Recent advances in duplex-specific nuclease-based signal amplification strategies for microRNA detection
Introduction
MicroRNAs (miRNAs) were a class of endogenous non-coding single-stranded RNA (ssRNA) molecules with about 20–24 nucleotides, which played an important role in biological processes, cell differentiation and disease development (He and Hannon 2004). Plenty of studies had revealed that the aberrantly expressed miRNA signatures were closely related to many malignant health-threatening diseases, ranging from cancers to cardio-cerebrovascular diseases (Kristensen et al., 2018; Li et al., 2012; Shen et al., 2013a; Zhou et al., 2018). With the rapid development of emerging technology, especially in the field of in-vitro diagnosis (IVD) and nanobiotechnology, researchers gradually realized the great potential of miRNAs as excellent substitutes for some traditional protein biomarkers with poor specificity and sensitivity in the early screening of malignant diseases (Chen et al., 2008; Matin et al., 2018). Generally, miRNAs were abundant in tissues, but what's exciting was that there were also trace circulating miRNAs existed in biological fluids, such as serum, plasma, urine, peritoneal fluid, and saliva (Ortizquintero 2016; Scalici et al., 2016). More importantly, different types of the critical health-threatening diseases demonstrated their unique miRNA expression profilings. For example, hepatocellular carcinoma demonstrated its specific miRNA expression profiling with up-regulation miRNAs of let-7i*, miR-429, miR-672 and down-regulation miRNAs of miR-219, miR-369-5p, miR-193a-3p, miR-758, miR-15a* (Li et al., 2016a). According to the latest release of miRBase (v22), there were 48 860 mature miRNAs and 38 589 hairpin precursors expounded in the current database, some of which might be used as new biological candidates for disease diagnosis (Kozomara et al., 2019). Therefore, accurate detection of the levels of miRNA shown excellent prospects for early diagnosis of the health-threatening diseases, which was essential for effective therapeutic management prognosis of patients.
In general, the miRNA biosensors comprised two elements, a biological recognition for capturing target miRNA and a transducer to convert the recognition reaction into a measurable signal by using different signal readout including electrochemistry (Azimzadeh et al., 2016; Rafieepour et al., 2016), colorimetry (Miao et al., 2016a; Wu et al., 2016a), fluorescence (Ou et al., 2019; Yin et al., 2017), and so on. Considering miRNAs were relatively low expression levels in cells, leading to an ultralow content existed in biological fluids, and relatively short sequence in base length, leading to a highly homologues in sequence similarity (Zhao et al., 2015). Therefore, adequate sensitivity and excellent specificity were the first prerequisite to construct novel methods for miRNA analysis. In addition, many other factors such as non-invasive, point-of-care and multiplexed detection, reproducibility, cost, and detection time should also be considered, especially in practical clinical application.
Conventional strategies for miRNA detection including quantitative real-time polymerase chain reaction (qRT-PCR), northern blotting and oligonucleotide microarray, were limited by one or more factors, such as poor sensitivity, time-consuming process, labor-intensive steps and expensive reagents, which hindered their wide application in the field of IVD (Chen et al., 2018; Kilic et al., 2018). For example, the northern blotting method once attracted considerable attention due to its excellent specificity and rarely false positive cases, but it was failed to be widely used in clinical practice due to its time-consuming and sample-consuming process (Streit et al., 2009). The oligonucleotide microarray was another method developed on the basis of northern blotting for miRNA detection, which took full advantage of multiplex detection (Jaluria et al., 2007). Unfortunately, the oligonucleotide microarray method was still limited by the unsatisfactory sensitivity and requirements of complicated probe in clinical practice application. At present, the qRT-PCR method was considered as the gold standard for miRNA detection due to its high sensitivity and broad dynamic range (Smith and Osborn 2009). However, the qRT-PCR method was also suffered from tedious labor-intensive steps and necessary for large-scale instruments and well-trained professionals, which hindered their wide application in clinical practice (Ko et al., 1998). Hence, newly strategies for miRNA detection were urgently needed to remedy the shortcomings of conventional strategies.
