【JACS】高达1435倍！新型双官能团正交标记活细胞荧光染料实现激活倍数接近3个数量级
文章标题：Bis-Tetrazine Fluorogenic (Silicon)-Rhodamine Dyes for Live-Cell Labeling
通讯作者：Edward A. Lemke
文章概要
化学生物学与细胞生物学的发展高度依赖于高精度、低背景的荧光成像技术。为了突破传统荧光探针在活细胞成像中由于游离探针导致的背景干扰，德国美因茨大学的Edward A. Lemke教授团队成功开发出了一类创新的双四嗪功能化荧光探针平台。该研究巧妙地将四嗪生物正交反应基团通过极短的非共轭连接桥引入到（硅代）罗丹明染料的氧杂蒽核心氨基上，实现了前所未有的超高效率猝灭。此类染料在未反应状态下近乎完全“黑暗”，而一旦与靶向生物分子发生逆电子需求狄尔斯–阿尔德反应后，荧光发射将展现出惊人的增强，其中双功能化衍生物的荧光激活倍数接近3个数量级（高达1435倍）。该项成果不仅为常规的单靶点生物正交标记提供了极佳的对比度，更在肽链大环化监测、高精度蛋白质荧光各向异性分析以及活细胞特异性双位点标记中展现出颠覆性的应用潜力，为无标记高分辨光学成像及活细胞蛋白质动态结构研究开辟了全新通路。
引言
在现代生物学研究中，实现活细胞内蛋白质的空间定位和动态追踪是揭示生命奥秘的核心手段。长期以来，绿色荧光蛋白等基因编码的荧光蛋白凭借其优异的遗传靶向特异性成为最广泛使用的工具，但这类蛋白质高达25至30千道尔顿的庞大体积常常会引发显著的立体位阻，从而干扰目标蛋白质的天然三级结构、亚细胞定位以及相互作用网络。为了克服这一瓶颈，基于小分子荧光探针与生物正交化学相结合的化学标记技术应运而生。这种方法利用较小的自标记蛋白质标签或通过遗传密码扩展技术引入非天然氨基酸，进而与特异性的荧光探针进行快速偶联。然而，在免洗涤的活细胞环境中，游离的未反应探针通常会产生强烈的背景杂散光，严重恶化成像信噪比。尽管基于罗丹明螺内酯平衡的探针能在一定程度上缓解背景问题，但它们极度依赖蛋白质标签内部特定的微环境来驱动荧光团的开放。如果想越过笨重的蛋白质标签，直接在活细胞内的多肽链上进行精细标记，则需要探针本身具备极高的生物正交猝灭效率与响应速度。特别是针对光谱红移至红光或远红光区域的硅代罗丹明染料，传统的单四嗪基团修饰由于空间柔性大或距离较远，猝灭效率往往非常有限，荧光增强倍数通常仅有几倍至几十倍，远远无法满足复杂细胞内环境的高对比度成像需求。
Figure 1. State-of-the-art fluorogenic (silicon)-rhodamines incorporating a single tetrazine moiety, that serves as a fluorescence quencher and as a biorthogonal “click” handle (top). Rational design of the new mono- and bis-functional (silicon)-rhodamine dyes, (Si)Rh-monoCTet and (Si)Rh-bisCTet, respectively (bottom).
