SABER amplifies FISH: enhanced multiplexed imaging of RNA and DNA in cells and tissues

Abstract

Fluorescence in situ hybridization (FISH) reveals the abundance and positioning of nucleic acid sequences in fixed samples. Despite recent advances in multiplexed amplification of FISH signals, it remains challenging to achieve high levels of simultaneous amplification and sequential detection with high sampling efficiency and simple workflows. Here we introduce signal amplification by exchange reaction (SABER), which endows oligonucleotide-based FISH probes with long, single-stranded DNA concatemers that aggregate a multitude of short complementary fluorescent imager strands. We show that SABER amplified RNA and DNA FISH signals (5- to 450-fold) in fixed cells and tissues. We also applied 17 orthogonal amplifiers against chromosomal targets simultaneously and detected mRNAs with high efficiency. We then used 10-plex SABER-FISH to identify in vivo introduced enhancers with cell-type-specific activity in the mouse retina. SABER represents a simple and versatile molecular toolkit for rapid and cost-effective multiplexed imaging of nucleic acid targets.

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Fig. 1: SABER-FISH design and workflow.
Fig. 2: SABER effectively amplifies fluorescent signals.
Fig. 3: Transcript detection and quantification in retina tissue.
Fig. 4: SABER enables spectrally multiplexed imaging.
Fig. 5: Exchange-SABER for detection of cell types in retina tissue.
Fig. 6: Sequential imaging of chromosomal targets using Exchange-SABER.
Fig. 7: SABER-FISH enables detection of in vivo RNA reporters for analysis of enhancer activity.

Data availability

All raw and processed data are available from the authors upon reasonable request.

Code availability

The complete set of CellProfiler38,60 pipelines used and example input images for each are available at https://github.com/brianbeliveau/SABER. PD3D, a package of MATLAB functions for detecting SABER puncta (or other fluorescent puncta) in 3D and assigning puncta to cells in a watershed segmentation, is available at https://github.com/ewest11/PD3D. Functions used for image processing are available at http://saber.fish or http://saber-fish.net/.

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Acknowledgements

The authors thank B. Fields, S. Kennedy, J.A. Abed, T. Wu, T. Ferrante, N. Liu, F. Dannenberg, M. Cicconet, P.M. Llopis and the Microscopy Resources on the North Quad (MicRoN) at Harvard Medical School for discussions and technical support. We also thank the ENCODE consortium and J. Stamatoyannopoulos (University of Washington) for retina DHS data. This work was supported by the National Institutes of Health (under grants 1R01EB018659-01 to P.Y., 1UG3HL145600 to P.Y., 1R01GM124401 to P.Y., 1U01MH106011-01 to P.Y., 1DP1GM133052 to P.Y., 5K99EY028215-02 to S.W.L. and a T32 training grant GM096911 supporting E.R.W.), the Office of Naval Research (under grants N00014-16-1-2410 to P.Y. and N00014-18-1-2549 to P.Y.), the National Science Foundation (under grant CCF-1317291 to P.Y. and a Graduate Research Fellowship to J.Y.K.), the Howard Hughes Medical Institute (C.L.C.), the Damon Runyon Cancer Research Foundation (under a fellowship to B.J.B.), the Uehara Memorial Foundation (under a fellowship to H.M.S.), the Human Frontier Science Program (under fellowship LT000048/2016-L to S.K.S.), EMBO (under fellowship ALTF 1278-2015 to S.K.S.) and the Wyss Institute’s Molecular Robotics Initiative (MRI) (P.Y., J.Y.K. and B.J.B.).

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Contributions

J.Y.K., S.W.L., B.J.B., E.R.W., C.L.C. and P.Y. conceived the study. J.Y.K. and B.J.B. designed SABER probes, designed and executed cell experiments and analyzed cell data. S.W.L. designed and executed tissue experiments. E.R.W. developed the analytical pipeline and methods for tissue cell segmentation and puncta quantification. J.Y.K., S.W.L., B.J.B., E.R.W., A.Z., S.K.S., H.M.S. and Y.W. contributed to optimizing and performing experimental protocols and obtaining data. J.Y.K., S.W.L., B.J.B., E.R.W., C.L.C. and P.Y. wrote the manuscript. All authors edited and approved the manuscript. C.L.C. and P.Y. supervised the work.

Corresponding authors

Correspondence to Brian J. Beliveau or Constance L. Cepko or Peng Yin.

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Competing interests

A provisional US patent has been filed based on this work (PCT/US2018/013019). P.Y. is cofounder of Ultivue, Inc. and NuProbe Global.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–10 and Supplementary Note

Reporting Summary

Supplementary Protocols

Step-by-step instructions for designing and applying SABER-FISH

Supplementary Table 1

SABER sequences. PER, imager, and probe pool sequences.

Supplementary Table 2

SABER simulated parameters. Melting temperatures of PER, probe and bridge sequences under different formamide conditions, as well as cross-talk probabilities.

Supplementary Table 3

SABER experimental conditions. Detailed PER, ISH and fluorescent-hybridization conditions for each experiment.

Supplementary Table 4

SABER puncta counts. Cell counts, puncta counts, puncta sizes, signal-to-noise values, cross-correlation values and biological replicate information for experiments.

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Kishi, J.Y., Lapan, S.W., Beliveau, B.J. et al. SABER amplifies FISH: enhanced multiplexed imaging of RNA and DNA in cells and tissues. Nat Methods 16, 533–544 (2019). https://doi.org/10.1038/s41592-019-0404-0

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