscosities. (f) Viscosity sensitivity coefficients of the fluorogenic SiR derivatives. Data represent mean ± s.d. from three biologically independent replicates. (g) Molecular structure of CA-conjugated SiR-Tp for HaloTag labeling. (h) Normalized excitation and emission spectra of SF676 in the presence and absence of HaloTag. (i) Representative fluorescence images of live HEK293T cells that express HaloTag upon incubation with SF676. HEK293T cells transfected with mock plasmid and incubated with SF676 were used as controls. Scale bars, 20 μm. (j) Quantification of the fluorescence of the individual cells in i. Data represent mean ± s.d. (for HaloTag, n = 552 cells; for mock, n = 529 cells). Statistical analysis was performed via a two-tailed t test. ***P < 0.001. (k) Fluorogenic labeling of subcellular targeted HaloTag fusions recorded. Scale bars, 10 μm.#

Figure 2. Palette of NIR fluorogenic dyes for live-cell imaging. (a) Molecular structures, rotational energy barriers (in eV), and absorbance maximum values of various fluorogenic SiR derivatives. (b–d) Excitation and emission spectra of SF640 (b), SF720 (c), and SF760 (d) in the presence and absence of HaloTag. (e,g, and i) Representative fluorescence images of live HEK293T cells expressing H2B–HaloTag-TagBFP in the presence of 500 nM SF640 (e), 1 μM SF720 (g), and 1 μM SF760 (i). HEK293T cells expressing H2B-TagBFP and incubated with the corresponding dyes were used as controls. Scale bars, 20 μm. (f, h, and j) Quantification of fluorescence in the individual cells shown in (e, g, and i), respectively. Data represent mean ± s.d. (for HaloTag–SF640, n = 819 cells; for mock–SF640, n = 580 cells; for HaloTag–SF720, n = 612 cells; for mock–SF720, n = 569 cells; for HaloTag–SF760, n = 656 cells; and for mock–SF760, n = 598 cells). Statistical analysis was performed via a two-tailed t test. ***P < 0.001. (k–m) Photobleaching curves of SF640, SF676, SF720, and SF760 in mammalian cells upon continuous excitation by a 640 nm laser (k), a 670 nm laser (l), and a 734 nm laser (m). Cells expressing HaloTag and incubated with SiR, JF646 or BD666 were used as controls. The data were normalized to the initial fluorescence intensity for each fluorescent tag at time 0. Data represent mean ± s.d. from three biologically independent experiments.#

这种基于TICT机制的马达策略展现出了令人惊叹的家族普适性。如图2所示，研究团队成功将该策略推广至碳基罗丹明、传统罗丹明乃至荧光素等多种氧杂蒽核心结构中，一举构建了涵盖发光波长从黄绿光到近红外全谱段（650 nm至781 nm）的“上海红”系列光谱调色盘（SF640、SF676、SF720和SF760等）。这些新型探针在哺乳动物细胞中不仅表现出显著优于传统染料的抗光漂白特性，还可方便地与SNAP-tag等其他化学标签或绿色荧光蛋白进行多色、免洗的多靶点活细胞联合成像。

Figure 3. Directed evolution of HaloTag to increase the performance of NIR fluorogenic dyes. (a) The amino acid residues located near the protein-fluorophore interaction interface. (PDB: 6Y7B) (b) Representative fluorescence images of live HEK293T cells expressing HaloTag upon incubation with 500 nM SF676. Scale bars, 20 μm. (c) Quantification of the fluorescence intensity of the individual cells shown in (b). Data represent mean ± s.d. (for iHalo, n = 479 cells; for HaloTag, n = 477 cells; and for HaloTag–BD666, n = 494 cells). Statistical analysis was performed via a two-tailed t test. ***P < 0.001. (d) Photobleaching curves of HaloTag variants in mammalian cells upon continuous excitation by a 670 nm laser. Data represent mean ± s.d. from three biologically independent experiments. (e) STED (top) and confocal (bottom) images of COS-7 cells expressing MAP7-iHalo upon incubation with SF676. Scale bars, 5 μm (left) and 1 μm (right). (f) Quantitative analysis that shows that the FWHM of the tubulin filament in ROI-1 (white dotted line-indicated) was 28 nm, whereas it was 359 nm in the confocal image. (g) Effective differentiation of three parallel microtubules (indicated by a white dotted line in ROI-2) spaced at 82 nm using STED; these could not be resolved clearly in the confocal images. (h) Representative time-lapse STED images of HeLa cells expressing EB3-iHalo or EB3-HaloTag. Scale bar, 5 μm. (i) Emission intensities normalized to the first frame and plotted versus the image number. Data represent mean ± s.d. from four biologically independent experiments. (j) Workflow for imaging neurons in mouse brain. (k) Representative fluorescence images of neurons expressing iHalo in cortex after the administration of SF640. Inset: Image of a coronal brain slice from a wild-type mouse after SF640 administration. Scale bar, 50 μm. (l) Images of fixed coronal brain slices from mice expressing iHalo in cortical neurons. Dendritic spines are indicated by white arrowheads. Scale bar, 50 μm. In (k–l), the images show the pseudocoloured normalized fluorescence intensity per pixel. The color scale bar ranges from 0 to 1, where 0 represents the minimum fluorescence intensity and 1 represents the maximum intensity within each image.#

