Sensitive detection of mRNA by using specific cleavage-mediated isothermal exponential amplification reaction

Sensors and Actuators B 252 (2017) 215–221

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Sensitive detection of mRNA by using specific cleavage-mediated isothermal exponential amplification reaction Hui Wang, Honghong Wang, Xinrui Duan ∗∗ , Xiangdong Wang, Yuanyuan Sun, Zhengping Li ∗ Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an, Shaanxi, 710119, PR China, PR China

a r t i c l e

i n f o

Article history: Received 3 March 2017 Received in revised form 31 May 2017 Accepted 1 June 2017 Available online 3 June 2017 Keywords: mRNA detection Isothermal exponential amplification RNase H IEXPAR

a b s t r a c t Messenger RNA (mRNA) as a bridge links the organism’s genotypes to its phenotypes. Quantitative detection of mRNA is vital in basic studies of life science and medical diagnostics. In this work, a sensitive, specific, and reverse transcription-free approach for quantification of mRNA based on cleavage-mediated isothermal exponential amplification reaction (IEXPAR) is established. The target mRNA firstly hybridizes with the flexibly designed DNA probe to form a loop structure. RNase H specifically digests the hybridized mRNA and releases the loop part of the mRNA, which can serve as the trigger to initiate IEXPAR. In this design, the DNA probe and the IEXPAR templates can specifically recognize three regions on the target mRNA, so that the high specificity can be achieved. Moreover, the specificity of RNase H can avoid the contamination of genomic DNA. IEXPAR is a powerful nucleic acid amplification technique with rapid, simple and cost-effective features, making the proposed assay highly sensitive and fast. With this assay, as low as 100 zmol target mRNA can be quantitatively detected and wide linear range with six orders of magnitude can be obtained. The assay can also be successfully applied to determine p53 mRNA in 5 ng total RNA sample extracted from MCF-7 breast cancer cells. © 2017 Published by Elsevier B.V.

1. Introduction Gene expression is the most fundamental life process, in which the genetic code stored in DNA translates into messenger RNA (mRNA) and then mRNA works as a blueprint to guide proteincoding [1]. Therefore, mRNA as a bridge links the organism’s genotypes to its phenotypes. Recently, as more and more genomes are decoded and corresponding mRNAs’ function is being revealed through the massively parallel DNA sequencing technologies [2,3], mRNA analysis has become increasingly important because more and more mRNAs have been regarded as the signature of biochemical pathway [4], the biomarkers for human diseases [5,6] and pathogen identification [7]. However, the mRNA detection methods are lagging far behind their rapid growing demand. Currently, reverse transcription PCR (RT-PCR) is the most common technique for mRNA detection, which has shown some intrinsical limitations, such as requirement of precision thermal cycles as well as careful optimization of experiment setup, the false-

∗ Corresponding author at: School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an, 710119, Shaanxi Province, PR China. ∗∗ Corresponding author. E-mail addresses: [email protected] (X. Duan), [email protected] (Z. Li). http://dx.doi.org/10.1016/j.snb.2017.06.008 0925-4005/© 2017 Published by Elsevier B.V.

positive results arising from the amplification artifacts (such as the primer-dimers). Especially, RT-PCR is highly sensitive to the contamination of genomic DNA [8]. Several isothermal amplification techniques have also been developed for mRNA detection, such as nucleic acid sequence based amplification (NASBA) [9], loop-mediated isothermal amplification (LAMP) [6,10], and rollingcycle amplification (RCA) [11,12]. However, all of these isothermal methods also need reversely transcribe mRNA to cDNA which possesses the risk of cross-contamination of genomic DNA and results in false-positive results. More recently, Tang and co-workers have pioneered the DNAzyme cleavage-based RNA amplification for mRNA detection without requirement of the reverse transcription [8]. Unfortunately, the cleavage products cannot be directly amplified. So multiple amplification steps and additional steps are needed to remove the 2 , 3 -cyclic phosphate group at the 3 -end of the cleavage products. We have developed a reverse transcriptionfree method for mRNA quantification detection based on ligase chain reaction (LCR) [13]. However, the method also needs thermal cycling and at least five probes for one mRNA target detection, which increases the cost and the design complexity. Compared to the isothermal amplification techniques mentioned above and others [14–16], isothermal exponential amplification reaction (IEXPAR) is powerful because of the superior properties of high amplification efficiency, rapid amplification