主要实验及结论
针对这一长期悬而未决的科学问题，研究团队另辟蹊径，提出了一种颠覆性的“颠倒”分子设计策略。传统的设计通常是在罗丹明的下部羧基苯环进行修饰，而本研究选择在染料氧杂蒽核心的两个氨基取代基上直接引入四嗪基团。这种极短且低柔性的C1连接桥能够让强电子撤回能力的四嗪基团与荧光核心近距离接触，通过极强的空间轨道相互作用引发高效的德克斯特电子转移或四嗪促进的系间窜跃，从而在未反应前将荧光彻底锁定在“关闭”状态。如图1所示，通过精密控制反应物的化学计量比和碱的用量，该平台能够灵活地在单化学平台下合成单功能化衍生物与双功能化衍生物。这种新颖的化学结构不仅保持了良好的细胞渗透性，还在生物正交反应发生后展现出了非凡的光物理性能改变。
Scheme 1. (a) Synthetic Route for Accessing Bromomethyl Tetrazine. (b) Synthetic Approach Based on a Late-Stage Functionalization of the Amino Groups with Tetrazine to Provide Mono- and Bis-Functional Dyes, (Si)Rh-monoCTet and (Si)Rh-bisCTet; CCDC 2548790 Contains the Supplementary Crystallographic Data of Rh538bisCTet
为了系统评估这一平台的响应特性，研究人员首先在体外测试了这些染料与模型亲双烯体反应物在大分子偶联前后的吸收与发射光谱变化。如图2所示，单功能化的红色荧光探针在发生逆电子需求狄尔斯–阿尔德反应后表现出优异的响应，发射增强显著；而当引入第二个四嗪基团形成双功能化结构时，猝灭效果达到了极致。其中名为Rh518bisCTet的绿色荧光探针在未反应时几乎毫无发射，而在与亲双烯体完全反应转化为相应的哒嗪产物后，其光子产额暴增， emission最高点的荧光增强比率竟然直逼3个数量级，达到了惊人的1435倍，这在目前已知的可见光激发红移荧光响应探针中处于领先地位。为了深入探究猝灭的物理机制，研究团队特意合成了具有更长C3桥连链的对比分子，如图3所示，随着连接链的增长，猝灭效率出现了断崖式下跌，荧光增强仅剩3.4倍，这强有力地证实了该系统高度依赖短程的空间接触式猝灭机制。
Figure 2. Spectral properties of (Si)Rh-CTet upon click reaction. (a) Normalized absorbance of (Si)Rh-CTet after click reaction. (b) Normalized emission of (Si)Rh-CTet after click reaction. (c) Click reaction between Rh518bisCTet and 2 equiv of BCN–OH to provide the mono clicked intermediate Rh518bisCTet-SingleTet, followed by incubation with 25 equiv BCN–OH in PBS (pH = 7.2) to obtain the fully converted bis-derivative. (d) Normalized absorption spectra of Rh518bisCTet and Rh518bisCTet-BCN, indicating moderate bathochromic shift after click reaction with BCN–OH. (e) Emission spectra of Rh518bisCTet, Rh518bisCTet-SingleTet and Rh518bisCTet-BCN showing the contribution of each tetrazine to the fluorogenic response after click reaction with BCN–OH.
Figure 3. Linker effect on fluorescence quenching. (a) To test for a Dexter-type quenching the derivative with a longer linker was synthesized, SiRh634monoC3Tet. (b–d) SiRh634monoC3Tet displayed poor turn-on compared to its shorter derivative SiRh628monoCTet (3.4- versus 279-fold fluorescence enhancement, respectively, calculated in function of peak intensity). (e) Normalized absorbance after click reaction, showing the difference in absorbance maxima wavelength between the shorter derivative, SiRh628monoCTet (λmax = 628 nm) and the longer one, SiRh634monoC3Tet (λmax = 634 nm). (f) Normalized emission after click reaction, showing the less pronounced difference in emission maxima between SiRh628monoCTet (λmax = 654 nm) and SiRh634monoC3Tet (λmax = 653 nm).
随后，研究团队将目光投向了这一双功能化染料在多肽大环化修饰中的独特应用。通过合成含有两个亲双烯体修饰位点的小分子多肽并将其与双功能化探针混合，实验成功实现了高效的“两点式”桥连环化。如图4所示，动力学和质谱分析表明，由于首次点击反应后产生的邻近效应，探针的第二个四嗪基团会极其迅速地与多肽的第二个位点发生内环化反应，有效规避了多分子间的交叉副反应。而在将这种大环化多肽产物孵育至活细胞的实验中，如图5所示，借助于共聚焦显微镜与流式细胞术的定量分析，研究人员清晰地观察到环化后的多肽相比于线性对照组展现出了明显增强的细胞内吞摄取和内入率，并且整个环化结构在复杂的细胞培养基中表现出优秀的化学稳定性。
Figure 4. Click reaction studies on peptides bearing BCN as chemical handle. (a) Click reaction on Rh518bisCTet via a three-component reaction. (b) Click reaction on the Rh518bisCTet via a two-component reaction. (c) Fluorescence enhancement upon click reaction with Pep1–8. (d) Apparent reaction kinetics between Rh518bisCTet and BCNK or Pep1–4.
