Sensitive Quantification of MicroRNAs by Isothermal Helicase

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Rapid and Sensitive Quantification of MicroRNAs by Isothermal Helicase-Dependent Amplification Fei Ma, Meng Liu, Bo Tang, and Chun-yang Zhang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 11 May 2017 Downloaded from http://pubs.acs.org on May 12, 2017

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Analytical Chemistry

Rapid and Sensitive Quantification of MicroRNAs by Isothermal Helicase-Dependent Amplification Fei Ma,† Meng Liu,† Bo Tang,* and Chun-yang Zhang*

College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan 250014, China * Corresponding author. Tel.: +86 0531-86186033; Fax: +86 0531-82615258. E-mail: [email protected].

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ABSTRACT Dysregulation of microRNA expression levels is closely associated with a variety of human diseases, and their rapid and sensitive quantification is essential to clinical diagnosis and therapy. Due to their poor sensitivity, conventional quantification methods are unable to detect low abundant microRNAs. Alternatively, nucleic acid amplification approaches have been introduced to improve the detection sensitivity, but most of them involve complicated probe design and time-consuming procedures. Herein, we report a simple, rapid and sensitive fluorescent method for label-free detection of low abundant microRNAs based on isothermal helicase-dependent amplification. In this assay, the target microRNA may specifically hybridize with 3′-terminus of the linear probe to form a DNA-microRNA heteroduplex, protecting the probes from exonuclease I digestion. The remaining probes may be subsequently amplified by helicase-dependent amplification, generating an ultrahigh fluorescence signal within 30 min. This assay is very sensitive with a low detection limit of 12.8 fM and exhibits a large dynamical range from 100 fM to 10 nM. Moreover, this assay can discriminate different microRNA family members, and it can be used to absolutely quantify endogenous microRNA of total RNA samples extracted from cancer cells, providing a powerful tool for biomedical research and clinical diagnostics.

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INTRODUCTION MicroRNAs are a family of small (~ 22 nt), endogenous, non-protein-coding RNAs that can post-transcriptionally regulate gene expression.1,2 MicroRNAs play critical roles in a variety of cellular processes including cell proliferation,3 metabolism,4 and apoptosis5, while the alteration of microRNA expression levels has been implicated in various human diseases such as liver disease,6 cardiovascular disease7, Parkinson's disease,8 and cancers.9-11 Consequently, microRNAs have been regarded as potential biomarkers for disease diagnosis, prognosis and therapy,12,13 and rapid and sensitive quantification of microRNAs is of great importance to the understanding of their biological functions and clinical applications. Northern blot analysis has been considered as a standard method for microRNA assay, but it suffers from poor sensitivity (above nM), time-consuming steps, and large sample consumption, making it unsuitable for low-abundant microRNA assay.14 To improve the detection sensitivity, a variety of nucleic acid amplification strategies have been introduced, such as polymerase chain reaction (PCR) and isothermal nucleic acid amplification. PCR-based microRNA assays may achieve significantly high sensitivity (∼100 fM),15,16 but they involve reverse transcription step for the transferring of microRNA signal to cDNA signal, which inevitably increases the complexity of probe design.17 In addition, they require sophisticated thermocycler to precisely control the thermocycling, impeding their wide applications.18 Alternatively, isothermal amplification-based methods such as rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), and exponential amplification reaction (EXPAR) may measure microRNAs under constant temperature without the involvement of thermocycling.19-24 Despite their high sensitivity (fM ~ aM), most 3



