Journal
NATURE BIOTECHNOLOGY
Volume 37, Issue 1, Pages 84-+Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nbt.4286
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Funding
- NIH [1F32GM120929-01A1, F30 NS100595, 5T32DK007780-19, 4DN U01 HL129998, R33 EB019767]
- Human Frontier Science Program [LT000919/2016-l]
- NIH Center for Photogenomics [RM1 HG007743]
- Chan Zuckerberg Initiative [174285]
- HCA Pilot Project [174285]
- NSF CAREER [1350601]
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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 nonspecific probe binding. Here we present click-amplifying FISH (clampFISH), a method for fluorescence detection of nucleic acids that achieves high specificity and high-gain (>400-fold) 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 bio-orthogonal 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.
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