Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Quantitative imaging of single mRNA splice variants in living cells

Nature Nanotechnology volume 9, pages 474–480 (2014)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Schematic design of sequence-selective single mRNA detection.
Figure 2: Hybridization dynamics of nanoparticle monomers and dimers measured by imaging and spectral analysis.
Figure 3: Quantification of intracellular dimers.
Figure 4: Detection and quantification of mRNA by hyperspectral measurement of plasmonic dimer probes.

Similar content being viewed by others

References

  1. Orban, T. I. & Olah, E. Expression profiles of BRCA1 splice variants in asynchronous and in G1/S synchronized tumor cell lines. Biochem. Biophys. Res. Commun. 280, 32–38 (2001).

    Article  CAS  Google Scholar 

  2. Budhram-Mahadeo, V., Ndisang, D., Ward, T., Weber, B. L. & Latchman, D. S. The Brn-3b POU family transcription factor represses expression of the BRCA-1 anti-oncogene in breast cancer cells. Oncogene 18, 6684–6691 (1999).

    Article  CAS  Google Scholar 

  3. Xu, X. L. et al. Conditional mutation of Brca1 in mammary epithelial cells results in blunted ductal morphogenesis and tumour formation. Nature Genet. 22, 37–43 (1999).

    Article  CAS  Google Scholar 

  4. Wilson, C. A. et al. Differential subcellular localization, expression and biological toxicity of BRCA1 and the splice variant BRCA1-Delta 11b. Oncogene 14, 1–16 (1997).

    Article  CAS  Google Scholar 

  5. Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A. & Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nature Methods 5, 877–879 (2008).

    Article  CAS  Google Scholar 

  6. Trcek, T., Larson, D. R., Moldón, A., Query, C. C. & Singer, R. H. Single-molecule mRNA decay measurements reveal promoter-regulated mRNA stability in yeast. Cell 147, 1484–1497 (2011).

    Article  CAS  Google Scholar 

  7. Levsky, J. M., Shenoy, S. M., Pezo, R. C. & Singer, R. H. Single-cell gene expression profiling. Science 297, 836–840 (2002).

    Article  CAS  Google Scholar 

  8. Shav-Tal, Y. et al. Dynamics of single mRNPs in nuclei of living cells. Science 304, 1797–1800 (2004).

    Article  CAS  Google Scholar 

  9. Santangelo, P., Nitin, N. & Bao, G. Nanostructured probes for RNA detection in living cells. Ann. Biomed. Eng. 34, 39–50 (2006).

    Article  Google Scholar 

  10. Bratu, D. P., Cha, B-J., Mhlanga, M. M., Kramer, F. R. & Tyagi, S. Visualizing the distribution and transport of mRNAs in living cells. Proc. Natl Acad. Sci. USA 100, 13308–13313 (2003).

    Article  CAS  Google Scholar 

  11. Tsuji, A. et al. Direct observation of specific messenger RNA in a single living cell under a fluorescence microscope. Biophys. J. 78, 3260–3274 (2000).

    Article  CAS  Google Scholar 

  12. Prigodich, A. E. et al. Nano-flares for mRNA regulation and detection. ACS Nano 3, 2147–2152 (2009).

    Article  CAS  Google Scholar 

  13. Lee, S. E., Liu, G. L., Kim, F. & Lee, L. P. Remote optical switch for localized and selective control of gene interference. Nano Lett. 9, 562–570 (2009).

    Article  CAS  Google Scholar 

  14. Seferos, D. S., Giljohann, D. A., Hill, H. D., Prigodich, A. E. & Mirkin, C. A. Nano-flares: probes for transfection and mRNA detection in living cells. J. Am. Chem. Soc. 129, 15477–15479 (2007).

    Article  CAS  Google Scholar 

  15. Dubois, A., Canva, M., Brun, A., Chaput, F. & Boilot, J-P. Photostability of dyemolecules trapped in solid matrices. Appl. Opt. 35, 3193–3199 (1996).

    Article  CAS  Google Scholar 

  16. Jun, Y-w. et al. Continuous imaging of plasmon rulers in live cells reveals early-stage caspase-3 activation at the single-molecule level. Proc. Natl Acad. Sci. USA 106, 17735–17740 (2009).

