Magnetically Assisted Immobilization-Free Detection of microRNAs Based on the Signal Amplification of Duplex-Specific Nuclease

Abstract

The double specific nuclease (DSN)-based methods for microRNAs (miRNAs) detection usually require the immobilization of DNA probes on a solid surface. However, such strategies have the drawbacks of low hybridization and cleavage efficiency caused by steric hindrance effect and high salt concentration on the solid surface. Herein, we proposed an immobilization-free method for miRNA detection on the basic of DSN-assisted signal amplification. The biotin- and fluorophore-labeled probes were captured by streptavidin-modified magnetic beads through streptavidin-biotin interactions, thus producing a poor fluorescence signal. Once the DNA probes were hybridized with target miRNA in solution to form DNA-miRNA duplexes, DNA stands in the duplexes would be selectively digested by DSN. The released target miRNA could initiate the next hybridization/cleavage recycling in the homogeneous solution, finally resulting in the release of numerous fluorophore-labeled fragments. The released fluorophores remained in solution and emitted strong fluorescence after treatment by the streptavidin-modified magnetic beads. The immobilization-free method achieved the assays of miRNA-21 with a detection limit down to 0.01 pM. It was employed to evaluate the expression levels of miRNA-21 in different cancer cells with satisfactory results.

Keywords: double specific nuclease; homogeneous analysis; immobilization-free; magnetic bead; microRNAs.

Scheme 1

Schematic representation of the magnetically assisted immobilization-free method for miRNA detection.

Figure 1

Fluorescence spectra of Bio-DNA-FAM before (curve a) and after (curve b) incubation with MB-SA, as well as the mixture of Bio-DNA-FAM/miRNA-21 (curve c) and Bio-DNA-FAM/miRNA-21/DSN (curve d) after treatment by MB-SA. Curve corresponds to that of incubating the MB-SA-Bio-DNA-FAM conjugates with the mixture of miRNA/DSN. The used concentrations of Bio-DNA-FAM, DSN, and miRNA-21 were 500 nM, 0.1 U, and 100 pM.

Figure 2

Effect of probe concentration (A) and incubation time (B) on fluorescence intensity. The concentrations of Bio-DNA-FAM, miRNA-21, and DSN were 500 nM, 100 pM, and 0.1 U, respectively.

Figure 3

(A) Fluorescence spectra for the detection of different concentrations of miRNA-21 (from to bottom to top: 0, 0.01, 0.1, 1, 5, 10, 25, 50, and 100 pM). (B) Calibration curve between fluorescence intensity and miRNA-21 concentration. The errors were deduced from three replicate measurements. The inset shows the linear portion of the calibration curve.

Figure 4

Selectivity of the method. Bar 1, buffer blank; bar 2, single-base mismatch; bar 3, three-base mismatch; bar 4, non-complementary; bar 5, miRNA-21.

Figure 5

Results for the assays of miRNA-21 in lysates extracted from MCF-7 and Hela cells.