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

Jun Wang,Liyang Zheng,Kefeng Wu,Mingjian Chen,Changbei Ma,Haisheng Liu

doi:10.3390/s17040760

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.

Highlights

MicroRNAs are a class of small (19–23 nucleotides long), single-stranded, endogenous, non-coding RNAs that commonly exist in plant, animal, and virus genomes, and were first discovered in the 1990s [1,2,3]
Northern blotting is widely used in miRNA detection and quantitation, it is time-consuming and requires the use of radiolabels and large sample size [12,13]. quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a highly sensitive method; the methodology requires tedious preparation and highly-skilled personnel [12]
Hairpin probe (HP: 50 -GCG TCG TCA ACA TCA GTC TGA TAA GCT ACG /2-amp/CGC-30 ), which were purified by HLPC, were obtained from Sangon Biotechnology Co., Ltd. (Shanghai, China)

Summary

Introduction

MicroRNAs (miRNAs) are a class of small (19–23 nucleotides long), single-stranded, endogenous, non-coding RNAs that commonly exist in plant, animal, and virus genomes, and were first discovered in the 1990s [1,2,3]. Devising rapid and sensitive methods for the detection of miRNAs is important. Several traditional mainstream techniques for miRNA detection and quantitation are Northern blotting, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and microarray analysis; each of these methods have limitations which hinder their wide and convenient application [9,10,11]. Northern blotting is widely used in miRNA detection and quantitation, it is time-consuming and requires the use of radiolabels and large sample size [12,13]. QRT-PCR is a highly sensitive method; the methodology requires tedious preparation and highly-skilled personnel [12]. Microarray is a high-throughput method, but the analysis platform is not suitable for quantitating rare miRNAs, owing to its low sensitivity [13].

Methods

Results

Conclusion