Isothermal Amplification for MicroRNA Detection: From the Test Tube

Mar 29, 2017 - miRNAs present a huge challenge in detection because of their extremely short length (∼22 nucleotides) and sequence homology (even wi...
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Isothermal Amplification for MicroRNA Detection: From the Test Tube to the Cell Ruijie Deng, Kaixiang Zhang, and Jinghong Li* Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Tsinghua University, Beijing 100084, China CONSPECTUS: MicroRNAs (miRNAs) are a class of small noncoding RNAs that act as pivotal post-transcriptional regulators of gene expression, thus involving in many fundamental cellular processes such as cell proliferation, migration, and canceration. The detection of miRNAs has attracted significant interest, as abnormal miRNA expression is identified to contribute to serious human diseases such as cancers. Particularly, miRNAs in peripheral blood have recently been recognized as important biomarkers potential for liquid biopsy. Furthermore, as miRNAs are expressed heterogeneously in different cells, investigations into single-cell miRNA expression will be of great value for resolving miRNA-mediated regulatory circuits and the complexity and heterogeneity of miRNA-related diseases. Thus, the development of miRNA detection methods, especially for complex clinic samples and single cells is in great demand. In this Account, we will present recent progress in the design and application of isothermal amplification enabling miRNA detection transition from the test tube to the clinical sample and single cell, which will significantly advance our knowledge of miRNA functions and disease associations, as well as its translation in clinical diagnostics. miRNAs present a huge challenge in detection because of their extremely short length (∼22 nucleotides) and sequence homology (even with only single-nucleotide variation). The conventional golden method for nucleic acid detection, quantitative PCR (qPCR), is not amenable to directly detecting short RNAs and hardly enables distinguishing between miRNA family members with very similar sequences. Alternatively, isothermal amplification has emerged as a powerful method for quantification of nucleic acids and attracts broad interest for utilization in developing miRNA assays. Compared to PCR, isothermal amplification can be performed without precise control of temperature cycling and is well fit for detecting short RNA or DNA. We and other groups are seeking methods based on isothermal amplification for detecting miRNA with high specificity (single-nucleotide resolution) and sensitivity (detection limit reaching femtomolar or even attomolar level). These methods have recently been demonstrated to quantify miRNA in clinical samples (tissues, serum, and plasma). Remarkably, attributed to the mild reaction conditions, isothermal amplification can be performed inside cells, which has recently enabled miRNA detection in single cells. The localized in situ amplification even enables imaging of miRNA at the single-molecule level. The single-cell miRNA profiling data clearly shows that genetically identical cells exhibit significant cell-to-cell variation in miRNA expression. The leap of miRNA detection achievements will significantly contribute to its full clinical adoption and translation and give us new insights into miRNA cellular functions and disease associations.

1. INTRODUCTION MicroRNAs (miRNAs) are a class of short noncoding RNAs that act as post-transcriptional regulators of gene expression.1 They play key roles in many fundamental cellular processes such as cell proliferation, migration, and apoptosis. Much clinical evidence suggests that abnormal miRNA expression is closely related to serious diseases such as cancers.2 Recent research has demonstrated that miRNAs can circulate in the human peripheral blood in a remarkably stable form,3 implying that the miRNAs are potential biomarkers for liquid biopsy. This has led to considerable interest in the development of miRNA assay for clinical diagnosis. Furthermore, given the cellto-cell variations and complex stochastic nature of RNA expression in cells,4 considerable information connecting cell function and miRNA expression may be covered by cellpopulation based miRNA analysis. Accurate quantification of miRNAs in single cells is crucial for studying the exact © 2017 American Chemical Society

association between miRNA expression and cell function, as well as human diseases. Thus, the development of miRNA detection method, especially for clinical samples and single cells is in great demand. Compared to previous nucleic acid targets, such as genes or mRNA, the key differences for miRNAs are their extremely short length (∼22 nucleotide) and sequence homology (singlenucleotide difference). These attributes of miRNA pose grand challenges for miRNA detection. Traditionally used methods, Northern blot, microarray, RT-qPCR, and next generation sequencing (NGS), have all advanced our knowledge of the roles of miRNAs in regulating gene expression and showed the potential use of miRNA in clinical diagnostics. However, methods like Northern blot, microarray, and NGS all suffer Received: January 20, 2017 Published: March 29, 2017 1059

