Abstract
Small RNA molecules have an important role in gene regulation and RNA silencing therapy, but it is challenging to detect these molecules without the use of time-consuming radioactive labelling assays or error-prone amplification methods. Here, we present a platform for the rapid electronic detection of probe-hybridized microRNAs from cellular RNA. In this platform, a target microRNA is first hybridized to a probe. This probe:microRNA duplex is then enriched through binding to the viral protein p19. Finally, the abundance of the duplex is quantified using a nanopore. Reducing the thickness of the membrane containing the nanopore to 6 nm leads to increased signal amplitudes from biomolecules, and reducing the diameter of the nanopore to 3 nm allows the detection and discrimination of small nucleic acids based on differences in their physical dimensions. We demonstrate the potential of this approach by detecting picogram levels of a liver-specific miRNA from rat liver RNA.
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Acknowledgements
The authors would like to thank M. Fischbein for assistance with optimizing the plasma etch process, the laboratory of C. Johnson for the use of their plasma etcher, D. Yates for assistance with ADF-STEM, and G. Rosenbloom and B. Cooperman for the tRNA molecule. The authors also thank J. Bartel, J. Fairfield, M. Goulian, K. Healy, R. Johnson, C. Johnson, N. Peterman, G.V. Soni and L. Willis for helpful advice regarding the manuscript. This work was supported by the National Institutes of Health (grant no. R21HG004767) and the Penn Genomics Frontier Institute. T.D. acknowledges support from the Israel Science Foundation (Bikura postdoctoral fellowship).
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M.W. and L.M. conceived and designed the experiments. T.D., V.R. and M.W. designed, fabricated and characterized the nanopore devices. J.J. and L.M. developed and performed the miRNA enrichment protocols. M.W. performed the nanopore experiments and analysed the data. M.W., T.D., L.M. and M.D. wrote the manuscript, and all other authors commented on it.
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J.J. and L.M. are employees of New England BioLabs, a company that sells p19 and other proteins for RNA and DNA research.
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Wanunu, M., Dadosh, T., Ray, V. et al. Rapid electronic detection of probe-specific microRNAs using thin nanopore sensors. Nature Nanotech 5, 807–814 (2010). https://doi.org/10.1038/nnano.2010.202
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DOI: https://doi.org/10.1038/nnano.2010.202
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