Figure 5. Confocal microscopy and flow cytometry of HeLa cells incubated with Rh518-Pep conjugates. (a) Schematic structure and primary sequence of the peptide-dye adduct tested. (b) Confocal microscopy images of HeLa cells treated with 4 μM Rh518–Pep conjugates, including Rh518bisCTet-Pep1 and the monoclicked control Rh518bisCTet-SingleTet-Pep9. Conjugates were prepared by reacting the dye with 5 equiv of Pep1 (for the cyclic peptide) and Pep9 (for the linear peptide), respectively, in PBS or DMEM buffer for 2–3 h to ensure complete conversion prior to the addition to the cell culture medium, as monitored via LC-MS (see Figure S26). Cells were incubated for 3 h at 37 °C and subsequently stained with SiR-Hoechst for nuclear visualization. Scale bar 20 μm. (c) Median fluorescence intensity (MFI) as a function of conjugate concentration, shown as bar plots from three independent replicates, after subtraction of the signals obtained from untreated samples (the 488 nm laser with a 532/30 nm bypass filter was used; note, this setup is suboptimal for detecting Rh518). (d) MFI of HeLa cells treated with 5 μM of dye-peptide conjugate, in the absence or presence of Dynasore (80 μM), shown as bar plots from three independent replicates, after subtraction of the signals obtained from untreated samples and samples treated with Dynasore alone.
Figure 6. Fluorescence anisotropy measurements in vitro. (a) Schematic illustrations of the contribution of fluorophore rotational freedom to the measured protein rotation in fluorescence anisotropy as single-site or double-site-labeled. (b) Experimental design for site-specific double incorporation of ncAAs via two amber codons in the model protein MBP. (c) Visualization of MBP labeled with the fluorogenic dye through either single or double site-specific conjugation. (d) Anisotropy steady state values (calculated from at least three replicates) for the reference (YPF), the free-dyes, previously reacted with BCN–OH (ATTO488MeTet, Rh518bisCTet-SingleTet and Rh518bisCTet), single-labeled protein (1xBCN-MBP-ATTO488MeTet and 1xBCN-MBP-Rh518bisCTet-SingleTet) and double-labeled protein (2xBCN-MBP-Rh518bisCTet and 2xTCO-MBP-Rh518bisCTet). Median values are reported as red line, while mean values are reported as blue line. (e) Time-dependent steady-state fluorescence anisotropy measurements of TCO- and BCN-labeled proteins were performed to assess measurement stability over time. The results show that the BCN derivative exhibits consistently stable anisotropy values throughout the observation period, whereas the TCO-labeled proteins display greater temporal variability.