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Figure 1. Schematic representation of isothermal amplification-based methods for miRNA detection: (A) rolling circle amplification (RCA); (B) duplex-specific nuclease signal amplification (DSNSA); (C) catalytic hairpin assembly (CHA); (D) loop-mediated isothermal amplification (LAMP); (E) exponential amplification reaction (EXPAR); (F) strand-displacement amplification (SDA); (G) hybridization chain reaction (HCR).

Table 1. Summary of the Main Isothermal Amplification Methods for miRNA Detection method RCA EXPAR HCR CHA LAMP SDA DSNSA

amplification mechanism polymerase extension, strand displacement polymerase extension, nicking strand displacement strand displacement polymerase extension, strand displacement nicking, polymerase extension, strand displacement cleaving Taqman probe

enzyme

miRNA as primer, trigger, or template

linear or exponential amplification

demonstrated in situ miRNA detection

ref

DNA polymerase, ligase

primer

both

yes

9

DNA polymerase, nicking endonuclease enzyme-free enzyme-free DNA polymerase

primer or trigger

exponential

not yet

23

trigger trigger primer

linear linear exponential

yes yes not yet

43 10 24

DNA polymerase, nicking endonuclease duplex-specific nuclease

template

both

not yet

25

template

linear

not yet

21

have been proposed. Until 2004, Allawi et al. pioneered utilization of the invader assay, now a commercial isothermal amplification technique to detect miRNA.8 Since then, creative works in many laboratories have led to the development of various miRNA assays using isothermal amplification, achieving miRNA detection with high specificity (single-nucleotide resolution) and superior sensitivity (detection limit reaching femtomolar or attomolar level). These methods are sequentially demonstrated to quantify miRNA in clinical samples (tissues, serum, and plasma). Moreover, attributed to the mild reaction conditions and its ability of localized amplification, isothermal amplification is well suited for in situ detection. We first introduced isothermal amplification to in situ miRNA detection, enabling imaging of miRNA in single cells.9 Continuous works utilizing nonenzyme isothermal amplification and probe delivery system even achieve highly sensitive miRNA imaging in living cells.10,11 miRNA profiling at the single-cell level will greatly advance our understanding of miRNA cellular functions

from low detection sensitivity, and an amplification process is usually demanded for miRNA detection, especially in clinical application. PCR is the common amplification strategy for achieving highly sensitive nucleic acid detection, but it cannot directly detect miRNAs as miRNA is too short to act as a PCR template. The strategy for RT-qPCR to circumvent the obstacle is turning the short miRNA into a long target, either by adding poly(A) tails5 or a using stem−loop probe,6 thus requiring sophisticated and complex probe design. Besides, it is a challenge for RT-qPCR to discriminate miRNA family members with very similar sequences.6 Isothermal amplification has emerged as a powerful method for quantification of nucleic acids and has already proven its utility for developing highly specific and sensitive miRNA assays. Compared to PCR, isothermal amplification can be performed without precise control of temperature cycling and is well fit for detecting short RNA or DNA (Figure 1). Since the early 1990s,7 dozens of isothermal amplification techniques 1060

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3. ISOTHERMAL AMPLIFICATION FOR DETECTING miRNA IN THE TEST TUBE

and disease associations. In this Account, we will discuss how isothermal amplification based methods achieve ultrasensitive and specific detection of miRNA and highlight the key successes in the development of isothermal amplification for miRNA detection transition from the test tube to the clinical sample and single cells.