In the last decades, extensive newly strategies with various detection performance for sensitive, selective and high-throughput detection of miRNAs were reported (Deng et al., 2017; He et al., 2017a; Liu et al., 2017; Tavallaie et al., 2018). Among them, enzyme-based signal amplification strategies showed great potential in improving the sensitivity, mainly due to its unique characteristics of enzymolysis process (Hu et al., 2016; Wang et al., 2019). As shown in Fig. 1, Duplex-specific nuclease (DSN) was a nuclease purified from hepatopancreas of Kamchatka crab, which was capable of specifically cleaving double-stranded DNA (dsDNA) or DNA in DNA-RNA heteroduplexes, and was inactive toward single-stranded oligonucleotides or double-stranded RNA (dsRNA) (Shagin et al., 2002). The cleavage rate of DSN for short perfectly matched duplexes (10–12 bps) was essentially higher than that for non-perfectly matched duplexes of the same length, exhibiting excellent ability in discriminating between perfectly and non-perfectly matched duplexes. It should be noted that DSN exhibited its enzymatic activity in the presence of Mg2+ (at least 5 mM) and was inhibited by ethylene diamine tetraacetic acid (EDTA). The optimized pH and temperature for enzymatic activity were 7–8 and 55–65 °C, respectively. In addition, DSN was tolerant to proteinase K treatment (at 37 °C) (Zhulidov et al., 2004). All these characteristics endowed DSN a possibility for construction of the newly miRNA biosensors, namely DSN-based miRNA biosensors (Ma et al., 2018a; Pang et al., 2019; Wang et al., 2020b; Xu et al., 2020). The principle of DSN-based miRNA biosensors was mainly based on the DSN-assisted target recycling. With the DSN-assisted target recycling, trace miRNAs triggered the release of considerable amounts of “signal molecules”. The released “signal molecules” were proportional or inversely proportional to the amount of target miRNA, leading to a significant measurable signal for miRNA analysis. In general, the “signal molecules” were connected with single-strand DNA (ssDNA) as the detection probe to capture the target miRNA. The “signal molecules” in the detection system could be enzymes (such as horseradish peroxidase (HRP) (Peng et al., 2018b), alkaline phosphatase (ALP) (Liu et al., 2015; Shuai et al., 2017), phospholipase A2 (PLA2) (Cheng et al., 2019), DNAzyme moieties (Deng et al., 2014; Kim et al., 2016; Zhang et al., 2014)), fluorophores (such as organic dyes (Hu et al., 2015; Ma et al., 2017; Xi et al., 2014), quantum dots (QDs) (Shen and Gao 2015; Wang et al., 2020a), up-conversion nanoparticles (UCNPs) (Lu et al., 2019b), fluorescent nanosphere (FS) (Peng et al. 2019, 2020), aggregation-induced emission luminogens (AIEgens) (Min et al., 2015; Wang et al., 2018b)), nanoparticles (such as gold nanoparticles (AuNPs) (Han et al., 2019; Huo et al., 2018), magnetic nanoparticles (MNPs) (Lu et al., 2016; Tian et al., 2017)), nucleotide sequences (such as signal output sequence (Guo et al., 2018; Li et al., 2018a; Zhang et al., 2015), 2’-O-methyl RNA sequence (Lv et al., 2016; Zhang et al., 2017), recognition sequences or residual sequences (Fan et al., 2019; Ki et al., 2019; Qiu et al., 2015; Tao et al., 2017; Yu et al., 2018)), etc. There were plenty of DSN-based miRNAs biosensors had been reported, including DSN based-fluorescent biosensors (Yin et al., 2012), colorimetric biosensors (Guo et al., 2016), electrochemical biosensor (Zhang et al., 2016a), chemiluminescence biosensor (Wang et al., 2016b), surface-enhanced Raman scattering (SERS) biosensor (Sun et al., 2016), mass spectrometric (MS) biosensors (Li et al., 2020), flow cytometric biosensors (Qiu et al., 2017) and so on.
Although some review articles for miRNA detection had been published (Chen et al., 2018; Kilic et al., 2018; Labib and Berezovski 2015), the novel proposed DSN-assisted signal amplification strategies needed to be reviewed due to the fast-evolving miRNA sensing methods. This article provided an overview on the newly developed DSN-assisted signal amplification strategies for miRNA detection. The purpose of this article was to summarize the advantages and shortcomings associated with each method and make comparison between them to choose an appropriate strategy according to the need of the work conducted. Before jumping to DSN-based miRNA biosensor section, miRNAs as potential biomarkers for different health-threatening diseases were discussed. In newly DSN-based miRNA biosensors section, we classified them according to the “signal molecules” in the assay. Various of the “signal molecules”, including enzymes, fluorophores, nanoparticles, nucleotide sequences, were detailed with representative examples from recently published papers. Moreover, we also described many other representative DSN-based miRNA biosensors in addition to the examples mentioned above. Finally, the future trends in miRNAs detection were briefly discussed considering the needs of IVD field.
Section snippets
miRNAs as potential disease biomarkers
In the past decades, rapid progress and great discoveries on miRNAs had proved that, miRNAs showed great potential in the treatment and diagnosis of many health-threatening diseases (Mcguire et al., 2015; Urbich et al., 2008). The aberrantly expressed miRNA profilings were closely related to many malignant health-threatening diseases, ranging from cancers to cardio-cerebrovascular diseases (Du et al., 2020; Zhou et al., 2018). However, it was clear that the study of miRNAs as potential
DSN-based signal amplification strategies for miRNA detection
As we all known, miRNAs showed great potential as new biomarkers for the early diagnosis or monitoring the therapeutic efficacy. However, the intrinsic characteristics of miRNA such as highly susceptible to degradation, relatively low expression level and short sequence, posed a challenge for miRNA detection (Zhao et al., 2015).
DSN was capable of specifically degrading dsDNA or DNA-RNA duplexes and inactive toward single-stranded oligonucleotides or dsRNA (Shagin et al., 2002). The principle of
Conclusion and future perspectives
In recent years, a number of miRNAs had been identified to differentially expressed in diseased tissues, or patient's plasma, serum and other body fluids, suggesting that specific miRNA signatures as biomarkers were attractively conducive to diseases diagnosis, prognosis and prediction of therapeutic responses. However, the intrinsic characteristics of miRNA, such as relatively low expression level and short sequence, posed a challenge for miRNA detection. DSN-based signal amplification
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The authors gratefully acknowledge that this work was financially supported by the Qingdao Major Scientific and Technological Project for Distinguished Scholars (20170103), Laoshan Major Scientific and Technological Project for Distinguished Scholars (20181030), China Postdoctoral Science Foundation (2019M652331, 2018M642619), Shandong Postdoctoral Innovation Project (201903039), Qingdao Postdoctoral Application Project (2018121236, 2018121238), Youth Innovation Team Talent Introduction Program
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