【JACS】湖南大学蒋健晖、汪凤林联手湖南师范大学刘锋|收率约 50%！新型酮缩酮桥联罗丹明打造高性能近红外化学遗传荧光探针#

Figure 1. (a) Schematic of X-bridged-rhodamines showing the correlations between emission maxima wavelength (λem, max), _K_L-Z values, and bridging groups (denoted as X). Structural modifications at the 10′ position of rhodamines significantly alter their photophysical characteristics. (b) Chemical structures for KR1–4 derivatives. (c) DFT-optimized structures of SiRh, KR1, and PRh and the calculated electrophilicity of their 9′ carbon atoms (highlighted by red circles). Right row: frontier molecular orbital plots of SiRh, KR1, and PRh, showing the HOMO and LUMO energy levels and the corresponding energy gaps (ΔE). Atom color code: H (grayish white), C (gray), N (blue), O (red). (d) Modular “bridge addition” synthesis of ketal-bridged rhodamines with high synthetic yields. Reaction yields are listed as percentages below each molecule.#

Figure 2. Normalized absorption (a) and emission spectra (b) for dyes KR1–4 in ethanol containing 0.1% (v/v) trifluoroacetic acid (TFA). Data are normalized to the absorbance maxima and emission peaks for KR1–4. (c) Photobleaching profiles for dyes KR1–4 under continuous laser irradiation (660 nm, 300 mW/cm2) using Cy5 as reference. (d) Normalized maximal absorbance in the zwitterionic forms for KR1–4 (5 μM) in water-dioxane mixtures (v/v, 0/100–80/20) as a function of dielectric constants at 25 °C. (e) Summary of photophysical properties for dyes KR1–4. All properties were measured in ethanol containing 0.1% (v/v) TFA, except for D50 and log _K_L-Z, which were determined in water-dioxane mixtures. (f) Deriving KR1–4 with HaloTag substrate via click chemistry to obtain HTKR1–4. (g) Normalized absorption and emission spectra of HTKR1 (5.0 μM) in the presence or absence of purified HaloTag protein (10.0 μM). Data are normalized to the absorbance maxima and emission peaks for HTKR1 in the presence purified HaloTag protein.#

为验证该类染料作为化学遗传指示剂的实用价值，研究人员将 KR1KR4 分别修饰 HaloTag 标签底物，得到探针 HTKR1HTKR4，同时还制备了适配 SNAP 标签的探针 SNAP-KR1，并在体外体系中完成荧光激活性能评价。实验结果显示，游离状态下的探针几乎不存在荧光背景，当与纯化的 HaloTag 蛋白结合后，荧光强度最高能够提升23 倍，其中 HTKR1 的综合表现最为突出。该类探针仅对对应的自标记蛋白产生特异性荧光响应，对血清蛋白、各类生化小分子均无明显干扰，并且在生理常见 pH 范围内光学性能保持稳定，标记特异性十分出色。分子动力学模拟进一步揭示了荧光激活的内在机制，探针结合 HaloTag 之后，分子中的羧酸根、阳离子氮会和蛋白内部的氨基酸残基形成氢键与静电作用，以此稳定荧光型两性离子结构。和商用硅罗丹明探针 SiR-Halo 相对比，HTKR1 拥有更低的荧光背景和更高的激活倍率，体外应用性能实现了显著突破。

Figure 3. (a) No-wash live-cell confocal imaging of MCF-7 cells expressing H2B-HaloTag incubated with HTKR1–4 (1 μM) for 60 min. Scale bar, 20 μm. (b) Flow cytometry profiles of HEK-293T cells with or without H2B-HaloTag expressing labeled with HTKR1–4 (1 μM). (c) Confocal imaging and colocalization analysis of MCF-7 cells expressing ER, nucleus and plasma membrane localized HaloTag fusions labeled by HTKR1 (1 μM). Scale bar, 10 μm. (d) Confocal (left) and SIM (right) imaging of MCF-7 cells expressing TOMM20-HaloTag labeled with HTKR1 (1 μM). Right panels show magnified views of the boxed region. Scale bar, 5 μm. (e) Normalized fluorescence intensity profile along the dashed arrow in (d). Date are normalized to the maximum intensity of each profile. (f) No-wash, three-color SIM imaging of MCF-7 cells expressing COX8-EGFP and TOMM20-HaloTag, stained with Hoechst 33342 (2 μM) and HTKR1 (1 μM) for 60 min. Right panels show magnified views of the boxed region. Scale bar, 5 μm.#