3.1. Targeting Short RNA (miRNA)

The extremely short size of miRNA poses a significant challenge for miRNA detection. To circumvent the obstacle, RT-qPCR turns short miRNA into a long target either by adding poly(A) tails5 or using a stem−loop probe.6 However, the way for developing a miRNA assay with sensitivity, robustness, and simplicity is to use the short sequence directly as the template,21 primer,22 or trigger10 for amplification. Isothermal amplification is endowed with new opportunities for building these simple and sensitive miRNA assays. Many isothermal amplification techniques can be initiated with short sized target sequences, either acting as triggers or primers (Figure 1). Jonstrup et al. first introduced RCA for miRNA analysis in 2006.22 RCA provides a convenient way for building miRNA assays because the short miRNA are well suited for being the templates for ligating the padlock probes and can further prime the RCA process (Figure 1A). However, the miRNA assay has relatively low sensitivity.22 Subsequently, Li and co-workers developed a miRNA assay using EXPAR23 and LAMP,24 demonstrating the feasibility of exploiting isothermal amplification to construct ultrasensitive miRNA assays. The miRNA was designed to be the trigger of EXPAR (Figure 1E). Attributed to the high amplification efficiency of EXPAR, miRNAs can be detected in amounts as low as 0.1 zmol within 30 min.23 SDA utilizes polymerase extension-fueled strand displacement to recycle target miRNA, usually yielding linear amplification. To improve the amplification efficiency, the Zhong group developed an exponential SDA with two repeated cycles of displacement reactions and utilized miRNA to initiate the amplification process.25 The limit of detection of this onepot miRNA assay reached 10 zmol, and the dynamic range spanned over 9 orders of magnitude. Contributing to the high amplification efficiency and compatibility, diversified sensitive miRNA detection platforms based on isothermal amplification were developed. For example, Ge et al. have demonstrated an ultrasensitive electrochemical detection platform for miRNA by combining a tetrahedral DNA nanostructure probe and HCR, conferring a detection limit of 10 aM (about 60 copies/10 μL sample).26 By cascade with DNAzyme signal amplification, RCA was utilized to construct a colorimetric miRNA assay, enabling detection of miRNA as low as 2 aM.27 Thus, miRNAprimed or -triggered isothermal amplification methods confer highly sensitive miRNA detection performance comparable to RT-qPCR with much simplicity and operability. Multiplexed miRNA detection will confer higher confidence for diagnosis and facilitate the study of miRNA function network. Therefore, many isothermal amplification-based multiplexed miRNA assays were further developed. For instance, the Ye group utilized the DSNSA method to turn on different fluorophore-labeled Taqman probes, enabling simultaneous detection of 3 miRNA species.21 However, multiplexing ability is usually limited by the number of distinguishable signal tags, such as spectrally distinct fluorophores and electronically distinct active substrates. Chapin and Doyle improved the multiplexing capacity of a miRNA assay by coupling RCA with fluorescence-encoded hydrogel microparticles.28 This work indicated that spectroscopic or graphical encoding methods may be utilized to develop highly multiplexed miRNA assays. Besides, isothermal

2. ISOTHERMAL AMPLIFICATION FOR BUILDING A HIGHLY SENSITIVE ASSAY Alternative to PCR methods which rely on the precise and complex thermocycling, isothermal amplification can rapidly and efficiently amplify the target nucleic acids or the signal of a recognition event at constant temperature under simple conditions. A large family of isothermal amplification techniques has successively been developed, such as rolling circle amplification (RCA), exponential amplification reaction (EXPAR), hybridization chain reaction (HCR), catalytic hairpin assembly (CHA), strand-displacement amplification (SDA), duplex-specific nuclease signal amplification (DSNSA), and loop-mediated isothermal amplification (LAMP). These isothermal amplification methods are mainly based on enzymebased replication, digestion, or enzyme-free strand displacement processes (Table 1). Both linear (amplification efficiency of 103−104) and exponential (amplification efficiency over 106) amplification of the target sequence could be achieved within 3 h.12 The exponential amplification methods were usually built through layered or sequential amplification using multiple primers, and they confer high amplification efficiency comparable to PCR. The appealing attributes of isothermal amplification provide an excellent opportunity to build highly sensitive bioassays: (1) the capability for rapid and high amplification; (2) the programmability allowing for construction of cascade amplification; (3) the compatibility with diversified detection platforms such as fluorescence, electrochemistry, and colorimetry. By taking these advantages, we and other groups have built many bioassays. Benefiting from its rapid and efficient amplification ability, we utilized EXPAR, an exponential amplification method, to establish a rapid Cas9 cleavage assay for sgRNA prescreening.13 In addition, we constructed cascade amplification by encoding the circular probes of RCA with functional DNA sequence DNAzyme, which can lead to a second amplification process by catalyzing generation of a colorimetric signal.14 This assay achieved highly sensitive detection of platelet-derived growth factor at a concentration as low as 0.2 pg/mL. The Ellington group built multilayered isothermal amplification by utilizing the upstream amplification products to trigger a downstream amplification process, which has also achieved extraordinary amplification efficiency.15 In a continuous effort to seek highly sensitive assays, isothermal amplification has been introduced to fluorescent,16,17 electrochemical,18 and colorimetric19 bioassay. For example, we and the Ju group performed RCA in situ in the electrode to build a highly sensitive electrochemical nucleic acid biosensor with a detection limit of femtomolar and attomolar.18,20 An overview of isothermal amplification methods for detecting biomolecules can be read in a recently published review. 12 All these characters of isothermal amplification present its immense potential for building highly sensitive miRNA assay. 1061

