Fluolab【JACS】湖南大学聂舟、旷实|深度组织成像突破：PLQY 提升 10 倍的 DNA 纳米探针实现肝损伤精准监测
文章标题： Programmable DNA-Based NIR-II Molecular Probes for Hepatotoxicity-Associated MicroRNA Imaging In Vivo
通讯作者： Shi Kuang, Zhou Nie
文章概要
引言
DNA分子探针因其强大的可编程性和生物相容性，已成为分子传感领域的重要工具。然而，传统的 DNA 探针大多运行在可见光光谱范围内，由于光子散射严重且组织穿透力差，极大地限制了其在深层器官（如肝脏或大脑）中的成像应用。虽然近红外二区（NIR-II, 1000–1700 nm） 荧光成像具有深层组织穿透和高信噪比的显著优势，但开发基于 DNA 的 NIR-II 探针面临着巨大的技术瓶颈：一方面是缺乏高亮度的 NIR-II 荧光团，另一方面则是缺乏与之精准光谱匹配的高效率淬灭剂。为了攻克这一难题，湖南大学聂舟教授与旷实副教授团队提出了一种协同设计策略，通过共工程化高亮度发射器及其光谱匹配的淬灭剂，首次将可编程 DNA 分子探针的应用窗口扩展到了 NIR-II 区域。
Scheme 1. Schematic Illustration of (a) the Principle of DNA-Based Molecular Probes; (b) Synergistic Design of NIR-II Fluorophore-Quencher Combination; (c) the Construction of the DNA-Based NIR-II Molecular Probe (NMP) and Its Mechanism of Activation for In Vivo Fluorescence Imaging of miRNA-122
主要实验及结论
研究团队首先通过一种创新的三元架构设计，构建了高亮度的 DNA-白蛋白-染料嵌合体（DAC）。该设计巧妙地利用人血清白蛋白（HSA）作为保护支架，将 DNA 链和近红外染料 IR783 定位偶联。实验结果表明，这种封装效应显著增强了染料的光物理性质，其在 NIR-II 窗口的光致发光量子产率（PLQY）达到了 1.06%，比商业常用染料 ICG 高出 10 倍，能够实现超过 8 毫米的深层组织穿透。更重要的是，HSA 的空间位阻效应有效地保护了 DNA 链免受核酸酶的降解，显著提升了探针在复杂生物环境中的稳定性。
Figure 1. Development and characterization of high-brightness, stoichiometric DNA-albumin-dye chimeras (DACs). (A) Schematic of the DNA-based NIR-II Molecular Probe (NMP) design, which utilizes DAC as the NIR-II emissive module. (B) Comparison of fluorescence emission spectra of HSA@IR783, free IR783, Cy7, and Cy5 (1 μM each) in PBS (10 mM, pH 7.4). Spectra were acquired using a silicon (Si) detector for the visible/NIR-I region and an InGaAs detector to capture the NIR-II tail emission. (C) 8 mm Comparative fluorescence images of capillary tubes filled with HSA@IR783, IR783, Cy7, and Cy5 (5 μM each), embedded in a 1% Intralipid tissue phantom at increasing depths. Imaging conditions: NIR-II (IR783@HSA or IR783): ex = 808 nm, 1,040 nm long-pass (LP) filter; NIR-I (Cy5): ex = 640 nm, 680 nm LP filter; NIR-I (Cy7): ex = 710 nm, 760 nm LP filter. (D) General synthetic strategy for constructing the DACs, involving site-specific conjugation of a DNA strand to Cys34 and subsequent binding of a cyanine dye. (E) MALDI-TOF MS spectrum confirming the successful formation of the final DAC-783 product from the HSA@Ha precursor. (F) Comparative fluorescence emission spectra of DAC-783, DAC-775, and DAC-820 (1 μM each) in PBS (10 mM, pH 7.4), demonstrating the versatility of the platform.
