Barcoded DNA nanostructures for the multiplexed profiling of subcellular protein distribution

Abstract

Massively parallel DNA sequencing is established, yet high-throughput protein profiling remains challenging. Here, we report a barcoding approach that leverages the combinatorial sequence content and the configurational programmability of DNA nanostructures for high-throughput multiplexed profiling of the subcellular expression and distribution of proteins in whole cells. The barcodes are formed by in situ hybridization of tetrahedral DNA nanostructures and short DNA sequences conjugated with protein-targeting antibodies, and by nanostructure-assisted ligation (either enzymatic or chemical) of the nanostructures and exogenous DNA sequences bound to nanoparticles of different sizes (which cause these localization sequences to differentially distribute across subcellular compartments). Compared with linear DNA barcoding, the nanostructured barcodes enhance the signal by more than 100-fold. By implementing the barcoding approach on a microfluidic device for the analysis of rare patient samples, we show that molecular subtypes of breast cancer can be accurately classified and that subcellular spatial markers of disease aggressiveness can be identified.

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Fig. 1: DNA STAMP.
Fig. 2: Highly sensitive protein detection with STAMP.
Fig. 3: STAMP measurements of protein expression and subcellular distribution.
Fig. 4: Multiplexed STAMP for high-throughput cellular profiling.
Fig. 5: Protein typing of rare clinical samples.

Data availability

The authors declare that the main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are available for research purposes from the corresponding author on reasonable request.

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Acknowledgements

The authors thank Y. K. Sim, Y. P. Neo and C. Liu for experimental assistance, J. L.Y. Yap, R. Malathi and A. Franco-Obregón for assistance with clinical sample collection, NUH Tissue Repository for providing clinical samples and S. V. Sundararajan for device fabrication. This work was supported in part by funding from NUS Research Scholarship, Ministry of Education, National Medical Research Council, NUS iHealthtech, A*STAR IMCB Independent Fellowship and NUS Early Career Research Award.

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N.R.S. and H.S. designed the study, performed data analysis and wrote the manuscript. N.R.S., N.R.Y.H., G.S.L., A.N., X.D. and Y.L. performed the research. C.W.C. and T.P.L. provided clinical samples. J.E.S. provided pathology evaluation. All authors contributed to the manuscript.

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Correspondence to Huilin Shao.

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Sundah, N.R., Ho, N.R.Y., Lim, G.S. et al. Barcoded DNA nanostructures for the multiplexed profiling of subcellular protein distribution. Nat Biomed Eng 3, 684–694 (2019). https://doi.org/10.1038/s41551-019-0417-0

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