Signal amplification of microRNAs with modified strand displacement-based cycling probe technology

Abstract

Micro ribose nucleic acids (miRNAs) play an important role in biological processes such as cell differentiation, proliferation and apoptosis. Therefore, miRNAs are potentially a powerful marker for monitoring cancer and diagnosis. Here, we present sensitive signal amplification for miRNAs based on modified cycling probe technology with strand displacement amplification. miRNA was captured by the template coupled with beads, and then the first cycle based on SDA was repeatedly extended to the nicking end, which was produced by the extension reaction of miRNA. The products generated by SDA are captured by a molecular beacon (MB), which is designed to initiate the second amplification cycle, with a similar principle to the cycling probe technology (CPT), which is based on repeated digestion of the DNA-RNA hybrid by the RNase H. After one sample enrichment and two steps of signal amplification, 0.1 pM of let-7a can be detected. The miRNA assay exhibits a great dynamic range of over 100 orders of magnitude and high specificity to clearly discriminate a single base difference in miRNA sequences. This isothermal amplification does not require any special temperature control instrument. The assay is also about signal amplification rather than template amplification, therefore minimising contamination issues. In addition, there is no need for the reverse transcription (RT) process. Thus the amplification is suitable for miRNA detection.

Research advances in the detection of miRNA

“…Jia et al. [23] used a molecular beacon probe to capture the products generated from SDA; thus the second amplification was initiated, with a similar principle to the cycling probe technology, which was based on repeated digestion of the DNA–RNA hybrid by the RNase H. The limit of the quantification for this method reached 0.1 pM after one sample enrichment and two steps of signal amplification. With its isothermal and enzyme-free nature, qualitative or quantitative detections of nucleic acid especially miRNAs in vivo were attractive.…”

“…Traditional methods for miRNA detection include northern blotting [[10], [11], [12]], microarray analysis [13,14] and quantitative polymerase chain reaction (qPCR) [15,16]. For new technology methods, in order to improve the sensitivity and selectivity of miRNA detection, these methods always rely on signal amplification strategies, such as nanoparticle-based amplification [[17], [18], [19], [20], [21]], isothermal exponential amplification [[22], [23], [24]], rolling circle amplification (RCA) [[25], [26], [27]], hybridization chain reaction (HCR) [[28], [29], [30]] and combination of these amplification strategies [[31], [32], [33]]. Herein, we summarize some of them and discuss their advantages and limitations for improving miRNA detection design.…”

A dual discrimination mode for improved specificity towards let‐7a detection via a single‐base mutated padlock probe‐based exponential rolling circle amplification

“…This result suggested an enhanced and excellent discrimination power of our proposed strategy, by intentionally introducing one base mutation on the padlock probe. In comparison with previous methods of padlock probe‐based RCA, this method has improved selectivity for discriminating single‐base mismatch among the highly homologous let‐7 family members . We believe that this enhanced discrimination capability contributes to more accurate analysis of biological function for individual miRNAs.…”

“…In comparison with previous methods of padlock probe-based RCA, this method has improved selectivity for discriminating single-base mismatch among the highly homologous let-7 family members. [24,[30][31][32] We believe that this enhanced discrimination capability contributes to more accurate analysis of biological function for individual miRNAs.…”

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.…”