Figure 2. Design, characterization, and performance evaluation of the NIR-II fluorophore-quencher combination. (A) Schematic comparing two potential quenching strategies: matching the quencher’s absorption to the fluorophore’s peak emission (peak-matching) versus matching it to the imaging-relevant tail emission (tail-matching). (B) Molecular design of the peak-matched quencher Q783 via a symmetry-breaking strategy and (C) its synthetic route. (D) Fluorescence spectra of the assembled NMP, comparing the quenching effect on the Fs strand (1 μM) by Qs-Q783 (peak-matched, 1 μM) and Qs-FD-1080 (tail-matched, 1 μM). (E) Comparative quenching efficiencies (QE) in the NIR-II window. (F, G) Spectral overlap analysis between the emission of DAC-783 and the absorption of (F) FD-1080 and (G) Q783. (H) Enhancement of quenching efficiency using a dual-quencher modification (1 μM Fs hybridized with 1 μM QsQ783 and BsQ783). (I) Schematic of the deep-tissue imaging experiment. Capillary tubes filled with Ts-activated probes (the NIR-II NMP, the NIR-I Cy5-BHQ3 probe, and the visible FAM-BHQ1 probe, 5 μM each and incubated with 5 μM Ts) were embedded at a 7 mm depth in a 1% Intralipid phantom. Imaging parameters: NIR-II: ex = 808 nm, 1040 nm LP filter; Cy5: ex = 640 nm, 680 nm LP filter; FAM: ex = 465 nm, 520 nm LP filter. (J) Resulting fluorescence images from the experiment in (I), demonstrating the superior deep-tissue signal of the NMP compared to the conventional NIR-I and visible probes.
为了实现高效的信号控制，研究者提出了 “对称性破缺”的设计思路来开发匹配的淬灭剂。通过在 IR783 支架上引入不对称的推拉电子基团，成功合成了非荧光的新型淬灭剂 Q783。这种设计打破了分子的对称性，极大地促进了其内部电荷转移过程，使其吸收光谱精准覆盖了发射器的峰值发射区域。对比实验显示，这种峰值匹配策略产生的淬灭效率远高于传统的尾部匹配策略。基于此，团队构建了可编程的近红外二区分子探针（NMPs），并证明其在 7 毫米深的组织模型中仍能保持清晰的荧光信号，而传统的可见光和近红外一区探针在相同深度下则完全无法成像。
Figure 3. Versatility and Programmability of the NMP platform for detecting diverse miRNA targets. (A) Real-time fluorescence monitoring showing the activation kinetics of NMP-122 (300 nM) in response to its target miR-122 (500 nM). Imaging conditions: ex = 808 nm, em >1,040 nm. Data represent the mean ± SD (n = 3). (B) Dose-dependent fluorescence response of NMP-122 (200 nM) toward miR-122 (0 to 600 nM). LOD was determined by the 3σ rule. Data represent means ± SD (n = 3). (C) Specificity analysis shown as a heatmap of the orthogonal fluorescence responses of NMP-122 (300 nM) and NMP-21 (300 nM) to their cognate and noncognate miRNA targets (500 nM). (D) Design schematic for an OR logic gate that reports the presence of either miR-21 or miR-122. (E) Fluorescence output of the OR logic gate in response to the four possible input combinations. (F) Design schematic for an AND logic gate that activates only when both miR-122 and miR-21 are present. (G) Fluorescence output of the AND logic gate, demonstrating its response to the four input combinations.
