Abstract
Alternative messenger RNA (mRNA) splicing is a fundamental process of gene regulation, and errors in RNA splicing are known to be associated with a variety of different diseases. However, there is currently a lack of quantitative technologies for monitoring mRNA splice variants in cells. Here, we show that a combination of plasmonic dimer probes and hyperspectral imaging can be used to detect and quantify mRNA splice variants in living cells. The probes are made from gold nanoparticles functionalized with oligonucleotides and can hybridize to specific mRNA sequences, forming nanoparticle dimers that exhibit distinct spectral shifts due to plasmonic coupling. With this approach, we show that the spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene, BRCA1, can be monitored at single-copy resolution by measuring the hybridization dynamics of the nanoplasmonic dimers. Our study provides insights into RNA and its transport in living cells, which could improve our understanding of cellular protein complexes, pharmacogenomics, genetic diagnosis and gene therapies.
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Acknowledgements
The authors thank Z. Machaty and K. Lee for the initial discussion on microinjection. The authors acknowledge funding to J.I. from the National Science Foundation (award no. 1249315), the Indiana Clinical Translational Sciences Institute (NIH-TR000006) and the Purdue Center for Cancer Research (NIH-NCI CCSG CA23168) and funding to K.L. and L.L. from Samsung (GRO 20122166) and Stanford NIH (award no. U54CA151459).
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K.L. and J.I. conceived the original idea for this study. K.L., L.L. and J.I. designed the experiments. K.L. built the hyperspectral imaging system and developed the data analysis algorithm. K.L. performed the nanoparticle dimer probe design/synthesis/characterization, microinjection and optical measurements. Y.C. performed the PCR, flow cytometry and other supporting biological validations. K.L., L.L. and J.I. analysed the data and wrote the manuscript.
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Lee, K., Cui, Y., Lee, L. et al. Quantitative imaging of single mRNA splice variants in living cells. Nature Nanotech 9, 474–480 (2014). https://doi.org/10.1038/nnano.2014.73
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DOI: https://doi.org/10.1038/nnano.2014.73
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