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Figure 2. Highly specific isothermal amplification-based methods for discriminating miRNA family members. (A) The ligation reaction-dependent strategy using T4 RNA ligase 2. Reproduced from ref 30. Copyright 2009 John Wiley & Sons, Inc. (B) Hairpin probe-triggered RCA for miRNA detection. Reproduced from ref 20. Copyright 2015 Royal Society of Chemistry. (C) Multiple DNA strand displacement processes for miRNA recognition. Reproduced from ref 31. Copyright 2015 Royal Society of Chemistry.

(Figure 2A).30 The miRNA assay could even distinguish let-7a and let-7c of which the single-nucleotide mismatch is located near the 3′ terminus. Thus, the specificity of the miRNA assay is highly dependent on the performance of the enzyme. Alternatively, the competitive hybridization method can be optimized for specific miRNA recognition by just tuning the probe sequence. We engineered a hairpin probe for recognizing miRNA, followed by RCA to amplify the recognition process (Figure 2B).20 The stability of hairpin structure would restrain nontarget binding. By tuning the stem length of the hairpin probe, in turn modulating its stability, the highest specificity could be obtained, sufficiently resolving miRNA family members. Another competitive hybridization reaction is the toehold-initiated strand displacement process. It is a nonenzymatic process in which one strand of DNA in the doublestranded probe is competitively displaced by the target sequence driven by a more favorable binding energy (usually by hybridization with an extra short sequence termed the toehold). By elaborately tuning the toehold length or sequence, the process can exactly be induced by only the target sequence, thus conferring high specificity for target recognition. We first introduced the strand displacement reaction to recognize miRNA.9 Recently, we further exploited strand displacement cascades for miRNA detection by building a DNA walker (Figure 2C).31 The DNA walker miRNA assay efficiently restrained nonspecific amplification through multiple strand

amplification could be integrated with microfluidics to build a high-throughput miRNA profiling platform. 3.2. Discrimination among miRNA Family Members

Another challenge for accurate quantification of miRNA is its sequence homology. The miRNA members in the same family may share very similar sequences, such as let-7a and let-7c differing by a single-nucleotide, thus calling for highly specific miRNA assays. In addition, the location of the nucleotide difference can be vary, such as in the middle or near the 3′ terminus, further posing difficulty for discriminating singlenucleotide variation. Although the miRNA-primed or -triggered isothermal amplification methods can achieve ultrasensitive miRNA assays, most of the methods cannot resolve highly similar miRNAs. There are commonly two ways to achieve a specific recognition process: (1) enzyme-dependent recognition; (2) competitive hybridization. The padlock probe and RCA-based method utilizes DNA/RNA ligase to catalyze the ligation process for recognizing the target sequence. It is a highly selective method even exploited for detecting single nucleotide polymorphisms.29 However, with the commonly used DNA ligase, the method exhibits poor specificity toward RNA targets. Cheng et al. utilized T4 RNA ligase 2 capable of specific ligation of a padlock probe upon RNAs, which enabled accurate discrimination of one-nucleotide differences between miRNAs 1062

