Multiplexed nanoflares: mRNA detection in live cells

Abstract

We report the development of the multiplexed nanoflare, a nanoparticle agent that is capable of simultaneously detecting two distinct mRNA targets inside a living cell. These probes are spherical nucleic acid (SNA) gold nanoparticle (Au NP) conjugates consisting of densely packed and highly oriented oligonucleotide sequences, many of which are hybridized to a reporter with a distinct fluorophore label and each complementary to its corresponding mRNA target. When multiplexed nanoflares are exposed to their targets, they provide a sequence specific signal in both extra- and intracellular environments. Importantly, one of the targets can be used as an internal control, improving detection by accounting for cell-to-cell variations in nanoparticle uptake and background. Compared to single-component nanoflares, these structures allow one to determine more precisely relative mRNA levels in individual cells, improving cell sorting and quantification.

Figure 1

Schematic representation of target detection by multiplexed nano-flares. The multiplexed nano-flares bind different target nucleic acids (shown in red and green), displacing the corresponding flare. Once the flare is released, the fluorophore is no longer quenched by the gold surface, and an increase in fluorescence can be measured. By using two different fluorophores, the ratio of each target can be determined in cells.

Figure 2

Sequence specific target detection in extracellular experiments. (A) A scheme of sequence specific flare release in response to two different targets (green shown on the left and red shown on the right). (B, C) Fluorescence spectra of nano-flares in the presence of no target, actin target or survivin target. Actin and survivin flares are labeled with Cy5 and Cy3, respectively. (B) Normalized fluorescence spectra corresponding to Cy3 (survivin flare) signal, with excitation at 530 nm. (C) Normalized fluorescence spectra corresponding to Cy5 (actin flare) signal, with excitation at 630 nm.

Figure 3

Detection of actin and survivin mRNA expression in cells. Survivin-Cy5 and Actin-Cy3 flares were used. (A) In order to change the relative levels of survivin and actin mRNA the cells were treated with either actin or survivin targeted siRNA. Nonsense siRNA with no target in the cells was used as a negative control. The cell associated fluorescence intensities corresponding to each flare were measured by flow cytometry. (B) In a similar experiment, survivin levels were reduced using siRNA and the change in fluorescence from the multiplexed nano-flares was measured and assessed qualitatively by fluorescence confocal microscopy.

Figure 4

Quantitative and qualitative determination of mRNA expression in living cells. (A) The detection of intracellular survivin mRNA by traditional nano-flares was compared to detection by multiplexed nano-flares, expressed as the ratio of survivin to actin. The cell associated fluorescence intensities were measured in HeLa cells using flow cytometry and expressed as a histogram. (B) In a similar experiment, the multiplexed nano-flares were used to compare the ratio of survivin to actin expression in HeLa and Jurkat cells. (C) The expression of survivin mRNA was measured as a function of survivin siRNA delivered by both the multiplexed nano-flare and RT-PCR. Error bars represent standard error of the mean from at least 3 independent experiments.