Harnessing CRISPR/Cas12a Activity and DNA-Based Ultrabright FluoroCube for In Situ Imaging of Metabolically Labeled Cell Membrane Glycoproteins.

IF 9.6 1区材料科学 Q1 CHEMISTRY, MULTIDISCIPLINARY Nano Letters Pub Date : 2024-11-13 Epub Date: 2024-10-29 DOI:10.1021/acs.nanolett.4c03605

Jiajia Liu, Ziyan Zhou, Yifan Bo, Qiming Yan, Xin Su

{"title":"Harnessing CRISPR/Cas12a Activity and DNA-Based Ultrabright FluoroCube for In Situ Imaging of Metabolically Labeled Cell Membrane Glycoproteins.","authors":"Jiajia Liu, Ziyan Zhou, Yifan Bo, Qiming Yan, Xin Su","doi":"10.1021/acs.nanolett.4c03605","DOIUrl":null,"url":null,"abstract":"Fluorescence imaging of cell membrane glycoproteins based on metabolic labeling faces challenges including the sensitivity and spatial specificity and the use of a high concentration of unnatural sugars. To overcome these limitations, we developed a method for in situ imaging of cell membrane glycoproteins by operating Cas12a activity, and employing the ultrabright DNA nanostructure, FluoroCube (FC), as a signal reporter. Following Cas12a activation, we observed stable and intense fluorescence signals within 15 min. The combination of bright FC and Cas12a's amplification capability allows for effective imaging with only 5 μM of unnatural sugars and a brief 24-h incubation. Computational modeling demonstrates that Cas12a specifically cleaves FC in the 11-17 nm range of the glycosylation site, enabling spatially precise imaging. This approach successfully enabled fluorescence imaging of glycoproteins across various cell lines and the detection of changes in glycoprotein levels induced by drugs.","PeriodicalId":53,"journal":{"name":"Nano Letters","volume":null,"pages":null},"PeriodicalIF":9.6000,"publicationDate":"2024-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Nano Letters","FirstCategoryId":"88","ListUrlMain":"https://doi.org/10.1021/acs.nanolett.4c03605","RegionNum":1,"RegionCategory":"材料科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/10/29 0:00:00","PubModel":"Epub","JCR":"Q1","JCRName":"CHEMISTRY, MULTIDISCIPLINARY","Score":null,"Total":0}

引用次数: 0

Abstract

Fluorescence imaging of cell membrane glycoproteins based on metabolic labeling faces challenges including the sensitivity and spatial specificity and the use of a high concentration of unnatural sugars. To overcome these limitations, we developed a method for in situ imaging of cell membrane glycoproteins by operating Cas12a activity, and employing the ultrabright DNA nanostructure, FluoroCube (FC), as a signal reporter. Following Cas12a activation, we observed stable and intense fluorescence signals within 15 min. The combination of bright FC and Cas12a's amplification capability allows for effective imaging with only 5 μM of unnatural sugars and a brief 24-h incubation. Computational modeling demonstrates that Cas12a specifically cleaves FC in the 11-17 nm range of the glycosylation site, enabling spatially precise imaging. This approach successfully enabled fluorescence imaging of glycoproteins across various cell lines and the detection of changes in glycoprotein levels induced by drugs.

Abstract Image

查看原文

微信好友朋友圈 QQ好友复制链接

本刊更多论文

利用 CRISPR/Cas12a 活性和基于 DNA 的超亮荧光立方体对代谢标记的细胞膜糖蛋白进行原位成像。

基于代谢标记的细胞膜糖蛋白荧光成像面临着灵敏度和空间特异性以及使用高浓度非天然糖等挑战。为了克服这些局限性，我们开发了一种通过操作 Cas12a 活性对细胞膜糖蛋白进行原位成像的方法，并采用超亮 DNA 纳米结构 FluoroCube（FC）作为信号报告器。Cas12a 激活后，我们在 15 分钟内观察到了稳定而强烈的荧光信号。明亮的 FC 与 Cas12a 的放大能力相结合，只需 5 μM 的非天然糖和短暂的 24 小时培养就能实现有效成像。计算模型表明，Cas12a 能在糖基化位点的 11-17 纳米范围内特异性地裂解 FC，从而实现空间精确成像。这种方法成功地实现了对各种细胞系的糖蛋白进行荧光成像，并能检测药物诱导的糖蛋白水平变化。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文去求助

来源期刊

Nano Letters 工程技术-材料科学：综合

CiteScore

16.80

自引率

2.80%

发文量

1182

审稿时长

1.4 months

期刊介绍： Nano Letters serves as a dynamic platform for promptly disseminating original results in fundamental, applied, and emerging research across all facets of nanoscience and nanotechnology. A pivotal criterion for inclusion within Nano Letters is the convergence of at least two different areas or disciplines, ensuring a rich interdisciplinary scope. The journal is dedicated to fostering exploration in diverse areas, including: - Experimental and theoretical findings on physical, chemical, and biological phenomena at the nanoscale - Synthesis, characterization, and processing of organic, inorganic, polymer, and hybrid nanomaterials through physical, chemical, and biological methodologies - Modeling and simulation of synthetic, assembly, and interaction processes - Realization of integrated nanostructures and nano-engineered devices exhibiting advanced performance - Applications of nanoscale materials in living and environmental systems Nano Letters is committed to advancing and showcasing groundbreaking research that intersects various domains, fostering innovation and collaboration in the ever-evolving field of nanoscience and nanotechnology.