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kinetics, and simple design [17]. In the previous works, our group and others have confirmed that microRNA can effectually initiate the IEXPAR, and thus, microRNAs can be directly detected by IEXPAR with high sensitivity [18–21]. This technique has also been applied to sensitive detection of telomerase activity [22,23], platelet-derived growth factor BB [24], transcription factor [25], and thrombin [26]. Unfortunately, IEXPAR is only suited to amplify the short RNA/DNA targets. In this work, we rationally designed a DNA probe, which can specifically recognize the two parts of target mRNA sequences and form an RNA loop structure through the hybridization. RNase H specifically digests the hybridized mRNA and releases the loop part of the mRNA, which can serve as the trigger to initiate IEXPAR. The current design has several superior features due to the following points: 1) the cleavage-mediated IEXPAR offers high specificity for mRNA detection due to three specific recognition regions on the target mRNA corresponding to the DNA probe and the IEXPAR template. 2) Since the RNase H only digests RNA, genomic DNA cannot initiate the cleavage-mediated IEXPAR, which will avoid the contamination of genomic DNA and ensure the reliability for mRNA analysis. 3) The superior properties of IEXPAR will guarantee the proposed assay to have high sensitivity. 2. Experimental section 2.1. Materials and reagents RNase H, Hybrid RNA Degeneration buffer (100 mM Tris-HCl, 200 mM KCl, 40 mM MgCl2 and 5 mM DTT, pH 7.8 @ 25 ◦ C), Ribonuclease (RNase) inhibitor, dNTPs and RNase-free water were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). Vent exo− DNA polymerase, ThermoPol reaction buffer (200 mM Tris-HCl, 100 mM KCl, 100 mM (NH4 )2 SO4 , 20 mM MgSO4 and 1% Triton X-100, pH 8.8 @ 25 ◦ C), Nt.BstNBI Nicking Enzyme and NEBuffer (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 and 100 ␮g/mL BSA, pH = 7.9 @ 25 ◦ C), and ProtoScripIIreverse transcriptase and reverse transcription reaction buffer (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2 ) were purchased from New England Biolabs (Beijing, China). JumpStartTM Taq DNA Polymerase and PCR reaction buffer were purchased from Sigma-Aldrich (Shanghai, China). All of RNA and DNA were synthesized and purified by TaKaRa Biotechnology Co., Ltd. All the sequences of RNA and DNA were listed in Table S1. 2.2. Cell culturing and total RNA extraction Michigan Cancer Foundation-7 (MCF-7) cell line was cultured in the DMEM (GIBICO) medium supplemented with 10% fetal calf serum (GIBICO), 1% NaHCO3 , 100 U/mL penicillin, 100 ␮g/mL streptomycin and 3 mmol/L L-glutamine under 5% CO2 at 37 ◦ C for 48 h. ® Total RNA was isolated from cells using TRIzol Reagent (Invitrogen, Beijing, China) following the manufacturer’s protocol. The concentration was determined from the absorption at 260 nm with NanoDrop 2000 (Thermo Fisher Scientific, USA). The concentration of total RNA was adjusted to 1 ␮g/␮L by RNase-free water and stored −80 ◦ C. 2.3. Digestion reaction by RNase H 1 ␮L p53 mRNA fragment or total RNA extracts were added to the mixture containing 1 ␮L 1 ␮M DNA Probe, 8 U RNase inhibitor, 2 ␮L hybridization reaction buffer (100 mM Tris-HCl, 200 mM KCl, 40 mM MgCl2 , 5 mM DTT, pH 7.8), and 4.8 ␮L RNase-free water. The mixture was incubated at 65 ◦ C for 5 min and 37 ◦ C for 5 min. Subsequently, 5 U RNase H was added into the mixture with a final volume 10.0 ␮L. The digestion reaction was carried out at 37 ◦ C for 30 min and terminated by inactivating RNase H at 65 ◦ C for 10 min,