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Figure 3. Isothermal amplification-based methods for miRNA detection in clinic samples. (A) Quadratic isothermal amplification for miRNA detection in breast cancer patients. Reproduced from ref 35. Copyright 2013 American Chemical Society. (B) Hairpin-based RCA for sensitive detection of serum circulating miRNAs for lung cancer diagnosis. Reproduced from ref 36. Copyright 2013 American Chemical Society. (C) Digital quantification of circulating miRNA directly in plasma using EXPAR and comprehensive droplet digital detection system. Reproduced from ref 37. Copyright 2015 Royal Society of Chemistry.

microarray, Northern blotting, and NGS. However, methods like microarray and Northern blotting suffer from very low sensitivity. And these methods may require complex sample pretreatment, including RNA extraction and reverse transcription. For example, conventional PCR is not able to work directly with clinical samples because of the large amount of protein in tissue or blood, which significantly interferes with the PCR reaction efficiency; thus the preanalysis sample processing steps are indispensable.34 These preanalysis factors significantly affect assay robustness due to the loss of targets and variations in handling techniques. Thus, there is a great need for development of new technologies to directly detect miRNA in tissue or blood without the need for complex sample preparation. Isothermal amplification, which confers high specificity and sensitivity, can be well-suited to address the direct miRNA detection issue. The avoidance of heating and cooling processes makes the reaction more compatible for clinical sample analysis, and there have been many methods developed. For example, Duan et al. developed a one-pot hairpin-mediated quadratic isothermal amplification strategy for miRNA detection (Figure 3A), which achieved a detection limit of 10 fM at 37 °C and

displacement processes. Besides it involved multiple amplification processes, thus gaining high amplification efficiency of the target miRNA. It was able to resolve let-7a and let-7f, which differ by only a single-nucleotide. Mismatch in the terminus of miRNAs will lead to even lower binding energy difference, making them more difficult to discriminate by DNA hybridization-based methods. The Zhang group proposed a reversible strand displacement reaction, termed toehold exchange, where the binding energy of the input target to the probe is close to that of the double-stranded probe, ensuring that mutations anywhere in the incoming strand would severely suppress the recognition process.32 Chen and Seelig designed a two-step detection reaction involving a first reversible toehold exchange step to ensure specificity and a second nonreversible reaction to improve the conversion rate,33 constructing a highly selective miRNA assay insensitive to the position of nucleotide variation.

4. DETECTING miRNA IN CLINICAL SAMPLES miRNAs are now recognized as important biomarkers with potential for early diagnosis and prognosis monitoring. Currently, several miRNA detection methods are available for miRNA detection in clinical samples, including RT-qPCR, 1063

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Figure 4. Isothermal amplification-based methods for in situ imaging of miRNA in single cells. (A) Target-primed RCA strategy for in situ visualization of miRNA expression. Reproduced from ref 45. Copyright 2014 American Chemical Society. (B) miRNA imaging in single cells using RCA and functionalized triple-helix probes. Reproduced from ref 46. Copyright 2016 Royal Society of Chemistry. (C) Live cell miRNA imaging using CHA. Reproduced from ref 10. Copyright 2015 American Chemical Society. (D) Live cell miRNA imaging by HCR. Reproduced from ref 11. Copyright 2016 Royal Society of Chemistry.

