An Exonuclease I-Based Quencher-Free Fluorescent Method Using DNA Hairpin Probes for Rapid Detection of MicroRNA

Abstract

MicroRNAs (miRNAs) act as biomarkers for the diagnosis of a variety of cancers. Since the currently used methods for miRNA detection have limitations, simple, sensitive, and cost-effective methods for the detection of miRNA are required. This work demonstrates a facile, quencher-free, fluorescence-based analytical method for cost-effective and sensitive detection of miRNA using a super 2-aminopurine (2-AP)-labeled hairpin probe (HP) and exonuclease I activity. Specifically, the fluorescence of 2-AP is strongly quenched when it is incorporated within DNA. In the presence of a target miRNA, HP attains an open conformation by hybridizing with the target miRNA to form a double-stranded structure with a protruding 3′-terminus. Next, the digestion of the protruding 3′-terminus is triggered by exonuclease I, during which 2-AP is released free in solution from the DNA, thereby increasing fluorescence. This method is highly sensitive, with a detection limit of 0.5 nM—10 times lower than a previously reported quencher-free fluorescence method. Furthermore, this method has potential applications in clinical diagnosis and biomedical research.

Dual-Signal Amplification Strategy for Sensitive MicroRNA Detection Based on Rolling Circle Amplification and Enzymatic Repairing Amplification

“…The F / F 0 – 1 values were found to present a linear correlation with the logarithmic concentrations of miR-21 in the range from 100 pM to 5 nM (inset of Figure B), with a calibration equation of F / F 0 – 1 = 132.7145 + 12.7740 lg­( C miR‑21 / M ) ( R 2 = 0.9937), where F and F 0 are the fluorescence intensities in the presence and absence of miR-21, respectively. The limit of detection (LOD) was estimated to be 0.2 pM according to the 3σ rule, which was comparable to some previously reported fluorescence assays for miRNA. − Moreover, the analytical performance of the proposed method was also compared to other reported methods (Table S2, Supporting Information). The high sensitivity and the low background might attribute to the dual-signal amplification procedure and the high fidelity of Endo IV in specifically cleaving the TaqMan probe in dsDNA.…”

“…The limit of detection (LOD) was estimated to be 0.2 pM according to the 3σ rule, which was comparable to some previously reported fluorescence assays for miRNA. 26 − 28 Moreover, the analytical performance of the proposed method was also compared to other reported methods (Table S2, Supporting Information ). The high sensitivity and the low background might attribute to the dual-signal amplification procedure and the high fidelity of Endo IV in specifically cleaving the TaqMan probe in dsDNA.…”

Intracellular microRNA quantification in intact cells: a novel strategy based on reduced graphene oxide-based fluorescence quenching

“…Fluorescence spectroscopy, field effect transistor (FET) based electrical impedance spectroscopy (EIS), and Raman spectroscopy, are very sensitive assay readout platforms for measuring the presence and activities of various biomolecules from biological samples including microRNAs 3, 12, 13 . Especially promising is fluorescence spectroscopy and has the advantage of being a sensitive probe typically at with a detection limit in the range of 0.5 nM 14 . But to develop these assay systems, biomaterials with electrical, electronic and spectroscopic properties are needed.…”