Living-DNA Nanogel Appendant Enables In Situ Modulation and Quantification of Regulation Effects on Membrane Proteins.
Living-DNA Nanogel Appendant Enables In Situ Modulation and Quantification of Regulation Effects on Membrane Proteins.
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DOI：
10.1021/acsabm.1c00302
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发表时间：
2021-05
影响因子：
4.7
通讯作者：
Juan Song;Yinan Shen;Hansen Mou;Wen Li;Julie Brouchon;Bi-Yi Xu;X. Xia;Jing-Juan Xu;Hongyuan Chen
中科院分区：
文献类型：
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作者：
Juan Song;Yinan Shen;Hansen Mou;Wen Li;Julie Brouchon;Bi-Yi Xu;X. Xia;Jing-Juan Xu;Hongyuan Chen
筛选膜蛋白上的附属物，了解其不同的调节作用，是寻找膜蛋白靶向药物的理想选择。然而，大多数人工附属物不能在原位进化，也不能发出信号反映调制，很难支持原位条件筛选。在这里，我们设计了活DNA附件，使这样的筛选。首先，开发了活细胞滚环扩增（LCRCA）策略以延长用于自缠结物理纳米凝胶的DNA附件。纳米凝胶统一了膜蛋白调节和定量的功能：它们的大小随着LCRCA的时间长度增加而增加，这改变了对膜蛋白的调节作用;它们大量的重复短序列允许在互补荧光团标记的短DNA存在下定量它们的大小。然后，以α6β4整联蛋白为靶点检查了活DNA附件的性能，其中对肌动蛋白丝分布、细胞活力和失巢凋亡机会的有效调节都得到了验证。筛选也清楚地阐明了有趣的非线性关系之间的规定和影响。我们希望这种基于活DNA附加物的筛选策略能够为更深入地了解膜蛋白的自然行为提供原型，并有助于精确设计膜蛋白靶向药物。
Screening appendants on membrane proteins to understand their varied regulation effects is desirable for finding the potential candidates of the membrane-protein-targeted drugs. However, most artificial appendants can hardly support in situ condition screening because they cannot evolve in situ, neither can they send out signals to reflect the modulation. Here, we designed living-DNA appendants to enable such screening. First, the living-cell rolling-circle amplification (LCRCA) strategy was developed to elongate the DNA appendants for self-tangled physical nanogels. The nanogels unify both the functions of membrane-protein modulation and quantification: their sizes increase with the increased time length of LCRCA, which change the regulation effect on the membrane proteins; their large number of repeating short sequences allow quantification of their sizes in the presence of the complementary fluorophore-tagged short DNA. Then, the performance of the living-DNA appendants was examined taking α6β4 integrins as the target, where effective regulation over the distribution of actin filaments, cell viability, and chances of anoikis are all validated. The screening also clearly elucidates the interesting nonlinear relationships between the regulations and the effects. We hope this screening strategy based on living-DNA appendants can stand for a prototype for deeper understanding of natural behaviors of membrane proteins and help in the accurate designing of the membrane-protein-targeted drugs.