then, the products were immediately put on ice for IEXPAR reaction or gel electrophoresis analysis. 2.4. Gel electrophoresis analysis The products of the digestion reaction were analyzed by 16% non-denaturing polyacrylamide gel electrophoresis (PAGE) in 1 × TBE buffer at a 90 V constant voltage from 90 min at room temperature, the gel was taken photograph under GelDocTM EZ Imager (Bio-Rad) after staining with 2 × SYBR Gold for 10 min. 2.5. IEXPAR reaction and real-time measurement of fluorescent intensity The reaction mixture for IEXPAR reaction was respectively prepared on ice as part A and part B. Part A consisted of NEBuffer, dNTPs, template I, template II, RNase inhibitor and 1 ␮L products of the specific cleavage reaction. Part B consisted of ThermoPol reaction buffer, Nt.BstNBI Nicking Enzyme, Vent exo− DNA Polymerase, SYBR Green I. The IEXPAR reaction was carried out in 10 ␮L volume containing 100 nM template I, 100 nM template II, 250 ␮M dNTPs, 4 U Nt.BstNBI Nicking Enzyme, 0.04 U Vent exo− DNA Polymerase, 4 U RNase inhibitor, 0.4 ␮g/mL SYBR Green I, ThermoPol buffer (2.5 mM Tris-HCl, pH = 8.8 @ 25 ◦ C, 10 mM KCl, 2 mM MgSO4 , 10 mM (NH4 )2 SO4 , 0.1% Triton X-100), and NEBuffer (2.5 mM TrisHCl, 5 mM NaCl, 0.5 mMgCl2 , 5 ␮g/mL BSA). Part A and Part B were mixed immediately before being placed in the StepOne Real-Time PCR System (Applied Biosystems, USA). The IEXPAR amplification was performed at 55 ◦ C and the real-time fluorescence intensity was monitored at intervals of 1 min. 2.6. RT-PCR analysis The RT-PCR analysis included two steps. Firstly, a volume of 5 ␮L reverse transcription mixture containing 0.5 pmol RT primer (see the sequence in Table S1), 1 × RT buffer, 1 mM dNTPs, 40 U ProtoScripIIreverse transcriptase and 4 U RNase inhibitor was incubated for 30 min at 37 ◦ C. Subsequently, 5 ␮L PCR reaction mixture containing 2 pmol forward primer and reverse primer (see sequences in Table S1), 0.4 ␮g/mL SYBR Green I, 0.5 U JumpStartTM Taq DNA Polymerase and 1 × PCR buffer was added into the reverse transcription mixture. Then the mixture was carried out with a StepOne Real-Time PCR System (Applied Biosystems, USA) by using hot start of 94 ◦ C for 2 min, followed by 40 cycles of 94 ◦ C for 10 s, 63 ◦ C for 20 s and 72 ◦ C for 10 s. 3. Results and discussion 3.1. Principle of specific cleavage-mediated IEXPAR for mRNA detection The principle of specific cleavage-mediated IEXPAR for mRNA assay is illustrated in Fig. 1. Here, p53 mRNA was selected as a model target. Firstly, a DNA probe was designed according to the sequence of p53 mRNA. The DNA probe consisted of two specifically binding regions (I* and II*) which were complementary to two corresponding sequences (I and II) in the target mRNA. In the presence of target mRNA, two binding regions in the DNA probe hybridized with the two sequences in the target mRNA to form a loop structure in the resulting RNA/DNA hybrids. Since the RNase H only digests the RNA molecules in the RNA/DNA double helix, I and II of mRNA in RNA/DNA hybrids were specifically digested by RNase H and generated shorter mRNA trigger to initiate subsequently IEXPAR. Then, RNase H was inactivated to avoid its interference in further reaction. Subsequently, two amplification

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Fig. 1. Schematic overview of the principle of specific cleavage-mediated IEXPAR for mRNA detection.

templates (template I and template II) were used in the amplification reaction. The 3 terminal of two templates were modified with a phosphate group to avoid non-specific extension due to mis-priming. From 5 to 3 direction, the template I consisted of a target mRNA trigger binding domain (TBD), a nicking recognition site (NRS, 3 -NNNNCTCAG-5 ) of nicking enzyme, and an IEXPAR trigger released domain (X). Once the mRNA trigger released from target mRNA hybridized with the TBD section of the template I, it will be extended along the template I in the presence of Vent exo− DNA polymerase and deoxyribonucleotide triphosphate (dNTPs) to form double-stranded DNA (dsDNA). Meanwhile, the nicking enzyme (Nt.BstNBI) can recognize the NRS sequence and cleave the just extended single strand DNA to form a nick at 4 bases downstream of the recognition sequence. Then the 3 terminus of the resulting strand DNA will be extended again at the nick site by the DNA polymerase, and the short single-stranded DNA trigger X* will be replaced and released via the strand-displacement activity of Vent exo− DNA polymerase [15]. As the cycle of the extension, cleavage and strand displacement continues, one trigger X* will be released in each cycle, which resulted in a linear amplification of the trigger released from target mRNA. The template II consisted of the same trigger binding/releasing domain (X) at its 5 and 3 ends and a nicking recognition site (NRS) in the middle. Once the DNA trigger X* hybridized to the 5 end of the template II, the extension reaction will occur along the template II to form dsDNA. After each extension-nicking-releasing cycle, the amount of the trigger X* was doubled. As the cycling continues, chain reactions were sustaining to achieve an exponential amplification of the DNA trigger X* until the template II were completely consumed. Finally, the majority of template II was extended into dsDNA as IEXPAR products. Since SYBR Green I (SG) could intercalate into dsDNA and resulted in greatly enhanced fluorescence, IEXPAR products can be monitored with SG in real time without any labeled probes. 3.2. The effect of the template I design Theoretically, RNase H able to digest all of the RNA sequences in the RNA/DNA hybrids. But the location of the cutted site in RNA/DNA hydrides is unknown due to the random digestion nature