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of isothermal amplification-based methods still suffer from time-consuming procedures and complex reaction schemes. To achieve a detection limit of ∼10 fM, the branched RCA-based assay requires more than 8 h of reaction time,20 and the hairpin probe-mediated RCA-based assay needs more than 6 h of reaction time.25 The LAMP-based assay involve complicated design of 4 different primer sets to achieve one-step detection of microRNAs,19 while the EXPAR-based assay requires the special design of template to incorporate a site for specific restriction enzyme recognition.21 Therefore, there is an urgent need to develop rapid and sensitive method with a simple reaction scheme for microRNA assay. Herein, we develop a simple, rapid and sensitive fluorescent method for label-free detection of microRNAs based on helicase-dependent amplification (HDA). HDA is one of the most appealing isothermal amplification techniques, in which a DNA helicase is employed to generate single-stranded templates for primer hybridization and a DNA polymerase is used for subsequent primer extension.26,27 In comparison with other isothermal nucleic acid amplification approaches, HDA has significant advantages of simple reaction scheme and time-saving procedure.28 Especially, HDA can achieve over a million-fold amplification within a short reaction time (less than 1 h),26,29 and has been widely applied for the detection of DNA,30 telomerase,31 transcription factor,29 and pathogens.32 However, so far there is no report about the successful application of HDA for microRNA assay probably due to the unique characteristics of microRNAs different from DNAs.1,2 In this research, we design a simple linear probe to recognize target microRNAs. The specific binding of the probes with target microRNAs protects the probes from exonuclease I (Exo I) digestion, and the remaining probes can be subsequently amplified by HDA to generate a distinct fluorescence 4


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signal with SYBR as the indicator. This assay is very rapid with only 30 min amplification time, and it exhibits high sensitivity with a detection limit of 12.8 fM and a large dynamic range of 5 orders of magnitude from 100 fM to 10 nM. Moreover, it can be applied for absolute quantification of target microRNAs in total RNA extracted from cancer cells.

EXPERIMENTAL SECTION Materials. The oligonucleotides (Table 1) were synthesized and purified by HPLC (TaKaRa Bio. Inc., Dalian, China). The Exonuclease I (Exo I), RNase inhibitor and IsoAmp II Universal tHDA kit were obtained from New England Biolabs (Ipswich, MA, USA). SYBR Gold was obtained from Life Technologies (Carlsbad, CA, USA). All other reagents were of analytical grade and used as received without further purification. Ultrapure water obtained from a Millipore filtration system was used throughout all experiments.

Table 1. Sequence of Oligonucleotides note

sequence (5’-3’)

forward primer

TAG CTT ATC AGA CTG ATG TTG ACT GAG G

reverse primer

AAT ATT TTC CAA CAA CGC TTC TGC AAT

probe

AAT ATT TTC CAA CAA CGC TTC TGC AAT CGG ATA TTG GCC TCT CAA TGC TTT TTC GTA CCA ACT TAT CAA ATC ATC CTC AGT CAA CAT CAG TCT GAT AAG CTA

miR-21

UAG CUU AUC AGA CUG AUG UUG A

miR-141

UAA CAC UGU CUG GUA AAG AUG G 5



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let-7a

UGA GGU AGU AGG UUG UAU AGU U

Exonuclease I Digestion. The indicated-concentration microRNA and 1 µM probe were incubated in 19 µL of reaction solution containing 1 mM DTT, 1× Exonuclease I reaction buffer, 1 U/µL RNase inhibitor, 10 mM NaCl and 10 mM Tris-HCl (pH 8.0) at 95 °C for 5 min, followed by slowly cooling to room temperature to form microRNA-DNA hybrid duplexes. Subsequently, 1 µL of Exo I (20 U/µL) was added to digest the free single-stranded probes at 37 °C for 20 min. The digestion reaction was terminated by heating at 90 °C for 10 min.

Helicase-Dependent Amplification Reaction and Real-Time Fluorescence Measurement. IsoAmp II universal tHDA kit was used for the helicase-dependent amplification reaction. The reaction was performed at 65 °C in a total volume of 50 µL of reaction solution containing 5 µL of digestion mixture, 5 µL of 10× annealing buffer II, 2 µL of 100 mM MgSO4, 4 µL of 500 mM NaCl, 3.5 µL of IsoAmp dNTP solution, 1 µL of 10 µM forward primer, 1 µL of 10 µM reverse primer, 1× SYBR gold, and 3.5 µL of IsoAmp enzyme mix (i.e., Bacillus stearothermophilus (Bst) DNA polymerase and Thermoanaerobacter tengcongensis UvrD (Tte-UvrD) helicase). The real-time fluorescence measurements were performed in a Bio-Rad CFX connectTM Real-Time System, and the fluorescence intensity was monitored at intervals of 30 s.

Gel Electrophoresis. The digestion products of Exonuclease I and the amplification products 6


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of HDA reaction were analyzed by 2% agarose gel electrophoresis in 1× TAE buffer (40 mM Tris-acetic acid, 2 mM EDTA, pH 8.0) at a 120 V constant voltage at room temperature for 40 min. The gels were stained by SYBR Gold and analyzed by a Bio-Rad ChemiDoc™ MP Imaging System.

