As a regulatory molecule of post-transcriptional gene expression, microRNA (miRNA) is a class of endogenous, non-coding small molecule RNAs. MiRNA detection is essential for biochemical research and clinical diagnostics but challenging due to its low abundance, small size, and sequence similarities. In this chapter, traditional methods of detecting miRNA like polymerase chain reaction (PCR), DNA microarray, and northern blotting are introduced briefly. These approaches are usually used to detect miRNA in vitro. Some novel strategies for sensing miRNAs in vivo, including hybridization probe assays, strand-displacement reaction (SDR), entropy-driven DNA catalysis (EDC), catalytic hairpin assembly (CHA), hybridization chain reaction (HCR), DNAzyme-mediated assays, and CRISPR-mediated assays are elaborated in detail. This chapter describes the principles and designs of these detection technologies and discusses their advantages as well as their shortcomings, providing guidelines for the further development of more sensitive and selective miRNA sensing strategies in vivo.
- hybridization probe assays
- strand-displacement reaction (SDR)
- entropy-driven DNA catalysis (EDC)
- catalytic hairpin assembly (CHA)
- hybridization chain reaction (HCR)
MicroRNA (miRNA) is a kind of endogenous non-coding RNA with a length of 18–25 nucleic acid sequences. It is usually integrated into the RNA-induced silencing complex (RISC) to execute its biological function of degrading mRNA or inhibiting transcription. MicroRNA is highly conservative and has strict temporal and spatial specificity. It plays a key regulatory role in the development of animals and plants, cell proliferation, differentiation and apoptosis, immunity and metabolism, angiogenesis, tumor invasion, and metastasis. Mature miRNA has the disadvantages of small fragment, no poly (A), high similarity among family members, and low expression level. As a result, it is difficult to sensitively and accurately detect miRNA. Therefore, it is very important to establish fast and simple methods with high sensitivity and specificity for miRNA detection .
Many miRNA analysis methods, including polymerase chain reaction (PCR), DNA Microarray, and Northern blotting have been developed. Although these traditional strategies are the gold standard methods for miRNA identification, detection, and analysis
2. Overview of traditional miRNA detection methods
2.1 Northern blotting
Northern blotting, invented by Alwine in 1979, is the first established method to identify and detect miRNA. It is widely used to detect the expression of miRNAs of various sizes from long primitive miRNAs to mature miRNAs. In the process, the miRNA was separated by polyacrylamide gel electrophoresis in total RNA, then transferred to the imprinted membrane, hybridized with the radionuclide labeled probe. The RNA molecule of interest is detected by the signal of labeled probe. This method can detect both the quantity and the length of miRNA, but it has some defects such as cumbersome operation, low sensitivity, time-consuming, and large sample consumption, which limits its application in clinical diagnosis.
To improve the detection sensitivity, Válóczi
The microarray, developed in the early 1990s, enables the high-throughput miRNA detection in a parallel fashion. In this method, the target miRNA is incubated and hybridized with multiple probes (complementary to the target miRNA sequence) on a chip. After removing the non-hybridized part, the signal can be detected and analyzed by fluorescence scanning or northern blotting . DNA probe-based microarray usually consumes a large amount of samples, and has disadvantages such as low sensitivity and specificity and false positives caused by cross-reactions. However, LNA probe can reduce the consumption of starting materials and improve the sensitivity and accuracy of microarray . Furthermore, liquid suspension microsphere hybridization can effectively avoid cross-reaction in the solid chip to decrease the occurrence of false positives.
Quantitative real-time polymerase chain reaction (qRT-PCR) is one of the main methods to detect low abundance miRNA with high sensitivity and accuracy . The principle of this method is to reversely transcribe miRNA into the corresponding cDNA that is used as a template to initiate real-time PCR, and then indirectly analyze miRNA by detecting the signal of amplified products .
Because the miRNA sequence is short (18 ~ 25 nt) and similar to the length of PCR primers, researchers overcome this shortcoming by introducing stem-ring primers for reverse transcription  or adding poly (A) into RNA to initiate reverse transcription and dyeing with SYBR Green. Besides, pri-miRNA and pre-miRNA can be introduced into qRT-PCR, causing inaccurate quantification . qRT-PCR usually requires complex primer design and precise reaction temperature control, thereby greatly increasing the cost and complexity of the experiment.