was capable of distinguishing miRNA among family members.35 More importantly, the developed method was able to analyze crude extractions from a carcinoma tissue sample. It has been demonstrated that miRNAs can circulate in the blood in a remarkably stable form.3 Since blood is the perfect liquid biopsy, quantification of circulating miRNA is of great utility for clinic diagnosis. Li et al. developed a hairpin probebased rolling circle amplification (HP-RCA) method for sensitive detection of serum miRNA with a detection limit of 10 fM (Figure 3B).36 The developed method could distinguish the expression of serum miRNA between cancer patients and normal donors. However, HP-RCA requires miRNA extraction, which may affect assay robustness. Our group developed an Integrated Comprehensive Droplet Digital Detection (IC 3D) system, which could specifically quantify target miRNA directly from blood plasma at extremely low concentrations ranging from 10 to 10000 copies/mL in ≤3 h without the need for sample pretreatment (Figure 3C).37 The IC 3D approach for miRNA quantification is achieved by combining micro-

encapsulation, EXPER, and digital counting using a highthroughput 3D particle counter. Using this new tool, we found that the concentration of miRNA let-7a content in colon cancer patient blood was significantly higher than that in healthy donor samples. Although isothermal amplification is a competitive method for miRNA detection compared with PCR, especially in the point of care application, all these methods face limitations at present. There is so far no standardized housekeeping RNA can be used as the standard endogenous reference gene for normalization, especially in the blood. Nevertheless, the use of a combination of multiple reference genes for normalizing miRNA concentration will improve the accuracy of miRNA quantification in the clinical samples.38 Besides, the methods capable of absolutely quantifying miRNA such as IC 3D could circumvent the need for endogenous reference, and the ability to perform robustly in crude and complex matrices, avoiding the necessity for lengthy purification steps, would further favor accurate miRNA detection in clinical samples. 1064

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Figure 5. Isothermal amplification for imaging of individual miRNAs in single cells. (A) Schematic representation of toehold-initiated RCA (TIRCA) for visualizing individual miRNAs in situ inside cells. (B) Comparison of the specificity of TIRCA, RCA, and padlock probe-based RCA methods. (C) The specificity of TIRCA using seal probes for let-7a, let-7d, and let-7g. (D) Imaging of individual let-7a in A549 cells by FISH, TIRCA, TIRCA after let-7a was blocked, and TIRCA using perfectly matched (Mis-0), single mismatched (Mis-1), and double mismatched (Mis-2) seal probes. Reproduced from ref 9. Copyright 2014 John Wiley & Sons, Inc.

5. ISOTHERMAL AMPLIFICATION FOR DETECTING miRNA IN SINGLE CELLS

phosphate of the miRNA and protein side chains to eliminate miRNA diffusion out of the cells39 and in turn enhanced the miRNA detection signal. However, these methods exhibit limited improvement of miRNA detection sensitivity with less than an order of magnitude. The in situ amplification will provide a significant increase in the hybridization signals and thereby leading to enhanced sensitivity as required for low-abundance miRNA detection. We and Ge et al. first introduced RCA for in situ amplified target miRNA.9,45 Ge et al. used a circular probe for in situ hybridization with the target miRNA molecules45 and initiated an RCA reaction to amplify the fluorescence signal (Figure 4A). Low-expressing miRNA that could not be detected by FISH was distinguishably visualized inside cells with the help of the amplification process. Zhang et al. later utilized a strategy in which nucleic acid molecular aggregates (NAMAs) resulting from RCA could self-assemble on graphene oxide nanoplates (GONPs) (Figure 4B).46 miRNA was recognized by a difunctional triple-helix probe and triggered RCA for producing the NAMAs. In contrast to conventional RCA, the NAMAs self-assembled on GONPs could form large and congregated fluorescence bright spots. This miRNA imaging approach could distinguish between cancer cells and normal cells. All these miRNA imaging approaches show great potential for unraveling miRNA disease associations. Imaging of miRNA in living cells could facilitate monitoring of the dynamic expression of miRNA and research on miRNArelated cellular processes. However, to date, miRNA remains as one of hardest target molecules to visualize in living cells, both because of its short size and the difficulty of labeling RNAs, let alone for low abundance miRNAs. Remarkably, as the nonenzyme isothermal amplification method is performed only with DNA probes, which can be delivered into cells, it