Preparation of Total RNA Samples from HeLa Cells. HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies, USA) with 10% fetal bovine serum (FBS, Life Technologies, USA) and 50 U/mL penicillin plus 50 µg/mL streptomycin at 37 °C with 5% CO2. The total RNA was obtained by miRNeasy Mini Kit (Qiagen, German) according to the manufacturer’s handbook, and its concentration was determined by the NanoDrop 2000c Spectrophotometer (Thermo Scientific, Wilmington, Delaware, USA).

RESULTS AND DISCUSSION Scheme 1. Scheme of Isothermal Helicase-Dependent Amplification for MicroRNA Assay

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Principle of microRNA Assay. As shown in Scheme 1, target microRNA can specifically bind to the 3'-terminus of the probe to form a DNA-microRNA heteroduplex via Watson-Crick base pairing, protecting the probe from Exo I digestion (note: Exo I is an exonuclease enzyme that can efficiently degrade single-stranded DNA in the 3' to 5' direction).33,34 Subsequently, the forward primer may hybridize with the remaining probe and is extended by DNA polymerase to form a double-stranded DNA (dsDNA). The resultant dsDNA may be unwound in the presence of single-stranded DNA-binding protein (SSB) and helicase, enabling the forward primer and the reverse primer to hybridize with each border of dsDNA. As a result, both primers can be extended by DNA polymerases to form new dsDNAs, which may function as new substrates to trigger next round reaction, leading to exponential amplification. In this research, SYBR Gold is used as the fluorescent dye to monitor HDA 8


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products.33 While in the absence of target microRNA, the probes are completely digested by Exo I, and neither HDA reaction nor signal amplification occurs. Due to the introduction of HDA,26 the reaction scheme of current assay is much simpler than EXPAR- and LAMP-based assays,19,21 and it takes less time to achieve high amplification efficiency than RCA-based assay.26,29 As a proof of concept, miR-21 is used as a model target. The miR-21 is a key regulator of oncogenic processes and is closely associated with various tumors such as lung cancer35 and breast cancer.36

Figure 1. Gel electrophoresis analysis of Exo I digestion. Lane M, DNA marker; lane 1, 1 µM probe; lane 2, 1 µM probe + 10 U Exo I; lane 3, 1 µM probe + 20 U Exo I + 0.2 µM miR-21; lane 4, 1 µM probe + 10 U Exo I + 0.5 µM miR-21; lane 5, 1 µM probe + 20 U Exo I + 1 µM miR-21.

MicroRNA-Directed Probe Protection. The protection of the probes from Exo I digestion by target microRNA is the initial step in this assay. We incubated the probes and different-concentration target miR-21 with Exo I, and employed 2% agarose gel electrophoresis to analyze the products. As shown in Figure 1, the band of the probes (Figure 1, lane 1) disappears after incubation with Exo I (Figure 1, lane 2), indicating that the probes can be completely digested by Exo I. On the contrary, the addition of target miR-21 may 9



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induce the appearance of the probe band (Figure 1, lanes 3, 4 and 5). Interestingly, the band intensity of the probes improves in a miR-21concentration-dependent manner (Figure 1, lanes 3, 4 and 5), and reaches the same level of the probes only (Figure 1, lane 1) when equal amount of miR-21 is added (Figure 1, lane 5). These results clearly demonstrate that the target miR-21 can efficiently protect the probes from Exo I digestion, and that the complete digestion of excess probes by Exo I may greatly contribute to the low background signal.

Figure 2. (A) Real-time fluorescence measurement of HDA reaction in the presence of 50 nM miR-21 (red line) and in the absence of miR-21 (black line), respectively. (B) Gel electrophoresis analysis of helicase-dependent amplification products in the absence of miR-21 (line 1) and in the presence of 50 nM miR-21 (lane 2), respectively. Lane M is the DNA marker.

