Zhao Xueqi, Zhao Yue, Li Zhu, Liu Huan, Fu Wenhao, Chen Feng, Sun Ying, Song Daqian, Fan Chunhai, Zhao Yongxi
Jilin Province Research Center for Engineering and Technology of Spectral Analytical Instruments, College of Chemistry, Jilin University, Changchun 130012, Jilin, People's Republic of China.
Institute of Analytical Chemistry and Instrument for Life Science, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an 710049, Shaanxi, People's Republic of China.
Proc Natl Acad Sci U S A. 2025 Apr 15;122(15):e2425000122. doi: 10.1073/pnas.2425000122. Epub 2025 Apr 10.
Cellular structure maintenance and function regulation critically depend on the composition and spatial distribution of numerous membrane proteins. However, current methods face limitations in spatial coverage and data scalability, hindering the comprehensive analysis of protein interactions in complex cellular nanoenvironment. Herein, we introduce roximity-ctivated NA canning ncoded sequencing (PADSE-seq), an innovative technique that utilizes flexible DNA probes with adjustable lengths. These dynamic probes are anchored at a single end, enabling free swings within a nanoscale range to perform global scanning, recording, and accumulating of information on diverse proximal proteins in random directions along unrestricted paths. PADSE-seq leverages the autonomous cyclic cleavage of single-stranded DNA to sequentially activate encoded probes distributed throughout the local area. This process triggers strand displacement amplification and bidirectional extension reactions, linking proteins barcodes with molecular barcodes in tandem and further generating millions to billions of amplicons embedded with the combinatorial identifiers for next-generation sequencing analysis. As a proof of concept, we validated PADSE-seq for mapping the distribution of over a dozen kinds of proteins, including HER1, EpCAM, and PDL1, in proximity to HER2 in breast cancer cell lines, demonstrating its ability to decode multiplexed protein proximities at the nanoscale. Notably, we observed that the spatial distribution of proximal proteins around low-abundance target proteins exhibited greater diversity across regions with variable proximity ranges. This method offers a massive access for high-resolution and comprehensive mapping of cellular molecular interactions, paving the way for deeper insights into complex biological processes and advancing the field of precision medicine.
细胞结构的维持和功能调控严重依赖于众多膜蛋白的组成和空间分布。然而，目前的方法在空间覆盖范围和数据可扩展性方面存在局限性，阻碍了对复杂细胞纳米环境中蛋白质相互作用的全面分析。在此，我们介绍了邻近激活核酸扫描编码测序（PADSE-seq），这是一种创新技术，它利用了长度可调的柔性DNA探针。这些动态探针一端固定，能够在纳米尺度范围内自由摆动，沿无限制路径在随机方向上对各种邻近蛋白质进行全局扫描、记录和信息积累。PADSE-seq利用单链DNA的自主循环切割来依次激活分布在整个局部区域的编码探针。这个过程触发链置换扩增和双向延伸反应，将蛋白质条形码与分子条形码串联连接，进一步生成数百万到数十亿个嵌入组合标识符的扩增子，用于下一代测序分析。作为概念验证，我们验证了PADSE-seq可用于绘制乳腺癌细胞系中与HER2邻近的十几种蛋白质（包括HER1、EpCAM和PDL1）的分布图谱，证明了其在纳米尺度上解码多重蛋白质邻近关系的能力。值得注意的是，我们观察到低丰度靶蛋白周围邻近蛋白质的空间分布在不同邻近范围的区域表现出更大的多样性。这种方法为细胞分子相互作用的高分辨率和全面映射提供了大量途径，为深入了解复杂生物过程和推动精准医学领域发展铺平了道路。
