【JACS】国家蛋白质科学中心秦伟捷联手东北大学舒杨|突破20毫米深度！刊登近红外触发系统，实现活体小鼠深度组织内生物大分子原位交联与标记#

文章标题：A Near-Infrared-Triggered Luminescence-Activated System for In Vivo Biomacromolecular Tagging and Photocatalytic Crosslinking for Large-Scale Investigation of RNA-Protein Complexes in Living Mice

通讯作者：Yang Shu, Weijie Qin

文章链接：https://doi.org/10.1021/jacs.6c06480

文章概要#

引言#

细胞与器官的功能受到蛋白质、核酸等生物大分子复杂相互作用网络的精确调控，其中RNA与蛋白质的相互作用在基因表达、免疫调控及多种疾病的发生发展中扮演着关键角色。尽管基于光催化的生物大分子原位交联与标记技术在细胞层面取得了显著进展，但由于紫外或可见光在生物组织中的穿透深度极其有限，且容易造成辐射损伤，如何在活体动物体内特别是深层器官中， spatiotemporally 捕捉和冻结这些动态、瞬时的生物大分子相互作用，一直是该领域面临的巨大挑战。

Scheme 1. Schematic Illustration of the IVCT-LAUC System for RNA-Protein Complexes (RPCs) Crosslinking and Tagging by NIR-Triggered in Situ Upconversion Photocatalysis#

主要实验及结论#

研究团队巧妙设计并合成了一种亲水性核壳多壳层上转换纳米颗粒（UCNPs），并将其与光敏剂脱镁叶绿酸A及呋喃-生物素探针相结合，成功构建了名为IVCT-LAUC的近红外触发活体交联与标记系统。在808纳米近红外激光的激发下，该纳米颗粒能够在组织内部原位发射出254纳米的紫外光和450/650纳米的可见光。其中，高能紫外光直接触发RNA与近端RNA结合蛋白的高效共价交联；而可见光则激活光敏剂产生局域化的单线态氧，驱动呋喃-生物素探针对交联后的复合物进行生物正交标记，在活体小鼠的肾脏和脾脏等深层器官中实现了秒级的时间分辨率捕获，且系统具有优异的体内生物相容性，不引起明显的组织损伤或热应激。

Figure 1. (a) Schematic illustration showing the synthesis process of the hydrophilic core–multishell UCNPs. (b) TEM and (c) HAADF-STEM images of the UCNPs. (d) STEM image and elemental mapping of a single UCNP. (e) Emission spectra of the UCNPs with and without 808 nm excitation.#

Figure 2. Confocal fluorescence microscopy images of HeLa cells showing internalization of (a) UCNP (red Cy3 channel) and (b) Pheo A (red fluorescence channel). (c) Intracellular 1O2 generation visualized by green DCF (scale bar = 50 μm) under different treatments. (d) Fluorescence intensity of DCF in the treated HeLa cells relative to the untreated controls for 1O2 quantification (cell number = 105, error bars represents mean ± SD, n = 3). (e) Fluorescence images showing the cellular internalization of the furan-biotin probe (scale bar = 100 μm). (f) Specificity of RNA labeling by furan-biotin evaluated via RNase digestion (blue Hoechst 33342 as a nuclear marker, the furan-biotin probe visualized by green streptavidin-FITC. scale bar = 25 μm).#

Figure 3. IVCT-LAUC system and 808 nm laser for global enrichment and identification of RBPs in HeLa cells. SDS-PAGE characterization of RBPs obtained (a) using different 808 nm irradiation times (15–60 s) and (b) under varying conditions. (c) Western blot analysis of Nucleolin, PTBP1, ELAVL1, β-actin, and β-tubulin in the enrichment product. The numbers below each lane indicate the relative optical density of the corresponding protein bands.#

Figure 4. IVCT-LAUC and 808 nm laser irradiation for global enrichment and identification of RBPs in HeLa cells. (a) Volcano plot displaying -log10(P value) (y-axis) and log2 fold change (x-axis) for identifying RBPs by quantitative differential proteomic comparison between the experimental group and the control group. Each red dot represents an RBP. (b) Quantification reproducibility evaluation of RBP enrichment in three tests. (c) GO enrichment analysis for molecular function and biological process. (d) Box plot of the abundance of the candidate and reported RBPs identified by the IVCT-LAUC strategy (*** p < 0.001).#