Verification of Assay Feasibility. After the Exo I digestion, the products were subjected to helicase-dependent amplification (HDA) reaction. In the presence of target miR-21, the fluorescence intensity increases rapidly and reaches a plateau within 10 min (Figure 2A, red line). In the control group without miR-21, no distinct fluorescence signal is observed (Figure 2A, black line) due to the complete digestion of the probes by Exo I. We further employed 2% 10


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agarose gel electrophoresis to verify the feasibility of this assay (Figure 2B). A distinguished band of HDA amplification products is observed in the presence of miR-21 (Figure 2B, lane 2), while no distinct band is observed in the absence of miR-21 (Figure 2B, lane 1). These results (Figure 2B) are in good agreement with the real-time fluorescence measurements (Figure 2A), indicating that the proposed method can be used to rapidly measure target microRNA with extremely low background signal.

Figure 3. (A) Real-time fluorescence measurement of HDA in the presence of different-concentration miR-21. (B) Linear relationship between ∆POI and the logarithm of miR-21 concentration. Error bars show the standard deviation of three experiments.

Sensitivity of the Assay. To investigate the sensitivity of the proposed assay, we monitored 11



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the real-time fluorescence signal in response to different-concentration miR-21 (Figure 3A). We used the point of inflection (POI) value to quantitatively measure microRNA concentration.21 The POI is defined as the time corresponding to the maximum slope of the fluorescence curve, and the ∆POI is defined as the difference in the POI value between the measured sample and the control group. As shown in Figure 3B, the ∆POI exhibits a linear correlation with the logarithm of miR-21 concentration in the ranges from 100 fM to 10 nM. The correlation equation is ∆POI = 36.35 + 2.58 log10C, with a correlation coefficient of R2=0.9971, where C is the miR-21 concentration (M). Notably, this assay has a large dynamic range of 5 orders of magnitude from 100 fM to 10 nM. The detection limit is calculated to be 12.8 fM (or 640 zmol) based on 3σ/K, where σ is the standard deviation of the control group and K is the slope of the linear regression curve.37 The sensitivity of this assay has improved by 4 orders of magnitude as compared with that of the quenched Staudinger-triggered probe-based RCA assay (0.2 nM),38 1 orders of magnitude as compared with those of duplex-specific nuclease signal amplification-based assay (0.3 pM)39 and hairpin probe-based EXPAR assay (0.38 pM).40 The ultrahigh sensitivity may be ascribed to the highly efficient HDA-mediated signal amplification and the low background signal resulting from the complete digestion of excess probes by Exo I. It is worth noting that such high sensitivity may be achieved within 30 min amplification, which is much shorter than the reported isothermal microRNA assays including the hairpin probe-based RCA assay (4 h),25 duplex-specific nuclease signal amplification-based assay (2 h)41 and hairpin probe-based EXPAR assay (more than 100 min).40

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Figure 4. (A) Gel electrophoresis analysis of Exo I digestion products. Lane M, DNA marker; lane 1, 1 µM miR-21 + 1 µM probe; lane 2, 1 µM miR-21 + 1 µM probe + 20 U Exo I; lane 3, 1 µM miR-141 + 1 µM probe; lane 4, 1 µM miR-141 + 1 µM probe + 20 U Exo I; lane 5, 1 µM let-7a + 1 µM probe; lane 6, 1 µM let-7a + 1 µM probe + 20 U Exo I. (B) The comparison of ∆POI values in the absence of target microRNA (blank), in the presence of 1 nM miR-21 (red color), 1 nM miR-141 (green color), and 1 nM let-7a (blue color), respectively. Error bars show the standard deviation of three experiments.

Specificity of the Assay. The capability to distinguish different microRNA family members is a great challenge for microRNA assay. To investigate the specificity of the proposed assay, we used the designed probes to detect target miR-21 and two non-target microRNAs including miR-141 and let-7a (Figure 4). The miR-141 belongs to the miR-200 family which is expressed in a variety of human cancers such as colon and prostate cancer,42,43 and let-7a is a 13