除了多肽环化，双功能基团带来的超低柔性 attachment 还为高精度荧光各向异性分析提供了绝佳的硬件基础。常规的单官能团染料在链接蛋白质后，由于连接键的自由旋转，往往会损失大量的各向异性分辨率。而如图6所示，将双官能团罗丹明染料跨越偶联在具有双非天然氨基酸位点的麦芽糖结合蛋白上时，这种严格受限的“刚性锁定”使稳态各向异性数值直接跃升至接近荧光蛋白的水平，且长时间监测显示其结构刚性与荧光输出具有极高且恒定的耐久度，这为探测活细胞内部蛋白质的微小构象剪切或解离提供了全新工具。
Figure 7. Live-cell labeling of HEK293T cells transiently expressing the protein of interest. (a) Double-amber GCE for site-specific incorporation of two ncAAs into the Vim–mCer construct, followed by (b) dual-tagging labeling with Rh518bisCTet and (c) dual-tagging labeling with Rh518bisCTet, and BCN–OH treatment. Same well images were acquired before and after addition of BCN–OH. Media was replaced with fresh FluoroBrite DMEM containing BCN–OH and images were acquired after 20 min at RT. Exemplary white arrows highlight representative regions with increased background signal. Brightness/contrast in (c) has been adjusted consistently for better visualization between ± BCN–OH. Scale bars: 10 μm. Colors have been adapted. (d) Representative flow cytometry dot plot showing dye fluorescence plotted against iRFP fluorescence for individual cells after gating on iRFP positive cells (see Figure S40). Each event corresponds to a single cell, enabling assessment of dye-positive (488 nm channel), iRFP-positive (630 nm channel) and double-positive populations among Bock (light blue) and BCNK (red) conditions for Rh506monoCTet and Rh518bisCTet. (e) Bar plot shows the MFI of the dye channel in iRFP positive cells for each condition, together with the reported MFI ratio between the corresponding BocK and BCNK samples. Because the dyes display different excitation efficiencies depending on the laser used, MFI values were corrected according to the relative excitation at the selected laser wavelength. Specifically, excitation was normalized to the excitation maximum, with relative excitation values of 0.58 for Rh506monoCTet, 0.40 for Rh518bisCTet for the green laser, and 0.33 for Rh538bisCTet for the yellow laser. For Rh538bisCTet, an additional correction was applied because the detector voltage was set to 200 instead of 250; this correction was used only to improve visualization and comparison across samples. All corrections were applied consistently across BocK and BCNK replicates.
Figure 8. Live-cell labeling of HEK293T cells transiently expressing the proteins of interest. HEK293T cells were cotransfected with either (a) two single-amber constructs (singleTAG-Vim-mCer and single-TAG-Vim-iRFP) and labeled with the monofunctional dye Rh506monoCTet, or (b) a combination of single- and double-amber constructs (singleTAG-Vim-iRFP and double-TAG-Vim-mCer) and labeled with the bis-functional dye Rh518bisCTet. White arrows in (a) highlight stochastic labeling arising from nonselective labeling of both singleTAG proteins by the monofunctional dye. In contrast, diamonds in (b) indicate the absence/reduce labeling on the singleTAG protein when using the bis-functional dye. Consequently, a higher degree of colocalization between the dye and mCer channels is observed specifically for the doubleTAG protein. Brightness and contrast have been consistently adjusted. Scale bar 10 μm. (c) Representative flow cytometry dot plot showing dye fluorescence plotted against iRFP fluorescence for individual cells. Each event corresponds to a single cell, enabling assessment of dye-positive, iRFP-positive and double-positive populations. (d) Flow cytometry data shown as the ratio of MFI in the dye channel (488 nm excitation for Rh518bisCTet or 561 nm excitation for Rh538bisCTet) to the iRFP channel (640 nm) in iRFP-positive cells. These results demonstrate the stronger fluorescence turn-on of the bis-functional dye in the double-amber construct, whereas the same dye remains comparatively dim in the single-amber construct. Normalization to the iRFP channel was necessary to account for the higher expression levels observed in the single-amber construct relative to the double-amber construct.
最终，团队挑战了最为严苛的活细胞内源原位蛋白质双位点标记成像。如图7和图8所示，研究人员在人类胚胎肾细胞中通过遗传密码扩展技术，在细胞骨架波形蛋白的特定位置同时引入了两个含有亲双烯体取代基的非天然氨基酸。当向活细胞中注入双功能化探针时，只有当两个位点同时存在且距离匹配时，探针才会依次触发两次生物正交点击并全面解除猝灭，亮起高对比度的荧光纤维网络。与之形成鲜明对比的是，如果细胞内仅表达含有单个非天然氨基酸的突变蛋白，或者遭遇了未反应完全的游离非天然氨基酸碎片，探针在发生单次点击后依然能维持极强的猝灭暗态。这种独特的“双重确认”机制 stochastically 消除了因非特异性位点引入或翻译提前终止导致的背景假阳性，流式细胞术也进一步从单细胞层面上量化证实了该双位点标记系统在复杂活细胞环境中无与伦比的背景抑制能力与靶向精准度。