5.1. In Situ Isothermal Amplification

Compared to PCR, isothermal amplification methods require no temperature control and can be performed inside fixed cells or even living cells, which is hardly feasible with PCR. The high temperature conditions required by PCR may contribute to the damage to cell morphology and target loss (especially miRNA with poor fixation stability).39 Isothermal amplification is thus a versatile in situ amplification strategy, which may enable the quantification and imaging of RNA or DNA in single cells. The Ward group at Yale University first used RCA for in situ imaging of point mutations in the interphase nuclei.29 The method has later been applied for imaging of mitochondrial genomes40 and mRNA41,42 in single cells. Besides RCA, Pierce and co-workers exploited HCR to image mRNAs in zebra fish embryos.43 These applications of in situ amplification implied great potential of its utility for highly sensitive miRNA imaging in single cells. 5.2. Detecting miRNA at Low Expression Levels in Single Cells

One of the most frequently used techniques to image miRNA expression in single cells is fluorescence in situ hybridization (FISH). However, some miRNA species have low expression levels, even with only few copies per cell.1 Because of the relatively high background inside cells, FISH can only detect abundantly expressed miRNAs.39 To solve this issue, some strategies were proposed to improve its sensitivity. For instance, locked nucleic acid (LNA) was utilized to enhance the binding strength of the probe and improve the hybridization efficiency, therefore increasing the sensitivity.44 Pena et al. proposed a fixation method for irreversible cross-linking between the 5′ 1065

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6. CONCLUSIONS AND PROSPECTS Isothermal amplification has fueled miRNA detection from the test tube to cells. Being well fit for the short size of the nuclei acid target, isothermal amplification overmatches the PCR method in some respects in miRNA detection. miRNAs can simply act as primers or triggers to initiate isothermal amplification. Upon coupling with enzyme-dependent recognition or a competitive hybridization process, miRNAs in the same family with even single-nucleotide variation can be distinguished. Besides, the diversity, feasibility, and robustness of isothermal amplification enable it to be readily programmed and adapted to various miRNA assays including fluorescence, electrochemistry, or colorimetry. Some of the miRNA assays have been demonstrated to be amenable for directly detecting miRNA in clinical samples including tissues and peripheral blood without the need for complex sample pretreatment, which is hardly achieved by traditional methods. These highly sensitive and selective miRNA assays show great promise for applications in early diagnose and prognosis monitoring. More importantly, the mild reaction conditions and capability of localized detection of isothermal amplification offer unprecedented opportunities for its adoption for imaging miRNA in situ in single cells. Attributed to the isothermal amplification, breakthrough for precise measurement of the spatial and expression information has been achieved by single-molecule level miRNA imaging. Furthermore, low expression level miRNA that is amplified can be distinguishably detected in living cells, enabling dynamic monitoring of miRNA expression in single cells. These achievements will no doubt facilitate exploring miRNA functions and its disease associations. The dynamic expression of miRNAs may further uncover their roles in cell functions. Though miRNA quantification at the single-molecule level has been achieved in fixed cells, in living cells, it may be more challenging for the following reasons: (1) efficient probe delivery to cytoplasm; (2) turn-on fluorophore-labeled probes designed to respond to isothermal amplification reaction; (3) sufficient stability of fluorophorelabeled probes. One potential way is performing in situ nonenzyme isothermal amplification such as HCR because the amplification process is only dependent on the hybridization reaction with the nucleic acid probe. The high efficiency of probe delivery and stability of probes will be prerequisite for achieving single-molecule miRNA imaging in living cells. Besides, as miRNAs mainly function as post-transcriptional regulators by interacting with other RNAs, the simultaneous determination of the spatial and expression information of miRNAs and their targeted RNAs in single cells will directly exhibit its roles in the post-transcriptional process. Especially, new noncoding RNAs such as circular RNAs (circRNA) and long-coding RNAs (lncRNA) have been discovered, and their function remains to be explored. Thus, the quantification and imaging of mRNA, circRNA, or lncRNA accompanied by miRNAs in single cells by isothermal amplification or smFISH will be appealing for RNA function study and can provide us new insights about miRNA roles in the complex interaction network in the cells.

