Fang Yuxin, Zou Peng
College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, PKU-IDG/McGovern Institute for Brain Research, Peking University, Beijing, 100871, China.
Academy for Advanced Interdisciplinary Studies, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100871, China.
Acc Chem Res. 2025 Aug 5;58(15):2526-2534. doi: 10.1021/acs.accounts.5c00390. Epub 2025 Jul 21.
ConspectusEngineered photosensitizer proteins, such as miniSOG, KillerRed, and SuperNova, have long been used for light-triggered protein inhibition and cell ablation. Compared to synthetic organic dyes, these genetically encoded tags provide superior spatial precision for subcellular targeting. More recently, the photochemistry of miniSOG has been repurposed for subcellular omics studies. Upon light activation, miniSOG generates reactive oxygen species (ROS) that oxidize nearby nucleic acids or proteins. These oxidized biomolecules can then react with exogenously supplied nucleophilic probes, which introduce bio-orthogonal handles for downstream enrichment and analysis.This labeling strategy, known as photocatalytic proximity labeling (PPL), has emerged as a powerful approach for profiling the molecular architecture of subcellular compartments and identifying RNA or protein interactors of specific targets. The use of light provides exceptional temporal control, enabling labeling windows as short as 1 s. Moreover, PPL readily supports pulse-chase experiments through simple light on/off switching, an advantage not easily achievable with conventional platforms such as APEX or TurboID.In this account, we highlight our recent developments and applications of genetically encoded PPL tools. These include CAP-seq for RNA/DNA labeling, RinID for protein labeling, and LAP-seq/MS/CELL for bioluminescence-activated multi-omic profiling. Together, these tools enable detailed mapping of the cellular biomolecular landscape. For example, CAP-seq revealed enrichment of transcripts encoding secretory and mitochondrial proteins near the endoplasmic reticulum membrane and outer mitochondrial membrane, supporting models of localized translation. Additionally, pulse-chase labeling using RinID in the ER lumen uncovered distinct decay kinetics of secretory proteins.Looking forward, future efforts may focus on developing low-toxicity and low-background chemical probes, engineering red-shifted photosensitizers for deep-tissue and applications, and integrating multiple proximity labeling (PL) platforms to study organelle contact sites and interorganelle molecular trafficking.
综述工程化光敏蛋白，如miniSOG、KillerRed和SuperNova，长期以来一直用于光触发的蛋白质抑制和细胞消融。与合成有机染料相比，这些基因编码标签在亚细胞靶向方面提供了更高的空间精度。最近，miniSOG的光化学已被重新用于亚细胞组学研究。在光激活后，miniSOG产生活性氧（ROS），氧化附近的核酸或蛋白质。然后，这些氧化的生物分子可以与外源供应的亲核探针反应，引入生物正交手柄用于下游富集和分析。这种标记策略称为光催化邻近标记（PPL），已成为描绘亚细胞区室分子结构和识别特定靶标的RNA或蛋白质相互作用体的有力方法。光的使用提供了出色的时间控制，使标记窗口短至1秒。此外，PPL通过简单的光开/关切换很容易支持脉冲追踪实验，这是传统平台（如APEX或TurboID）不易实现的优势。在本综述中，我们重点介绍了基因编码PPL工具的最新进展和应用。这些工具包括用于RNA/DNA标记的CAP-seq、用于蛋白质标记的RinID以及用于生物发光激活的多组学分析的LAP-seq/MS/CELL。这些工具共同实现了细胞生物分子景观的详细图谱绘制。例如，CAP-seq揭示了在内质网膜和线粒体外膜附近编码分泌蛋白和线粒体蛋白的转录本的富集，支持了局部翻译模型。此外，在内质网腔中使用RinID进行脉冲追踪标记揭示了分泌蛋白不同的降解动力学。展望未来，未来的努力可能集中在开发低毒性和低背景的化学探针、设计用于深部组织和应用的红移光敏剂，以及整合多个邻近标记（PL）平台以研究细胞器接触位点和细胞器间分子运输。