    Article  CAS  Google Scholar 

  17. Cognet, L. et al. Single metallic nanoparticle imaging for protein detection in cells. Proc. Natl Acad. Sci. USA 100, 11350–11355 (2003).

    Article  CAS  Google Scholar 

  18. Mirkin, C. A., Letsinger, R. L., Mucic, R. C. & Storhoff, J. J. A DNA-based method for rationally assembling nanoparticles into macroscopic materials. Nature 382, 607–609 (1996).

    Article  CAS  Google Scholar 

  19. Alivisatos, A. P. et al. Organization of ‘nanocrystal molecules' using DNA. Nature 382, 609–611 (1996).

    Article  CAS  Google Scholar 

  20. Elghanian, R., Storhoff, J. J., Mucic, R. C., Letsinger, R. L. & Mirkin, C. A. Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles. Science 277, 1078–1081 (1997).

    Article  CAS  Google Scholar 

  21. Yguerabide, J. & Yguerabide, E. E. Light-scattering submicroscopic particles as highly fluorescent analogs and their use as tracer labels in clinical and biological applications: II. Experimental characterization. Anal. Biochem. 262, 157–176 (1998).

    Article  CAS  Google Scholar 

  22. Lee, K. & Irudayaraj, J. Periodic and dynamic 3-D gold nanoparticle–DNA network structures for surface-enhanced Raman spectroscopy-based quantification. J. Phys. Chem. C 113, 5980–5983 (2009).

    Article  CAS  Google Scholar 

  23. Graham, D., Thompson, D. G., Smith, W. E. & Faulds, K. Control of enhanced Raman scattering using a DNA-based assembly process of dye-coded nanoparticles. Nature Nanotech. 3, 548–551 (2008).

    Article  CAS  Google Scholar 

  24. Bao-An, D., Zheng-Ping, L. & Cheng-Hui, L. One-step homogeneous detection of DNA hybridization with gold nanoparticle probes by using a linear light-scattering technique. Angew. Chem. Int. Ed. 45, 8022–8025 (2006).

    Article  Google Scholar 

  25. Storhoff, J. J., Lucas, A. D., Garimella, V., Bao, Y. P. & Muller, U. R. Homogeneous detection of unamplified genomic DNA sequences based on colorimetric scatter of gold nanoparticle probes. Nature Biotechnol. 22, 883–887 (2004).

    Article  CAS  Google Scholar 

  26. Taton, T. A., Mirkin, C. A. & Letsinger, R. L. Scanometric DNA array detection with nanoparticle probes. Science 289, 1757–1760 (2000).

    Article  CAS  Google Scholar 

  27. De La Rica, R. & Stevens, M. M. Plasmonic ELISA for the ultrasensitive detection of disease biomarkers with the naked eye. Nature Nanotech. 7, 821–824 (2012).

    Article  CAS  Google Scholar 

  28. Reinhard, B. r. M., Sheikholeslami, S., Mastroianni, A., Alivisatos, A. P. & Liphardt, J. Use of plasmon coupling to reveal the dynamics of DNA bending and cleavage by single EcoRV restriction enzymes. Proc. Natl Acad. Sci. USA 104, 2667–2672 (2007).

    Article  CAS  Google Scholar 

  29. Sonnichsen, C., Reinhard, B. M., Liphardt, J. & Alivisatos, A. P. A molecular ruler based on plasmon coupling of single gold and silver nanoparticles. Nature Biotechnol. 23, 741–745 (2005).

    Article  Google Scholar 

  30. Liu, G. L. et al. A nanoplasmonic molecular ruler for measuring nuclease activity and DNA footprinting. Nature Nanotech. 1, 47–52 (2006).

    Article  CAS  Google Scholar 

  31. Raschke, G. et al. Biomolecular recognition based on single gold nanoparticle light scattering. Nano Lett. 3, 935–938 (2003).

    Article  CAS  Google Scholar 

  32. Lee, K. & Irudayaraj, J. Correct spectral conversion between surface-enhanced Raman and plasmon resonance scattering from nanoparticle dimers for single-molecule detection. Small 9, 1106–1115 (2012).

    Article  Google Scholar 

  33. Lee, K., Drachev, V. P. & Irudayaraj, J. DNA–gold nanoparticle reversible networks grown on cell surface marker sites: application in diagnostics. ACS Nano 5, 2109–2117 (2011).