为了进一步压榨该系统的发光潜力，研究人员通过活细胞流式细胞术对HaloTag蛋白与染料相互作用的界面残基进行了饱和突变与定向进化。如图3所示，筛选出的新型工程化蛋白标签iHalo（L161I/E170I双突变体） 能够为SF系列染料提供更紧密、更强空间位阻的结合口袋，使复合体的细胞荧光亮度直接飙升了3倍，并大幅延长了荧光寿命。凭借这一卓越的亮度和无与伦比的光稳定性，iHalo-SF676系统在受激发射损耗（STED）超分辨率显微镜下大放异彩，成功将活细胞中微管长丝的半高全宽（FWHM）精细解析至微弱的28纳米尺度，清晰辨别出常规衍射极限下根本无法分出的并行微管。同时，该系统在小鼠大脑皮层神经元的在体标记与突触棘超微结构成像中也展现了极高的实用价值。

Figure 4. Development of NIR Ca2+ indicators (a) Schematic diagram of the Ca2+ indicator SFCa. The synthetic indicators include the calmodulin binding peptide (CBP), the circularly permuted HaloTag variant, and calmodulin (CaM). (b) Normalized Ca2+ titers of SFCa676. Data represent mean ± s.d. from four biologically independent experiments. (c, d) Normalized fluorescence excitation (dashed lines) and emission (solid lines) spectra of SFCaL676 (c) and SFCaL720 (d) in the presence (magenta) and absence (gray) of Ca2+. (e, g) Representative images of SFCaL–SF676 (e) and SFCaL–SF720 (g) for imaging of Ca2+ dynamics in single HeLa cells treated with histamine. Scale bars, 30 μm. (f, h) Fluorescence traces F/F0 of SFCaL–SF676 (f) and SFCaL–SF720 (h) in the histamine-stimulated HeLa cells in e and g, respectively. (i) Normalized fluorescence excitation (dashed lines) and emission (solid lines) spectra of SFCaS–SF640 in the presence (red) and absence (gray) of Ca2+. (j) Fluorescence lifetime of SFCaS–SF640 in the presence (τ = 3.58 ns, χ2 = 1.10) and absence (τ = 2.81 ns, χ2 = 1.03) of Ca2+, fitted to a three-component fluorescence decay. (k) Pseudocoloured fluorescence intensity (top) and fluorescence lifetimes (bottom) of SFCaS–SF640 in HeLa cells before and after histamine stimulation. Scale bars, 50 μm. The color bars indicate the normalized fluorescence intensity and fluorescence lifetimes, respectively. (l, m) Quantitative fluorescence intensity (l) and fluorescence lifetime (m) in the histamine-stimulated HeLa cells in k. (n) Workflow for imaging neurons activity in zebrafish brain. (o) Micrographs of zebrafish neurons using a spinning-disk confocal microscope. Scale bars, 50 μm. (p) Quantitative analysis of the indicated cells in o.#

马达探针对微环境改变的高度敏感性，还为其向功能化生物传感器的衍生铺平了道路。研究团队通过将圆排列化的iHalo标签与钙调蛋白及其靶肽融合，创造性地开发出了新一代近红外化学遗传钙离子指示剂SFCa。如图4所示，在结合钙离子引发结构改变后，限制马达旋转的程度发生剧烈跃变，使得SFCaL-SF676和SFCaL-SF720在活细胞内表现出高达5倍以上的荧光强度动态响应。此外，该系统与荧光寿命成像（FLIM）完美兼容，在结合钙离子后复合体的荧光寿命可发生显著延长。通过将该传感器特异性表达于斑马鱼的神经元系统中，研究人员成功在单细胞水平上实时捕捉到了活体斑马鱼大脑中自发的、高灵敏度的钙离子动力学振荡轨迹。