offers unprecedented opportunities to build highly sensitive miRNA imaging approaches in living cells. Most recently, Weizmann and co-workers first demonstrated the utility of isothermal amplification for miRNA detection in living cells.10 The strategy based on CHA achieved real-time imaging of lowabundance miRNA hsa-miR-21 inside live cells (Figure 4C). Binary-fluorophore-labeled hairpin probes were delivered into cells using transfection reagent Lipofectamine 2000. After the probes enter the cells, miRNA acted as reaction initiators and activated CHA, constructing multiple DNA repeating units to produce in situ amplified signal of miRNA. Instead using the commercial transfection reagent, Li et al. recently constructed a nanodelivery system based on graphene oxide as the carrier of HCR probes.11 By utilizing enzyme-free HCR, this approach conferred both specific recognition and signal enhancement of target miRNAs in living cells (Figure 4D). Although these methods based on in situ isothermal amplification have been demonstrated with the ability for detecting low abundance miRNA in living cells, the obstacles to monitor the dynamic miRNA expression in living cells remain unsolved. Probe delivery and amplification processes would account for relatively long time. As demonstrated by Li et al., amplification of miRNA by HCR would finish at a time of 8 h.11 Thus, isothermal amplification-based miRNA imaging in living cells now cannot allow for prompt response to miRNA expression variation. However, it can be implemented for studying relatively long-term processes or factors that influence the miRNA expression. 5.3. Imaging of Individual miRNA in Single Cells

The miRNA profiling ability with the highest spatial resolution and quantification precision can be achieved by imaging of single-molecule miRNAs in single cells. Single-molecule fluorescence in situ hybridization (smFISH) has emerged as a powerful tool for studying the expression level and spatial organization of single-cell RNAs. However, the short nature of miRNAs prevents signal amplification by multiple probe binding, which is exerted to obtain enhanced and detectable signals by smFISH. RCA can achieve localized amplification of the target molecule, turning the target sequence into a long DNA product with thousands of tandem repeats. Thus, it is a one target−one amplicon amplification process, which may be applied to achieve single-molecule target RNA/DNA detection or imaging. We first utilized RCA to image individual miRNAs in single cells.9 A DNA strand-displacement process was introduced to initiate RCA of specific miRNAs (Figure 5A), which achieved both stringent recognition and in situ amplification of the target miRNA. This assay could be utilized to identify miRNAs at physiological temperature with high specificity (Figure 5B,C) and to visualize individual miRNAs in situ in single cells within 3 h (Figure 5D). This is the first report that single-molecule miRNA can be specifically imaged, conferring specificity sufficient to discriminate miRNA family members even with one-nucleotide variation. The single-cell miRNA profiling clearly showed that genetically identical cancer cells exhibited significant cell-to-cell variation in miRNA expression. This further confirmed the necessity for accurate quantification of miRNAs in a single cell to investigate the exact association between miRNA expression and cell function, as well as human diseases.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Jinghong Li: 0000-0002-0750-7352 1066

DOI: 10.1021/acs.accounts.7b00040 Acc. Chem. Res. 2017, 50, 1059−1068

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The authors declare no competing financial interest. Biographies Ruijie Deng is a Ph.D. candidate under the supervision of Professor Jinghong Li in Tsinghua University, China. His research interests include isothermal amplification and its application in single-cell RNA profiling. Kaixiang Zhang is a Ph.D. candidate under the supervision of Professor Jinghong Li in Tsinghua University, China. His research interests include DNA based biosensors and single-cell analysis. Jinghong Li is a Cheung Kong Professor in the Department of Chemistry at Tsinghua University, China. His current research interests include electroanalytical chemistry, bioanalysis, biosensors, nanoelectrochemistry, and single-cell analysis.

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ACKNOWLEDGMENTS This work was financially supported by National Natural Science Foundation of China (No. 21621003, No. 21235004, and No. 21327806) and Tsinghua University Initiative Scientific Research Program.

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DOI: 10.1021/acs.accounts.7b00040 Acc. Chem. Res. 2017, 50, 1059−1068

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DOI: 10.1021/acs.accounts.7b00040 Acc. Chem. Res. 2017, 50, 1059−1068