    Article  CAS  Google Scholar 

  34. Jain, P. K., Huang, W. & El-Sayed, M. A. On the universal scaling behavior of the distance decay of plasmon coupling in metal nanoparticle pairs: a plasmon ruler equation. Nano Lett. 7, 2080–2088 (2007).

    Article  CAS  Google Scholar 

  35. Mie, G. Contributions to the optics of diffusing media. Ann. Physik 25, 377 (1908).

    Article  CAS  Google Scholar 

  36. Debye, P. Light scattering in solutions. J. Appl. Phys. 15, 338–342 (1944).

    Article  CAS  Google Scholar 

  37. Capecchi, M. R. High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell 22, 479–488 (1980).

    Article  CAS  Google Scholar 

  38. Feng, L., Huang, J. & Chen, J. MERIT40 facilitates BRCA1 localization and DNA damage repair. Genes Dev. 23, 719–728 (2009).

    Article  CAS  Google Scholar 

  39. Wang, B., Hurov, K., Hofmann, K. & Elledge, S. J. NBA1, a new player in the Brca1 A complex, is required for DNA damage resistance and checkpoint control. Genes Dev. 23, 729–739 (2009).

    Article  CAS  Google Scholar 

  40. Shao, G. et al. MERIT40 controls BRCA1–Rap80 complex integrity and recruitment to DNA double-strand breaks. Genes Dev. 23, 740–754 (2009).

    Article  CAS  Google Scholar 

  41. Orban, T. I. & Olah, E. Emerging roles of BRCA1 alternative splicing. Mol. Pathol. 56, 191–197 (2003).

    Article  CAS  Google Scholar 

  42. Sun, L. & Irudayaraj, J. PCR-free quantification of multiple splice variants in a cancer gene by surface-enhanced Raman spectroscopy. J. Phys. Chem. B 113, 14021–14025 (2009).

    Article  CAS  Google Scholar 

  43. Sun, L. & Irudayaraj, J. Quantitative surface-enhanced Raman for gene expression estimation. Biophys. J. 96, 4709–4716 (2009).

    Article  CAS  Google Scholar 

  44. Sun, L., Yu, C. & Irudayaraj, J. Raman multiplexers for alternative gene splicing. Anal. Chem. 80, 3342–3349 (2008).

    Article  CAS  Google Scholar 

  45. Seetin, M. G. & Mathews, D. H. in Bacterial Regulatory RNA 99–122 (Springer, 2012).

  46. Rouskin, S., Zubradt, M., Washietl, S., Kellis, M. & Weissman, J. S. Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo. Nature 505, 701–705 (2013).

    Article  Google Scholar 

  47. Krude, T. Mimosine arrests proliferating human cells before onset of DNA replication in a dose-dependent manner. Exp. Cell Res. 247, 148–159 (1999).

    Article  CAS  Google Scholar 

  48. Grunwald, D. & Singer, R. H. In vivo imaging of labelled endogenous beta-actin mRNA during nucleocytoplasmic transport. Nature 467, 604–607 (2010).

    Article  Google Scholar 

  49. Larson, D. R., Zenklusen, D., Wu, B., Chao, J. A. & Singer, R. H. Real-time observation of transcription initiation and elongation on an endogenous yeast gene. Science 332, 475–478 (2011).

    Article  CAS  Google Scholar 

  50. Yu, C., Nakshatri, H. & Irudayaraj, J. Identity profiling of cell surface markers by multiplex gold nanorod probes. Nano Lett. 7, 2300–2306 (2007).

    Article  CAS  Google Scholar 

Download references

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).

Author information

Authors and Affiliations

  1. Department of Bioengineering, Department of Electrical Engineering and Computer Science, University of California Berkeley, California, 94720, Berkeley, USA

    Kyuwan Lee & Luke P. Lee

  2. Department of Agricultural and Biological Engineering, Bindley Bioscience Center, Purdue University, 225 South University Street, West Lafayette, 47907, Indiana, USA

    Kyuwan Lee, Yi Cui & Joseph Irudayaraj

Authors
  1. Kyuwan Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yi Cui
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Luke P. Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Joseph Irudayaraj
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding authors

Correspondence to Luke P. Lee or Joseph Irudayaraj.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary Information (PDF 2141 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nnano.2014.73

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing