Dual FRET molecular beacons for mRNA detection in living cells

Abstract

The ability to visualize in real-time the expression level and localization of specific endogenous RNAs in living cells can offer tremendous opportunities for biological and disease studies. Here we demonstrate such a capability using a pair of molecular beacons, one with a donor and the other with an acceptor fluorophore that hybridize to adjacent regions on the same mRNA target, resulting in fluorescence resonance energy transfer (FRET). Detection of the FRET signal significantly reduced false positives, leading to sensitive imaging of K-ras and survivin mRNAs in live HDF and MIAPaCa-2 cells. FRET detection gave a ratio of 2.25 of K-ras mRNA expression in stimulated and unstimulated HDF, comparable to the ratio of 1.95 using RT-PCR, and in contrast to the single-beacon result of 1.2. We further revealed intriguing details of K-ras and survivin mRNA localization in living cells. The dual FRET molecular beacons approach provides a novel technique for sensitive RNA detection and quantification in living cells.

Figure 1

A schematic illustration showing the concept of dual FRET molecular beacons. Hybridization of donor and acceptor molecular beacons to adjacent regions on the same mRNA target results in FRET between donor and acceptor fluorophores upon donor excitation. By detecting FRET signal, fluorescence signals due to probe/target binding can be readily distinguished from that due to molecular beacon degradation and non-specific interactions.

Figure 2

Solution studies of probe–target hybridization of dual FRET molecular beacons. (A) Fluorescence emission spectra of K-ras targeting molecular beacons under Cy3 excitation (545 nm). The blue curve was generated by having donor beacons only in the presence of target, representing the signal of a single beacon assay. The red curve was a result of having donor beacons only without target, representing the background of a single-beacon assay. The green curve was due to both donor and acceptor beacons in solution with target, representing the FRET signal. The light blue curve was due to both donor and acceptor beacons in solution without target, representing the background of a FRET assay. The black curve was a result of having acceptor beacons only in the presence of target, representing the background signal of single acceptor beacons. A high signal-to-background ratio was obtained at the peak FRET signal (∼665 nm). (B) Emission spectra for survivin-targeting molecular beacons, with the four curves defined as in (A). (C) Emission spectra for ‘random sequence’ molecular beacons (RBs) with, respectively, complementary targets (blue curve), survivin targets (red curve), K-ras targets (green curve) and no target (light blue curve), indicating very high hybridization specificity.

Figure 3

Detection of K-ras mRNA expression in normally-growing and stimulated HDF cells using single donor molecular beacons only (A–D), or dual FRET molecular beacons (E–H). (A and C) Fluorescence signal of single ‘random’ sequence molecular beacons in (A) normally-growing and (C) stimulated HDF cells, respectively, representing the background due to beacon degradation and non-specific interactions. (B and D) Fluorescence signal due to single K-ras targeting donor beacons in (B) normally growing and (D) stimulated HDF cells under Cy3 excitation (545 nm) and emission detection (570 nm). Note that when single beacons were used with unstimulated cells the K-ras signal level in (B) was similar to that of the background in (A). (E and G) Fluorescence signal of two ‘random’ sequence molecular beacons in (E) normally growing and (G) stimulated HDF cells, respectively, under Cy3 excitation (545 nm) and FRET detection (665 nm), representing the background in dual FRET beacon assays. (F and H) Fluorescence signal due to K-ras targeting dual FRET molecular beacons in (F) normally growing and (H) stimulated HDF cells using FRET optics (i.e. 545 nm excitation and 665 nm emission detection). The dual FRET molecular beacons gave a much better signal-to-background ratio, and a more quantitative measure of mRNA expression level (I).

Figure 4

Detection of survivin mRNA expression in MIApaCa-2 and normal HDF cells using single donor molecular beacons only (A–D), or dual FRET molecular beacons (E–H). (A and C) Fluorescence signal of single ‘random’ sequence molecular beacons in (A) MIAPaCa-2 and (C) normal HDF cells, respectively, representing the background due to beacon degradation and non-specific interactions. (B and D) Fluorescence signal in Cy3 channel due to single survivin-targeting donor beacons in (B) MIAPaCA-2 and (D) HDF cells. Note that single survivin-targeting molecular beacons gave a signal level similar to the background. (E and G) Fluorescence signal of two ‘random’ sequence molecular beacons in (E) MIAPaCA-2 and (G) HDF cells, respectively, using FRET optics, representing the background signal in FRET-based assays. (F and H) Fluorescence signal due to survivin-targeting dual FRET molecular beacons in (F) MIAPaCa-2 and (H) HDF cells using FRET optics. The dual FRET molecular beacons gave a much better signal-to-background ratio, and could give better quantification in survivin mRNA expression level in MIAPaCa-2 and HDF cells.

Figure 5

mRNA localization in HDF and MIAPaCa-2 cells. (A and B) Fluorescence images of K-ras mRNA in stimulated HDF cells. Note the filamentous K-ras mRNA localization pattern. (C) A fluorescence image of survivin mRNA localization in MIAPaCa-2 cells. Note that survivin mRNAs localized to one side of the nucleus of the MIAPaCa-2 cells.

Figure 6

Fluorescence in situ hybridization (FISH) studies. (A) Detection of K-ras mRNA in fixed HDF cells using fluorescently labeled linear probes. Note the filamentous localization pattern near the cell peripheral region. (B) A negative control study of the FISH assay using fluorescently labeled linear Poly-A probes resulted in very low background.