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member of let-7 miRNAs which are associated with cancer development.44 We employed 2% agarose gel electrophoresis to analyze the products of Exo I digestion in the presence of target miR-21, miR-141, and let-7a (Figure 4A). In the presence of target miR-21, the formation of miR-21-probe heteroduplex protects the probes form Exo I digestion, and the band intensity remains unchanged before (Figure 4A, lane 1) and after Exo I digestion (Figure 4A, lane 2). However, in the presence of miR-141, the band intensity significantly decreases after Exo I digestion (Figure 4A, lane 4) as compared with the control group without Exo I digestion (Figure 4A, lane 3). Similar result is observed for let-7a, with the decrease in the band intensity after Exo I digestion (Figure 4A, lane 6) as compared with the control group without Exo I digestion (Figure 4A, lane 5). Consistent with the results of agarose gel electrophoresis (Figure 4A), real-time fluorescence measurement indicates that the ∆POI obtained in the presence of target miR-21 is 4.07-fold higher than that obtained in the presence of miR-141, and 9.89-fold higher than that obtained in the presence of let-7a (Figure 4B). These results

clearly demonstrate that the proposed method possess high specificity with the capability to discriminate target microRNA from irrelevant microRNAs.

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Figure 5. (A) Real-time fluorescence measurement of amplification reaction in the presence of 0.5 µg of total RNA (red line), 0.5 µg of total RNA + 0.5 fmol of synthesized miR-21 (blue line), and the control group without any sample (black line). (B) Comparison of ∆POI values in the presence of control group without any sample (blank, black column), 0.5 µg of total RNA (red column), and 0.5 µg of total RNA + 0.5 fmol of synthesized miR-21 (blue column). Error bars show the standard deviation of three experiments.

Real sample analysis. To investigate the feasibility of the proposed method in real sample analysis, we measured the absolute amount of endogenous miR-21 in total RNA samples extracted from human epithelioid cervix carcinoma (HeLa) cells. As shown in Figure 5A, the real-time fluorescence signal produced by 0.5 µg of total RNA (Figure 5A, red line) can be distinguished well from that produced by the control group without total RNA (Figure 5A, 15



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black line), and the ∆POI value produced by 0.5 µg of total RNA is much higher than that produced by the control group without total RNA (Figure 5B). The absolute amount of miR-21 in total RNA is quantified to be 0.181 fmol / 0.5 µg (or 2.18 × 105 copies/ng) based on the correlation equation in Figure 3B, which is in good agreement with the results obtained by DNA-gold nanoparticle probe-based assay (2.4 × 105 copies/ng)41 and multi-amplified enzymatic cascade assay (2.13 × 105 copies/ng).45 To further evaluate the performance of the proposed method in real sample analysis, 0.5 fmol of synthesized miR-21 is spiked into 0.5 µg of total RNA, and the absolute amount of miR-21 in the spiked sample is quantified to be 0. 695 fmol with a recovery rate of 102.8%. These results clearly demonstrate the feasibility of the proposed method in real sample analysis.

CONCLUSIONS In conclusion, we have developed a simple, rapid and sensitive fluorescent method for label-free detection of microRNAs based on isothermal helicase-dependent amplification (HDA) without the involvement of complicated procedures and precise temperature control. The binding of target microRNAs with the probes may protect the probes from Exo I digestion, and the remaining probes can be subsequently amplified by HDA to generate a distinct fluorescence signal. Due to the complete digestion of excess probes by Exo I, extremely low background-signal may be achieved. In addition, the introduction of HDA may significantly improve the detection sensitivity. In comparison with the reported isothermal amplification-based assays,19,21,25,40,41 the proposed method possesses significant advantages of simple reaction scheme and time-saving procedure, enabling the achievement of high 16


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sensitivity within only 30 min amplification time. This assay is very sensitive with a detection limit of 12.8 fM, and it exhibits a large dynamic range of 5 orders of magnitude from 100 fM to 10 nM. Importantly, this assay can be applied for absolute quantification of endogenous microRNA in cancer cells, holding great potential for further applications in clinical diagnosis and microRNAs-related biomedical researches.

AUTHOR INFORMATION Corresponding Author *Tel.: +86 0531-86186033. Fax: +86 0531-82615258. E-mail: [email protected]. . *Tel.: +86 0531-86180010. Fax: +86 0531-86180017. E-mail: [email protected]. Author Contributions † These authors contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grant Nos. 21325523 and 21527811), Shandong Province Science Foundation for Youths (Grant No. ZR2016HQ07), and the Award for Team Leader Program of Taishan Scholars of Shandong Province, China,

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For TOC only

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Sensitive Quantification of MicroRNAs by Isothermal Helicase

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