Figure 4. Live-cell imaging of miR-122 using the hepatocyte-targeted NMP-122-MET probe. (A) Schematic illustration of the probe anchored on the cell membrane, undergoing activation in response to extracellular miR-122 by the injured hepatocytes. (B) Quantitative Western blot analysis of MET receptor expression in BRL, HEK293T, and NIH/3T3 cells. (C) Confocal microscopy images of the “always-on” (quencher-free) version of NMP-122-MET (200 nM), demonstrating its specific targeting to MET-expressing BRL cells versus control cell lines (HEK293T and NIH/3T3). Scale bar: 20 μm. Red channel (IR783): ex = 785 nm, 835 nm LP filter. (D) Quantitative fluorescence analysis of the images in (C). Data represent mean ± SD (n = 7); ***p < 0.001. (E) Confocal images demonstrating specific activation of the probe. BRL cells were incubated with either the functional NMP-122-MET (200 nM) or a mutated control probe (mNMP-122-MET, 200 nM) and treated ± miR-122 or a miR-122 antisense oligonucleotide (400 nM). Scale bar: 20 μm. Red channel (IR783): ex = 785 nm, 835 nm LP filter. (F) Quantitative fluorescence analysis of the images in (E). Data represent mean ± SD (n = 5); ****p < 0.0001. (G) Confocal images demonstrating activation of the probe to the miR-122 released by the injured cells. BRL cells were incubated with 9 mM APAP for 6 h or pretreated with NAC (1 mM) for 1 h, followed by the addition of the functional NMP-122-MET (200 nM) and treated ± APAP or NAC. Scale bar: 20 μm. Red channel (IR783): ex = 785 nm, 835 nm LP filter. (H) Quantitative fluorescence analysis of the images in (G). Data represent mean ± SD (n = 5); ****p < 0.0001. (I) Schematic of the deep-tissue imaging comparison. Capillary tubes containing miR-122 (5 μM)-activated probes (NIR-II NMP-122-MET or NIR-I Cy5-BHQ3 probe, 5 μM each) were embedded in a 1% Intralipid phantom at depths ranging from 0 to 8 mm. Imaging conditions: NIR-II: ex = 808 nm, 1040 nm LP filter; Cy5: ex = 640 nm, 680 nm LP filter. (J) Resulting fluorescence images at different depths from the setup in (F), comparing the signal penetration of the NIR-II and NIR-I probes.
在应用层面，研究团队开发了针对肝毒性相关微 RNA（miR-122） 的响应探针 NMP-122。该探针集成了 c-MET 靶向适配体，能够精准锚定在肝细胞膜上。在活细胞实验中，探针能够敏锐地捕捉到受损肝细胞释放的胞外 miR-122 信号。随后在药物诱导性肝损伤（DILI） 的小鼠模型中，通过静脉注射 NMP-122，研究者成功实现了对肝脏损伤过程的实时、无创监测。成像结果清晰地显示了肝脏中 miR-122 水平的动态波动，且荧光强度与生化指标及 qPCR 检测结果表现出极高的相关性，证明了该平台在深层组织分子诊断中的可靠性。
Figure 5. Noninvasive NIR-II imaging of drug-induced liver injury using NMP-122-MET. (A) Schematic depicting the diagnosis of DILI via tail vein-injected NMP-122-MET. (B) Experimental timeline for the DILI model, outlining the APAP, NAC, and probe administration schedule. (C) Validation of the DILI model by qPCR, showing elevated serum miR-122 levels in APAP-treated mice and their reduction by NAC pretreatment. (D–E) Left: Representative whole-body NIR-II fluorescence images at indicated time points postinjection of the functional NMP-122-MET (5 μM, 100 μL) or the mutant probe (mNMP-122-MET, 5 μM, 100 μL). Right: Quantified hepatic fluorescence intensity over time (imaging parameters: λex = 808 nm, 1040 nm LP filter). (F–G) Representative ex vivo images and corresponding fluorescence quantification of excised livers from mice in all treatment groups. *p < 0.05, **p < 0.01, ***p < 0.001.
总结及展望
这项工作不仅开发出了一套性能优异的 DNA 纳米探针工具箱，更重要的是建立了一套通用的近红外二区荧光团与淬灭剂共设计蓝图。通过将白蛋白的生物活性与 DNA 的编程逻辑相结合，研究人员展示了复杂逻辑运算（如 AND/OR 门）在深层组织成像中的可能性。这一突破性的进展为 DNA 纳米技术从体外检测走向临床转化铺平了道路。未来，随着 AI 蛋白质设计工具的介入，这种定制化的蛋白质笼有望进一步提升探针的超稳定性和环境适应性，为癌症转移监测和各类器官损伤的早期诊断提供更强大的技术支撑。