Quantum dot-based molecular beacon to monitor intracellular microRNAs

Abstract

Fluorescence monitoring of endogenous microRNA (miRNA or miR) activity related to neuronal development using nano-sized materials provides crucial information on miRNA expression patterns in a noninvasive manner. In this study, we report a new method to monitor intracellular miRNA124a using quantum dot-based molecular beacon (R9-QD-miR124a beacon). The R9-QD-miR124a beacon was constructed using QDs and two probes, miR124a-targeting oligomer and arginine rich cell-penetrating peptide (R9 peptide). The miR124a-targeting oligomer contains a miR124a binging sequence and a black hole quencher 1 (BHQ1). In the absence of target miR124a, the R9-QD-miR124a beacon forms a partial duplex beacon and remained in quenched state because the BHQ1 quenches the fluorescence signal of the R9-QD-miR124a beacon. The binding of miR124a to the miR124a binding sequence of the miR124a-targeting oligomer triggered the separation of the BHQ1 quencher and subsequent signal-on of a red fluorescence signal. Moreover, enhanced cellular uptake was achieved by conjugation with the R9 peptide, which resulted in increased fluorescent signal of the R9-QD-miR124a beacons in P19 cells during neurogenesis due to the endogenous expression of miR124a.

Keywords: microRNA; molecular beacon; neurogenesis; quantum dot.

Figure 1

A schematic diagram of the R9-QD-miR124a beacons to image miR124a. The amine-terminated oligonucleotide containing the miR124a recognition sequence was annealed at the region with partial complementary sequences attached to the quencher molecules, which was designated as the miR124a-targeting oligomer. The carboxyl-terminated QD655 was conjugated with the miR124a-targeting oligomer and the R9 peptide. When the mature miR124a was hybridized with the miR124a binding region in the R9-QD-miR124a beacons, the quencher molecules were separated, resulting in an increase in the fluorescence signal.

Figure 2

Characterization of the R9-QD-miR124a beacons. (A) Transmission electron microscopy (TEM) images; (B) electrophoretic shift assay; and (C) dynamic light scattering (DLS) analysis of the unconjugated QDs and the R9-QD-miR124a beacons; (D) Coupling efficiencies of the miR124a-targeting oligomer and the R9 peptide to QDs.

Figure 3

(A) Quenching efficiency of the R9-QD-miR124a beacons in the tube. A fixed concentration of the QDs (10 pM) were conjugated with various concentrations (0–300 pM) of the miR124a-targeting oligomer to determine the optimal concentration of miR124a needed to achieve the best quenching effect. The fluorescence activity of the R9-QD-miR124a beacons gradually decreased as the concentration of miR124a-targeting oligomer increased. ROI analysis from the fluorescence tube image showed that the fluorescence signal decreased in a dose-dependent manner (* p < 0.05); (B) The fluorescence recovery effect of the R9-QD-miR124a beacons after treatment of an exogenous miR124a in the tube. The R9-QD-miR124a beacons were treated with an exogenous (300 pM) or a mutant (300 pM) miR124a for 1 h at 37 °C. The quenched fluorescence intensity was activated in the conjugate group in the presence of the miR124a; however, no fluorescence recovery was observed in the mutant oligonucleotide group (** p < 0.005).

Figure 4

The activation of fluorescence intensity of the R9-QD-miR124a beacon in C6 cells. (A) In vitro cytotoxicity of QDs and the R9-QD-miR124a beacons in cells; (B) R9 peptide conjugation improved internalization efficiency and miR124a specificity of the R9-QD-miR124a beacons in C6 cells. The fluorescence signal from the quenched R9-QD-miR124a beacons was activated in the exogenous miR124a-treated group (300 pM), unlike in the mutant-treated group (300 pM); (C) The visualization of fluorescence recovery of the R9-QD-miR124a beacons in C6 cells. The R9-QD-miR124a beacons were incubated with C6 cells for 1 h 30 min, and exogenous miR124a or the miR124a mutant was added to the pre-treated C6 cells. Confocal microscopy imaging showed that the fluorescence signal of the R9-QD-miR124a beacons was significantly activated in C6 cells after treatment with exogenous miR124a. In contrast, induction of the miR124a mutant showed a weak fluorescence signal of the R9-QD-miR124a beacon in C6 cells, suggesting maintenance of the quenched fluorescence signal.

Figure 5

The activation of fluorescence intensity of the R9-QD-miR124a beacons in differentiated P19 cells. The P19 cells were induced to neuronal differentiation for 3 days. (A) qRT-PCR analysis of the expression of miR124a during neuronal differentiation in the P19 cells; (B) Immunocytostaining of the P19 cells by Oct3/4 (stem cell marker) and NeuroD (neuronal marker) antibodies. Red fluorescence indicates Oct3/4 or NeuroD expression, and blue fluorescence indicates DAPI, which stains the nucleus; (C) Undifferentiated and differentiated P19 cells were incubated with the R9-QD-miR124a beacons. Confocal microscopy imaging showed that the fluorescence signal of the R9-QD-miR124a beacons in differentiated P19 cells was significantly activated by endogenous miR124a. Images were merged with DAPI; (D) Fluorescence intensity of the R9-QD-miR124a beacons in P19 cells during neuronal differentiation. Data are displayed as means ± standard error of triplicate samples (* p < 0.05, ** p < 0.005).