Although the traditional miRNA detection methods are widely used in miRNA detection, there are still some shortcomings such as complex operation, low sensitivity, poor specificity, and large sample consumption. These shortcomings greatly limited the application of these methods in clinical diagnosis and treatment. Importantly, these approaches are only applied to the
3. Emerging sensing techniques in microRNA detection
3.1 Hybridization probe assay
Hybridization probe assay is a simple and direct detection method without amplification of target miRNA. The principle of this method is as follows: firstly, miRNA is fixed in the tissue or cell. Secondly, signal-labeled nucleic acid probes are added and hybridized with the miRNA based on the principle of complementary pairing. Finally, the position of target miRNA to be detected in the tissue or cell is displayed by certain detection means.
With the principle of hybridization, Wang
To further improve the detection and imaging sensitivity, fluorescence double-labeling and double-quenching strategy is undoubtedly a good design. Molecular beacon (MB) is hairpin-structure DNA probe. Its two ends are labeled with two identical fluorescent molecules. When MB is in a close state, self-quenching effect between two fluorescent molecules occurs. Graphene oxide (GO) is a good fluorescence quencher. The electrostatic interaction between DNA probe and GO also quenches effectively the fluorescence of DNA probe. Based on the aforementioned properties of MB and GO, Yang
Another approach to enhance the sensitivity of hybridization probe assay is to use the plasmon coupling effect of assembled nanostructures, especially, dissymmetric nanostructures. Xu
Although the introduction of nanomaterials (gold nanoparticles, graphene oxide, upconversion nanoparticles, MnO2 nanosheets, quantum dots, silver nanoparticles, noble metal nanoclusters, and silica nanoparticles) remarkably enhanced the sensitivity of hybridization probe assay, the lack of signal amplification limits its practical application in the sensitive detection of intracellular miRNA.
3.2 Strand displacement reaction (SDR)
Strand displacement reaction (SDR) is a dynamic process of hybridization-driven DNA strand exchange accompanied by branch migration . In this process, a single-stranded reactant (input, target miRNA) reacts with multi-stranded DNA complex and releases another single-stranded product (output signal) and a new DNA complex. This process operates autonomously through a series of reversible DNA hybridization and dissociation steps to produce numerous output strands, thereby generating cascaded signal amplification [15, 16].
Using SDR, Wang
In recent years, the combination of SDR and nanomaterials for miRNA detection has become a hot spot. Li
Surface-enhanced Raman spectroscopy (SERS) refers to that when molecules approach the metal surface of nanostructures, the Raman signal of molecules is enhanced. Gold nanoparticles with the core-satellite structure are important SERS substrate structures, which are composed of a single Au (or Ag) inside and multiple Au (or Ag) linked outside. The core-satellite structure is combined with SDR to detect miRNA and produce the SERS signal.
Gold nanorods have strong and controllable plasma resonance properties, which can be widely used in photothermal therapy and amplification detection of miRNA. Qu
Inspired by Qu’s work, Yan
In addition to the strand displacement reaction catalyzed by fuel strand, toehold-mediated strand displacement is another commonly used amplification strategy. A novel catalytic self-assembly nanosensor based on quantum dots was constructed  to detect miRNA
Enzyme-mediated strand displacement reaction enables the exponential accumulation of DNA products through the continuous polymerization-nicking-displacement cycle process catalyzed by polymerases. Based on the amplification methods, Yang
Peng’s team  developed a telomerase-catalyzed FRET ratio probe for accurate miRNA detection. AuNPs were modified with capture probe containing recognition sequence and telomerase primer located at the 5′ of capture probe strand. The detection probe (a molecular beacon labeled with donor FAM and acceptor TARMA) hybridized with the capture probe, separating the fluorescent donor and acceptor and causing low FRET signal. Once miRNA specifically recognized and hybridized the capture probe. The detection probe was then replaced by miRNA to form a stem-ring structure. Thus, the FAM and TAMRA were brought in close proximity to produce high FRET signal. In addition, the capture probe was extended with telomerase primers and hybridized with the catalytic strand to displace target miRNA. The released miRNA also triggered the above-mentioned detection system. This method had low background signal and can detect low abundance miRNA molecules in living cells.
3.3 Catalytic hairpin assembly (CHA)
Catalytic hairpin assembly (CHA) is an enzyme-free, hairpin fuel-driven, and automomous nucleic acid amplification technology. A CHA system needs to design two hairpin structures according to the sequence of target miRNA . One segment of the first hairpin is complementary to the target miRNA sequence . Its hairpin structure can be unfolded by miRNA, and then form a complementary structure with another hairpin probe. The target miRNA will be replaced and dissociated, which can further catalyze CHA between other hairpin probes, forming a cycle to generate amplification signal. The catalytic hairpin assembly has been widely used in nucleic acid detection due to its enzyme-free and target-recyclable advantages.
