Abstract

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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Acknowledgements

We thank F. Tuluc from the CHOP flow cytometry core facility for discussions and assistance with flow cytometry. We also thank J. Peterson for his early contributions and the many bioRxiv readers who reached out with feedback and suggestions. S.H.R. acknowledges support from NIH 1F32GM120929-01A1; I.A.M. acknowledges support from NIH F30 NS100595; O.S. acknowledges support from the Human Frontier Science Program LT000919/2016-l; C.L.J. acknowledges support from NIH 5T32DK007780-19; and A.R. from NIH 4DN U01 HL129998, NIH Center for Photogenomics RM1 HG007743, the Chan Zuckerberg Initiative and HCA Pilot Project 174285, NSF CAREER 1350601 and NIH R33 EB019767.

Author information

Affiliations

  1. Department of Bioengineering, University of Pennsylvania, Philadelphia Pennsylvania, USA.

    • Sara H Rouhanifard
    • , Ian A Mellis
    • , Margaret Dunagin
    • , Sareh Bayatpour
    • , Connie L Jiang
    • , Ian Dardani
    • , Orsolya Symmons
    • , Benjamin Emert
    • , Eduardo Torre
    • , Allison Cote
    •  & Arjun Raj
  2. Genomics and Computational Biology Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Ian A Mellis
    • , Benjamin Emert
    •  & Arjun Raj
  3. Cell and Molecular Biology Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Connie L Jiang
  4. Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Eduardo Torre
  5. Altius Institute for Biomedical Sciences, Seattle, Washington, USA.

    • Alessandra Sullivan
    •  & John A Stamatoyannopoulos
  6. Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Arjun Raj

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Contributions

S.H.R. and A.R. designed the study and wrote the manuscript. I.A.M. performed statistical analysis. S.H.R., M.D., S.B., C.L.J., I.D., O.S., B.E., E.T., A.C., A.S. and J.A.S. designed and performed the experiments.

Competing interests

A.R. receives royalties related to Stellaris RNA FISH probes.

Corresponding author

Correspondence to Arjun Raj.

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    Supplementary Text and Figures

    Supplementary Figures 1–14

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    Life Sciences Reporting Summary

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    Supplementary Protocol

    ClampFISH working protocol

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    Supplementary Table 1

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DOI

https://doi.org/10.1038/nbt.4286