AgoFISH: cost-effective in situ labelling of genomic loci based on DNA-guided dTtAgo protein
AgoFISH: cost-effective in situ labelling of genomic loci based on DNA-guided dTtAgo protein
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AgoFISH：基于 DNA 引导的 dTtAgo 蛋白的经济高效的基因组位点原位标记
DOI：
10.1039/c9nh00028c
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发表时间：
2019-07-01
影响因子：
9.7
通讯作者：
Sun, Yujie
中科院分区：
文献类型：
--
作者：
Chang, Lei;Sheng, Gang;Sun, Yujie
DNA荧光原位杂交(FISH)技术已被广泛应用于真核细胞基因组区域的空间定位。目前的FISH探针制备方法存在合成成本高、实验步骤复杂等问题，限制了其适用性。在这里，我们报告了一种新的FISH方法，称为AgoFISH，该方法基于DNA引导的嗜热嗜热菌核酸酶缺陷蛋白(DTtAgo)的DNA结合活性。荧光标记的dTtAgo与5‘-磷酸化的单链引导DNA相结合，可以将荧光标记引入内源DNA序列，与引导DNA互补。我们证明了AgoFISH可以成功地标记人类和小鼠细胞中的重复序列，包括着丝粒、着丝粒周围和单拷贝串联重复序列。将dTtAgo与设计的单链引导DNA池组装在一起，能够可视化编码基因中的非重复序列。此外，AgoFISH还可以应用于无通道间串扰的双色标签。这种经济、简便的方法为染色质高阶结构的研究以及基于FISH的临床分子诊断提供了一种方便而有力的工具。
DNA fluorescence in situ hybridization (FISH) has been widely used for visualizing spatial localization of genomic regions in eukaryotic nuclei. Current probe preparation methods for FISH involve high probe synthesis cost and complex experimental procedures, thus limiting their applicability. Here, we report a new FISH method named AgoFISH based on the DNA-guided DNA binding activity of the nuclease-deficient Argonaute protein in Thermus thermophilus (dTtAgo). Fluorescently labelled dTtAgo combined with 5 '-phosphorylated single-stranded guide DNA can bring the fluorescent label to the endogenous DNA sequence that is complementary to the guide DNA. We demonstrated that AgoFISH can successfully label repetitive sequences in human and mouse cells, including the centromere, the pericentromere and single-copy tandem repeats. Assembling dTtAgo with a designed pool of single-stranded guide DNAs is able to visualize nonrepetitive sequences within coding genes. Furthermore, AgoFISH can also be applied to dual-color labelling without crosstalk between channels. This cost-effective and simple method provides a convenient and powerful tool for chromatin higher-order structure studies as well as FISH-based clinical molecular diagnosis.