Scheme 2. Schematic Illustration of NIR Photocatalytic Crosslinking and Tagging in Living Mice using 808 nm Laser and the IVCT-LAUC for RBP Enrichment#

利用该系统出色的全器官穿透深度（可达20毫米），研究人员对活体小鼠器官进行了大规模的RNA结合蛋白组学筛选。通过定量差异蛋白质组学分析，成功在活体小鼠肾脏中鉴定出1709个高置信度的RNA结合蛋白，并在脾脏中鉴定出563个蛋白，其中包含了大量在传统离体或细胞实验中难以捕获的低丰度关键催化酶和抗病毒信号蛋白。此外，该平台被成功应用于靶向调控剂Roc-A和MS-444的体内药理机制研究，精准绘制了药物触发的活体大分子相互作用网络动态重构图谱，揭示了药物在生物体内引发的潜在脱靶效应及代谢重新编程过程。

Figure 5. Evaluation of the IVCT-LAUC strategy for in vivo RPC tagging and RNA analysis. (a) Left: Fluorescence images of mouse kidney (blue Hoechst 33342 for nuclei staining and green DCF for intracellular 1O2). Right: The fluorescence intensity of DCF for intracellular 1O2 detection in tissue of kidney (error bars represent mean ± SD, n = 3, scale bar = 100 μm). (b) Temperature changes on the skin surface above the target organs of mice injected with UCNP, furan-biotin, and Pheo A. Inset: photothermal imaging of mice at 1 and 2 min. (c) Streptavidin-HRP dot blotting analysis of the furan-biotin-tagged biomolecules from mouse kidney tissue. The upper panel shows RNA dot blot results under different treatment conditions. The lower panel demonstrates the selectivity of the furan-biotin probe. RNA and DNA are extracted from mouse kidney tissue using commercial extraction kits, and BSA serves as a protein control. (d) Pie chart of the type and percentage of RNA isolated by the IVCT-LAUC (Experiment) and TRIzon (Control).#

Figure 6. IVCT-LAUC system and 808 nm irradiation for global analysis of kidney RBPs in living mice. (a) Comparison of the IVCT-LAUC with 808 nm irradiation and the psoralen probe with 254 nm UV irradiation for RPC cross-linking in living mouse kidney using SDS-PAGE. (b) Quantification reproducibility of kidney RBP enrichment in three tests. (c) GO enrichment analysis of the identified RBPs from mouse kidney using IVCT-LAUC. (d) For each term, the number of proteins associated with translation. (e) Number of identified RBPs with classical and nonclassical RBDs. (f) Pathway enrichment analysis of the identified RBPs from mouse kidney.#

Figure 7. IVCT-LAUC system and 808 nm irradiation for global analysis of Roc-A-mediated in vivo RPI alterations. (a) Schematic representation of drug and vehicle administration protocols and temporal regimen in mice. (b) Volcano plot displaying differential RNA-binding of the mouse RBPs after administration of Roc-A and vehicle. X-axis: log2 fold change, and y-axis: -log10(P value). (c) Western blot validation of Roc-A-induced RNA association changes of selected RBPs. Values shown below each lane are relative to untreated mice (set at a value of 1). (d) GO molecular function analysis, (e) GO biological process analysis, and (f) pathway analysis of the significantly up-regulated candidate off-targets.#

总结及展望#

本研究成功开发出一种基于上转换发光诱导光催化的新型近红外大分子活体交联与标记策略，首次在活体小鼠深层器官中实现了无须基因编辑的RNA-蛋白质复合物秒级原位冻结与高效富集。该技术不仅克服了传统光学标记方法在体内穿透深度不足的瓶颈，也为在复杂的活体生理环境下开展大规模、高时空分辨率的生物大分子相互作用研究提供了强有力的工具。未来，这一通用型光催化开关平台有望扩展至更多类型的生物大分子标记，并在活体药效评估、疾病新靶点发现以及临床前药物脱靶风险评价等领域展现出广阔的应用前景。