of the RNase H [27]. In other words, the length of the released RNA sequence that is used as mRNA trigger is uncertain after RNase H digesting. Therefore, the design of template I is a key issue for the mRNA assay. In order to address this issue, we designed a series of template I for the released mRNA triggers including of template I-0, template I-5, template I-10, template I-15, and template I-18. For template I-0, trigger binding domain (TBD) only can hybridize with the sequence of loop structure in the RNA/DNA hybrids. For template I-5, template I-10, template I-15 and template I-18, TBD not only can hybridize with the sequence of loop structure, but also can hybridize with the loop sequence and other additional 5, 10, 15 and 18 nucleotides of the target mRNA from the 3 terminus of the loop structure in the RNA/DNA hybrids, respectively. Synthesized 57 nt p53 mRNA fragment (the 759th to 815th nucleotides of p53 mRNA) were used as a model target to investigate the effect of the template I type for mRNA detection. According to the experimental section, we respectively used template I-0, template I-5, template I-10, template I-15, and template I-18 to detect the mRNA at 0 (blank), 10 fM, 100 fM and 1 pM. As shown in Fig. 2, when template I-0 was used (Fig. 2A), the proposed assay can only detect as low as 1 pM mRNA. When template I-5 was used (Fig. 2B), as low as 100 fM mRNA can be detected, and the detection limit was reduced one order of magnitude. When template I-10 (Fig. 2C), template I-15 (Fig. 2D) and template I-18 (Fig. 2E) were used, as low as 10 fM mRNA can be detected, the detection limit was reduced two orders of magnitude. These results demonstrated that the mRNA triggers released from the loop structures have a different length. Besides the mRNA sequence in the loop, several additional nucleotides exist in the mRNA triggers resulted from RNase H digestion. But the addition nucleotides should less than 10 nucleotides. These results are consistent with the property of RNase H digestion because the RNase H generally binds to at least 8 nucleotides [28,29] and duplex of ten base pairs (5 -3 ) in the RNA/DNA hydrides has a melting temperature approximate equals to reaction temperature (37 ◦ C). As shown in Fig. 2F, while the number of additional nucleotides in the template I-10 and I-15 was 10 or 15, the interval of POI (point of inflection, the time corresponding to the maximum slope of the fluorescence curve) between each the mRNA targets with different concentrations and blank reach the maximum. To sum up, template

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Fig. 2. (A–F): The effect of the template I design on the mRNA detection. The real-time fluorescence curves were produced by 1 pM, 100 fM, and 10 fM p53 mRNA and the blank (without p53 mRNA) with the cleavage-mediated IEXPAR. Error bars were estimated from the standard deviations of three repetitive tests. (G): Non-denaturing polyacrylamide gel electrophoresis (PAGE) analysis of the DNA probe, mRNA, and the products of digestion reaction. The concentration of the DNA probe and p53 mRNA were 200 nM.

I-10 can fully utilize mRNA triggers after RNase H digesting to initiate IEXPAR for sensitive detecting of mRNA. Therefore, template I-10 was employed for the further experiment. Furthermore, the DNA probe, mRNA, and the products of digestion reaction were analyzed by non-denaturing polyacrylamide gel electrophoresis (PAGE) (Fig. 2G). The results from the lane 1, lane 2, lane 4, and lane 5 showed that the RNase H didn’t digest the DNA probe and target mRNA. When DNA probe and mRNA presented, an obvious band of DNA/RNA hybrids can be found (lane 3). When RNase H presented, the band of DNA/RNA hybrids completely disappeared and a wider band appeared (lane 6). These results showed that RNase H specifically digested the RNA molecular in DNA/RNA hybrids and efficiently released a shorter RNA with different length between loop RNA (lane 7, 20 nt) and DNA probe (35 bp), which further verified that our template design was reasonable. 3.3. Influence of the amount of DNA polymerase and nicking enzyme on mRNA assay The amounts of DNA polymerase and nicking enzyme heavily affected the yield of IEXPAR product. Thus, it is necessary to find the optimal concentration ranges of both enzymes. To study the effect of the amount of DNA polymerase for mRNA detection, the IEXPAR were conducted according to the procedure in the experimental section except the amount of Vent exo− DNA polymerase. Different concentrations of p53 mRNA fragment and blank were simultaneously determined by using 0.02 U, 0.04 U, 0.08 U, and 0.2 U Vent exo− DNA polymerase, respectively. When the amount of the DNA polymerase was limited, extension step would slow down the whole amplification reaction and resulted in low sensitivity and prolonged the reaction time. As we can observe in Fig. 3A, when the amount of DNA polymerase was 0.02 U, the fluorescence signals of all the samples were not detectable in 90 min. With increasing the amount of DNA polymerase from 0.04 U to 0.2 U (Fig. 3 B to D), the IEXPAR was correspondingly accelerated, resulting in the decrease of POI values of real-time fluorescence curves produced by the 1 pM, 100 fM, 10 fM, and blank. The difference of the POI values between the target mRNA with different concentrations and the blank reach maximum when the amount of Vent exo− DNA polymerase was

