Proximity-activated DNA scanning encoded sequencing for massive access to membrane proteins nanoscale organization

IF 9.4 1区 综合性期刊 Q1 MULTIDISCIPLINARY SCIENCES
Xueqi Zhao, Yue Zhao, Zhu Li, Huan Liu, Wenhao Fu, Feng Chen, Ying Sun, Daqian Song, Chunhai Fan, Yongxi Zhao
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Abstract

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 p roximity- a ctivated D NA s canning e 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.
邻近激活的DNA扫描编码测序的大规模访问膜蛋白纳米级组织
细胞结构的维持和功能的调节主要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和数据可扩展性方面存在局限性,阻碍了复杂细胞纳米环境中蛋白质相互作用的全面分析。在这里,我们介绍了p接近-一种激活DNA序列编码测序(pase -seq),这是一种利用灵活的DNA探针和可调节长度的创新技术。这些动态探针锚定在单端,使其能够在纳米尺度范围内自由摆动,从而沿着不受限制的路径沿随机方向对不同近端蛋白质进行全局扫描、记录和信息积累。pase -seq利用单链DNA的自主循环切割来顺序激活分布在整个局部区域的编码探针。该过程触发链位移扩增和双向延伸反应,将蛋白质条形码与分子条形码串联起来,并进一步产生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pase -seq用于绘制乳腺癌细胞系中HER1、EpCAM和PDL1等接近HER2的十几种蛋白质的分布,证明了其在纳米尺度上解码多路蛋白接近度的能力。值得注意的是,我们观察到低丰度目标蛋白附近近端蛋白的空间分布在不同接近范围的区域中表现出更大的多样性。该方法为细胞分子相互作用的高分辨率和全面制图提供了大量访问,为深入了解复杂的生物过程铺平了道路,并推动了精准医学领域的发展。
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来源期刊
CiteScore
19.00
自引率
0.90%
发文量
3575
审稿时长
2.5 months
期刊介绍: The Proceedings of the National Academy of Sciences (PNAS), a peer-reviewed journal of the National Academy of Sciences (NAS), serves as an authoritative source for high-impact, original research across the biological, physical, and social sciences. With a global scope, the journal welcomes submissions from researchers worldwide, making it an inclusive platform for advancing scientific knowledge.
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