ClampFISH detects individual nucleic acid molecules using click chemistry-based amplification.
ClampFISH detects individual nucleic acid molecules using click chemistry-based amplification.
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夹鱼使用基于点击化学的扩增检测单个核酸分子。
DOI：
10.1038/nbt.4286
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发表时间：
2018-11-12
影响因子：
46.9
通讯作者：
Raj A
中科院分区：
文献类型：
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作者：
Rouhanifard SH;Mellis IA;Dunagin M;Bayatpour S;Jiang CL;Dardani I;Symmons O;Emert B;Torre E;Cote A;Sullivan A;Stamatoyannopoulos JA;Raj A
在细胞和组织中检测单个核酸的方法，如荧光原位杂交(FISH)，受到相对较低的信号强度和非特异性探针结合的限制。在这里，我们介绍了点击放大FISH(钳制FISH)，一种实现高特异性和高增益(>；400x)信号放大的核酸荧光检测方法。ClampFISH探针以双螺旋方式与目的序列杂交时形成“C”构型。使用生物正交点击化学将探针的末端连接在一起，有效地将探针锁定在靶子周围。反复的杂交和点击放大了荧光强度。我们发现，在低倍显微镜和基于RNA的流式细胞术中，夹持FISH能够检测RNA物种。此外，我们还证明了CLAMPFISH探针的模块化设计允许RNA和DNA检测的多路复用，锁定机制防止了扩张显微镜中的探针分离，并且CLAMPFISH可以应用于组织样本。
Methods for detecting single nucleic acids in cell and tissues, such as fluorescence in situ hybridization (FISH), are limited by relatively low signal intensity and non-specific probe binding. Here we present click-amplifying FISH (clampFISH), a method for fluorescence detection of nucleic acids that achieves high specificity and high-gain (>400x) signal amplification. ClampFISH probes form a “C” configuration upon hybridization to the sequence of interest in a double helical manner. The ends of the probes are ligated together using bioorthogonal click chemistry, effectively locking the probes around the target. Iterative rounds of hybridization and click amplify the fluorescence intensity. We show that clampFISH enables the detection of RNA species with low magnification microscopy and in RNA-based flow cytometry. Additionally, we show that the modular design of clampFISH probes allows multiplexing of RNA and DNA detection, that the locking mechanism prevents probe detachment in expansion microscopy, and that clampFISH can be applied in tissue samples.