A target-triggered dual amplification strategy for sensitive detection of microRNA

Abstract

The accurate and quantitative analysis of microRNA (miRNA) expression is critical for biomedical research and clinical theranostics. In this study, we report a novel sensor for the sensitive detection of miRNA based on a duplex-specific nuclease (DSN)-assisted dual signal amplification strategy. A chimeric probe (DNA/2-OMe-RNA) that consists of a miRNA recognition DNA sequence and a Taqman probe hybridization RNA sequence (2'-O-methyl RNA) was designed and synthesized. One molecule of target miRNA can trigger cyclical cleavage of the chimeric probes to produce 2'-O-methyl RNA by DSN in the first round of amplification. The 2'-O-methyl RNA molecules can subsequently hybridize with Taqman probes and initiate the second round of cyclical amplification to generate detectable fluorescence by DSN. The proposed strategy exhibits high specificity in discriminating cognate miRNAs, and the dual signal transduction process enables the detection of miRNA concentrations as low as 7.3fM. We further applied this assay to miRNA quantification in cancer cells to confirm its applicability. The present study provides a sensitive, specific and simple method for miRNA detection and holds great potential for further application in biomedical research and in the clinical laboratory.

A new graphene oxide enhanced fluorescence anisotropy strategy for microRNA detection

“…近年来研究发现, 在特定 组织中的miRNA表达失控与多种病变过程有关, 如病 毒性疾病、心血管疾病、免疫紊乱和癌症等 [22,23] . 并 且, 大多数胞外miRNA携带着特殊的信息, 与多种疾 病密切相关 [22] . miRNA-21含量在人体恶性胶质瘤中 的失调与多个胶质母细胞瘤细胞系凋亡细胞的死亡有 关 [24] .…”

Rapid and label-free fluorescence bioassay for microRNA based on exonuclease III-assisted cycle amplification

“…12 In order to overcome these disadvantages, some new signal amplication methods were developed to improve the sensitivity, reduce reaction time and step up the operation. These methods include rolling circle ampli-cation (RCA), 15,16 hybridization chain reaction (HCR), 17,18 strand displacement assay (SDA), 19,20 the enzyme-based cyclic amplication, [21][22][23][24][25][26] and the energy transfer-based assay. 27,28 Among them, the enzyme-based cyclic amplication has become a gradually popular strategy to detect miRNAs due to its special amplifying function and can be used for homogeneous liquids.…”

“…27,28 Among them, the enzyme-based cyclic amplication has become a gradually popular strategy to detect miRNAs due to its special amplifying function and can be used for homogeneous liquids. 29 According to previous reports, duplex specic nuclease (DSN), 21,22 l-exonuclease, 23 nicking enzyme, 24,25 and DNA polymerase 26 were oen used in enzyme-based biosensors for the detection of miRNA. For instance, Li et al 21 have developed a uorescence biosensor based on labeling-molecular beacon and DSN to detect miRNA.…”

Lateral flow nucleic acid biosensor for sensitive detection of microRNAs based on the dual amplification strategy of duplex-specific nuclease and hybridization chain reaction

“…DSN is well suitable for the detection of miRNAs based on the characteristic of DNA cleavage in the DNA−miRNA heteroduplex. Due to its unique enzymatic characteristics and great potential application in miRNA detection[ 10 , 25 , 26 ], DSN has attracted increasing interest. Despite their remarkable advantages, such as simple methodology, fast experimental protocols, and high specificity, the detection limits of DSN-assisted target recycling methods need further improvement for miRNA detection.…”