Quantitative imaging of single mRNA splice variants in living cells

Journal name:
Nature Nanotechnology
Volume:
9,
Pages:
474–480
Year published:
DOI:
doi:10.1038/nnano.2014.73
Received
Accepted
Published online

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.

At a glance

Figures

  1. Schematic design of sequence-selective single mRNA detection.
    Figure 1: Schematic design of sequence-selective single mRNA detection.

    a, Bottom: Spectral characteristics of different nanoparticle structures formed by DNA hybridization for sequence-specific detection. Monomers (light blue line) and dimers (purple line) can be differentiated from one another by their unique spectral characteristics. Top: Real-colour images (left panels) and finite-difference time-domain (FDTD) simulation results (right panels) of the nanoparticle monomer (i) and dimer (ii) showing the field intensities with (dimer) and without (monomer) plasmonic coupling between the particles. Scale bars, 1 µm (real colour images) and 20 nm (FDTD simulation results). b, Nanoparticle probes are injected into individual living cells to form dimers in a sequence-specific manner upon targeting a single mRNA. Hyperspectral imaging presents the full spectrum generated by each point in the whole image, so that splicing information can be analysed based on the changes in the spectral profile. Examples are shown of representative BRCA1 splicing structures (certain sequences are missing from wild type) and their spectral features when they are detected by nanoparticle dimers.

  2. Hybridization dynamics of nanoparticle monomers and dimers measured by imaging and spectral analysis.
    Figure 2: Hybridization dynamics of nanoparticle monomers and dimers measured by imaging and spectral analysis.

    Target molecules are quantified by the number of dimers formed in vitro. a, Dark-field images of nanoparticles after addition of target molecules show dimerization of nanoparticles over time. Bright yellow spots represent nanoparticle dimers; dim green spots represent nanoparticle monomers. b,c, Spectral quantification of monomers (b) and dimers (c), measured in a relatively noise-free region (red squares in a). Spectra measured on four pixels of a CCD camera reveal the intensity variation at 538 nm (b) and 612 nm (c), for monomer and dimer signals, respectively. The variation follows a Poisson distribution, providing the average number of particles in a confined volume. d, Quantification of images and spectral data, showing an inverse trend in the levels of monomers (blue dashed line) and dimers (red solid line). e, Single-molecule detection captured by CCD images (1 f.p.s., 100 ms integration). Two monomers (red arrows) in close proximity (i–iii) and formation of a dimer (iv). f, When the temperature is increased, the nanoparticle dimer is disassembled at the melting temperature of the hybridized DNA (64.7 °C).

  3. Quantification of intracellular dimers.
    Figure 3: Quantification of intracellular dimers.

    a, Approximately 12.5, 25, 37.5 or 50 dimers are injected into four different cells by microinjection (i–iv). Dark-field images (left) and dimer localization maps from a hyperspectral image (right) of each cell are shown. Scale bars, 10 µm. b, Calibration of the whole-cell spectral quantification technique. Inset: Dimer peak intensities of calibration (dimers) and control experiments (monomers only) are plotted after subtraction of the baseline. Error bars indicate the standard deviation of five independent measurements.

  4. Detection and quantification of mRNA by hyperspectral measurement of plasmonic dimer probes.
    Figure 4: Detection and quantification of mRNA by hyperspectral measurement of plasmonic dimer probes.

    a, Hyperspectral image of synchronized MDA-MB-231 cells containing injected nanoparticle probes. Dashed line: cell borders. Scale bar, 10 µm. b,c, BRCA1 alternative splicing variants missing exons 9 and 10 are targeted by plasmonic nanoparticle probes, and hyperspectral images of dimers (b, red dots) and monomers (c, green dots) are shown for several time points. Scale bars, 10 µm. d, A distinct increase in the number of dimers is observed over time. Number of dimers represents the quantification of the target variants missing exons 9 and 10. Error bars indicate the standard deviation of measurements from three cells. Inset: Wavelength window (blue column) for dimer detection, corresponding to the dimer spectrum (solid red line) and the description of length change by splicing event, and the wavelength window (red column) for monomer detection (green dashed line). e, (i) Multiplex detection of Δ(9,10,11q) and Δ(11q) using a single set of probes in MCF-7 cells. (ii) Trimodal distribution of the detected plasmon peaks, showing the existence of only two different dimer structures (signals at 584 nm and 612 nm corresponding to Δ(11q) and Δ(9,10,11q), respectively), and exhibiting minimal interference at other wavelengths, possibly indicating that non-targeted mRNA isoforms have negligible plasmonic interaction. Corresponding dimer localization maps (iii,v), as well as their wavelength windows and splicing variant conformations (iv,vi), are presented. Scale bars, 10 µm. f, Single-cell spectral quantification of three splicing variants Δ(9,10), Δ11q and Δ(9,10,11q) for the two cell lines (MCF-7 and MDA-MB-231) at asynchronous and G1/S synchronized growth stages. Error bars indicate the standard deviation of five independent measurements.

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Author information

Affiliations

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

    • Kyuwan Lee &
    • Luke P. Lee
  2. Department of Agricultural and Biological Engineering, Bindley Bioscience Center, Purdue University, 225 South University Street, West Lafayette, Indiana 47907, USA

    • Kyuwan Lee,
    • Yi Cui &
    • Joseph Irudayaraj

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.

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The authors declare no competing financial interests.

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