Figure 4. (a) Schematic of HTKR1-based chemigenetic indicator for fluorogenic imaging ROIs based on the FLIPs system. (b) Plasmid constructs and confocal fluorescence images of cells coexpressing HaloTag-fMCP and either circular RNA with MS2 tag or control circular RNA without MS2 tag, stained with Hoechst33342 (2 μM) and HTKR1 (1 μM). Scale bar, 50 μm. (c) Normalized fluorescence intensities from HTKR1 for transfected individual cells (100 cells from three independent experiments) in (c). Data are normalized to mean fluorescence intensity for cells expressing HaloTag-MCP and circular RNA with MS2 tag. (d) Schematic of the HaloTag-fMCP system for dynamic imaging of ActB mRNA translocation to SGs, marked by G3BP1-TurboRFP. (e) Confocal images of MCF-7 cells coexpressing 8 × MS2 tagged-ActB mRNA, HaloTag-fMCP, and G3BP1-TurboRFP, and labeled with HTKR1 (1 μM). Images were obtained before and 60 min after arsenite (500 μM) treatment. Zoomed-in views of the regions outlined by white boxes are shown on the right. Scale bar, 10 μm.#

细胞实验证明这类探针具备良好的生物相容性与极低的细胞毒性，可安全用于活细胞成像研究。将 HTKR1 用于标记融合 HaloTag 的靶蛋白，能够精准识别定位于细胞核、内质网、细胞膜、线粒体等不同亚细胞区域的蛋白，无需洗脱未结合的探针就能完成免洗成像，细胞核与细胞质的荧光比值最高可达 60，成像对比度极佳。依托探针优秀的荧光亮度与光稳定性，研究团队开展结构光照明显微镜（SIM）超分辨成像，成功清晰分辨出线粒体膜结构、细胞膜微绒毛、内质网网状结构以及肌动蛋白纤维等细微形貌，还可以实时追踪细胞丝状伪足的弯曲、伸缩动态变化，实现了亚细胞结构的动态超高分辨观测。除此之外，发射波长超过 700 nm 的 KR4 还能和其他荧光染料搭配使用，有效规避光谱串扰问题，可支撑多色同步成像工作的开展。

Figure 5. (a) Schematic of HTKR1-based chemigenetic indicator designed for detecting PKA activity using the HaloTag fused SPARK system. (b) Confocal images of HEK-293T cells expressing HaloTag-fused SPARK system labeled with HTKR1 (1 μM) before and after the addition of β-adrenergic agonist isoprenaline (10 μM). For inhibiting PKA activity, H89 (10 μM) was added 5 min before isoprenaline stimulation (denoted as “+H89”). Scale bars, 10 μm. (c) Confocal images of HEK-293T cells expressing HaloTag-fused SPARK system labeled with HTKR1 (1 μM), acquired before and 120 s after treatment with isoprenaline (10 μM). Scale bars, 10 μm. Right row: Normalized PKA-HaloTag-SPARK signals from cells depicted in the left images at different times. Data are normalized to the maximum SPARK signal after the addition of isoprenaline. (d) Time-lapse images of the zoomed-in region from (c). Lower row: Fluorescence intensity profiles along the dashed lines in the images above. (e) HaloTag fused SPARK system for reversible imaging of PKA activity. Time-lapse fluorescence imaging of HEK-293T cells expressing HaloTag-fused SPARK system labeled with HTKR1 (1 μM) treated with isoprenaline and H89 or isoprenaline only. Scale bars, 10 μm. Right panel: Normalized PKA-HaloTag-SPARK signals from the cells in left panels. Data are normalized to the maximum SPARK signal after the addition of H89.#

在蛋白成像的基础上，研究团队持续拓展该类探针的应用场景。结合自主研发的 FLIPs 体系，HTKR1 能够实现活细胞内RNA 的特异性荧光成像，还可以动态追踪 mRNA 在细胞受到外界刺激后，向应激颗粒发生转位的完整过程。同时，研究人员联合基于相位分离的 SPARK 传感体系，搭建起激酶活性检测平台，利用 HTKR1 实现了蛋白激酶 A（PKA）活性的动态成像，激酶被激活后短短数十秒内就能观测到荧光信号聚集，加入激酶抑制剂后信号又会逐步消散，完整还原出细胞内激酶活性可逆调控的特征。多项拓展实验充分证明，酮缩酮桥联罗丹明探针拥有极强的通用性，能够适配多种前沿生物检测体系。