Nano-flares: probes for transfection and mRNA detection in living cells

Abstract

We demonstrate that novel oligonucleotide-modified gold nanoparticle probes hybridized to fluorophore-labeled complements can be used as both transfection agents and cellular “nano-flares” for detecting mRNA in living cells. Nano-flares take advantage of the highly efficient fluorescence quenching properties of gold, cellular uptake of oligonucleotide nanoparticle conjugates without the use of transfection agents, and the enzymatic stability of such conjugates, thus overcoming many of the challenges to creating sensitive and effective intracellular probes. Nano-flares exhibit high signaling, have low background fluorescence, and are sensitive to changes in the number of RNA transcripts present in cells.

Figure 1

(a) Nanoparticles functionalized with a recognition sequence are hybridized with a short complementary Cy5 labeled reporter strand, which is capable of being displaced by the target. (b) Fluorescence spectra of 1 nM nano-flares alone (green), in the presence of 1 mM target (red), and in the presence of 1 mM non-complementary sequence (blue). (c) Oligonucleotide sequences.

Figure 2

Intracellular testing of nano-flares. Differential contrast and fluorescence image of survivin-expressing SKBR3 cells treated with survivin nano-flares (top left panel) and non-complementary nano-flares (top right panel). Analogously treated non survivin-expressing C166 cells (bottom panels). Scale bar is 20 mm. Flow cytometry data is shown below each image. The bold numbers to the right of the histogram are the total mean fluorescence of the cell populations. The background fluorescence (untreated cells) was 3.4, and 4.7 for the C166 and SKBR3 cells, respectively.

Figure 3

Quantification of survivin knockdown using nano-flares. (a) Flow cytometry data collected on siRNA treated SKBR3 cells. The siRNA concentration is given in the graph to the left of the histogram. In the untreated sample, half of the population exhibiting equal or greater fluorescence than the mean is shaded grey. Treated samples show a smaller fraction of the cell population exhibiting the mean fluorescence (declining grey, increasing black). (b) Plot of mean fluorescence (black circles) and survivin expression (grey bar-graph) as a function of siRNA concentration. The RT-PCR reactions were conducted in triplicate, and the error bars shown above are the standard deviations of those measurements.