Like SDR, CHA usually employs nanomaterials as a scaffold and carrier to deliver DNA probes into living cells. As shown in Figure 3, Liu
To improve the kinetics and efficiency of CHA in the complex intracellular environment, inspired by spatial-confinement effects of cells, Liu
3.4 Hybrid chain reaction (HCR)
The hybridized chain reaction proposed by Dirks and Pierce in 2004 is an isothermal signal amplification technology based on DNA strand displacement reaction . Single strand promoter DNA (target miRNA) binds to the stem-loop nucleic acid probe and causes conformation changing of hairpin DNA. The unfolded hairpin structure can unfold a new DNA hairpin. Two kinds of stem-loop probes were alternately hybridized to form double-stranded DNA containing a large number of repeat units . This method has the advantages of constant temperature, efficient signal amplification, and without the requirement of enzyme. It has been applied to the detection of DNA or RNA.
Exploiting the signal amplification function of protein with multiple binding sites, Huang
To improved the stability of miRNA sensing system, Wu and coworkers  designed a DNA probe composed of tripartite Y-shaped DNA structures, folate probe FAP and hairpin probe H1, H2. MiRNA triggered H1 and H2 hairpin probes to assemble HCR, separating Cy5 and BHQ-2 labeled on H1 to recover fluorescence signal. This method was proved to have high sensitivity with a sub-picomolar limit
3.5 Entropy-driven DNA catalysis (EDC)
Entropy-driven DNA catalysis (EDC) exponentially amplifies DNA signal by target-induced entropy change of pre-design sensing system . EDC is a simple, rapid, and enzyme-free isothermal signal amplification technology based on toehold exchange mechanism and adaptable to different low-abundance targets due to its modular design and tunability.
A EDC system usually is composed of a three-strand substrate complex (output strand and signal strand are complementary with link strand) and a fuel strand . In the absence of targets, the sensing system does not work because the toehold domain in substrate that binds to the fuel strand has been protected. As a catalyst, miRNA can combine with the substrate link strand to replace the signal strand, and then the fuel strand replaced miRNA to be recycled. The production of liberated molecules leads to the increase of entropy, repeating the abovementioned strand displacement reaction to generate amplified signals.
To avoiding the addition of external enzyme or fuel transfection, Lu
3.6 DNAzyme-mediated assays
DNAzyme is a kind of DNA with catalytic function and structure recognition ability. It was screened by Breaker and Joyce through the systematic evolution of ligands by exponential enrichment (SELEX) technology in 1994. The single strand, simulating the function of enzymes
Although AuNP-DNA probes are highly sensitive and selective, they suffer from the aggregation of AuNPs in the complex intracellular environment. To overcome this limitation, there is highly desirable for homogeneous DNA (composed entirely of DNAs) sensing system. Xue
To effectively protect the probe from degradation by nuclease and greatly improve its cell permeability, Li
The catalytic activity of DNAzyme depends on the concentration of its cofactor Mg2+. However, the content of Mg2+ in the cell is too low to support the long-time catalytic reactions used for the signal amplification. To circumvent this limitation, Wei
3.7 CRISPR-mediated assays
CRISPR/Cas9 system is a new gene-editing technology based on the bacterial adaptive immune defense system. It can insert or delete genes accurately to knock out target genes . CRISPR/Cas9 system consists of CRISPR RNA, transactivating crRNA, and endonuclease Cas9. Cas9 is specific to the protospacer adjacent motif with the guidance of crRNA and tracrRNA to form RNA–DNA complex. Cas9 can cut double-stranded DNA to complete gene editing.
For the convenience of operation, scientists fused the mature tracrRNA-crRNA dual structure into a sgRNA, its 5′ strand sequence is complementary to the target miRNA, and the 3′ stranded structure could bind to Cas9. Therefore, only one sgRNA needs to be designed to edit the related genes. This method has some advantages such as simple operation, high efficiency, low cost, and no introduction of foreign genes. So far, it has become the most popular gene-editing technology. Similarly, this method has been applied to the detection of miRNA.
An ideal method for detecting intracellular miRNA should possess high throughput, high specificity, high detection sensitivity, wide detection range, and low detection cost. To achieve this goal, a variety of miRNA detection methods have been developed, but there are many shortcomings, and the technology needs to be improved. The additionally introduced nanomaterials is self-aggregated and enriched in different tissues in a complex living environment. Hybridization probe assay lacks signal amplification capabilities. SDA, HCR, and CHA need to avoid high background signal caused by probe leakage. DNAzyme-mediated assays usually require exogenous cofactors to initiate signal amplification. Therefore, EDC and CRISPR-mediated assays are the most promising detection methods of miRNA
The authors gratefully acknowledge the National Natural Science Foundation of China (No. 31972772) and the Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, China (ZZ-A11).
Conflict of interest
There are no conflicts to declare.