0.04 U. Based on the consideration of both reaction time and detection sensitivity, 0.04 U was selected as the optimum amount of Vent exo− DNA polymerase in further mRNA detection. To investigate the influence of the amount of Nicking Enzyme used in IEXPAR on our mRNA detection, the real-time fluorescence curves produced by 1 pM, 100 fM and 10 fM target mRNA were recorded by using 3 U, 4 U, 5 U, 6 U Nt.BstNBI Nicking Enzyme. The blank treated the same way except did not contain any target mRNA. The IEXPAR and measurement of fluorescence were carried out according to the procedure described in the experimental section except the amount of Nicking Enzyme. As indicated in Fig. 4, the amount of Nt.BstNBI nicking enzyme only exhibited slight effect on our mRNA assay. However, when the amount of nicking enzyme was 4 U, the interval of POI between the target mRNA with different concentrations and blank reach maximum (Fig. 4E). Thus we used 4 U Nt.BstNBI Nicking Enzyme for mRNA detection in this work. 3.4. Effect of the concentration of template II for mRNA detection To evaluate the effect of the concentration of template II in IEXPAR for mRNA detection, the IEXPAR were carried out according to the procedure in the experimental section except the concentration of template II. 1 pM, 100 fM, 10 fM target mRNA and blank were simultaneously detected by using 10 nM, 50 nM, 100 nM, and 200 nM template II, respectively. As exhibited in Fig. 5, the concentrations of template II mainly affected the fluorescence intensity and had ignorable effect on the POI value of the real-time fluorescence curves. We selected 100 nM template II for further mRNA assay because it could produce enough fluorescence intensity. 3.5. Evaluation of the performance of cleavage-mediated IEXPAR for mRNA detection Under the optimized conditions mentioned above, the dynamic range and sensitivity of the proposed mRNA assay based on specific cleavage-mediated IEXPAR were firstly evaluated by using the synthetic 57 nt p53 mRNA fragment as target with serially diluting experiments. As demonstrated in Fig. 6A, the proposed assay was sensitive enough to detect the p53 mRNA fragment in the range

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Fig. 3. The influence of amount of Vent exo− DNA polymerase. The real-time fluorescence curves were produced by 1 pM, 100 fM, 10 fM p53 mRNA and the blank (without target mRNA) with IEXPAR. The amount of Vent exo− DNA polymerase was 0.02 U (A), 0.04 U (B), 0.08 U (C), and 0.2 U (D).

from 10 fM (100 zmol) to 10 nM (100 fmol). At the same time, the proposed assay shown an excellent linear relationship between the POI values were linearly dependent on the logarithm (lg) of the concentration of target mRNA in that ranges. The correlation

equation was POI = −33.2 − 7.0lgCmRNA (M) and the corresponding correlation coefficient was R2 = 0.998. The results indicated that as low as 10 fM can be accurately determined and the linear range spanned over at least six orders of magnitude. Furthermore, two

Fig. 4. The influence of the amount of Nt.BstNBI Nicking Enzyme. The real-time fluorescence curves were produced by 1 pM, 100 fM and 10 fM target mRNA and the blank (without target mRNA) with IEXPAR. The amount of Nt.BstNBI Nicking Enzyme was (A) 3 U, (B) 4 U, (C) 5 U, and (D) 6 U. Error bars were estimated from the standard deviations of three repetitive tests.

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Fig. 5. The effect of the concentration of template II for mRNA detection. The concentration of template II was 10 nM (A), 50 nM (B), 100 nM (C), and 200 nM (D), respectively.

random mRNA fragments (BRCA1 mRNA and beta-actin mRNA) were selected as challenges to investigate to the specificity of the proposed cleavage-mediated IEXPAR method for mRNA assay. The 1 pM p53 mRNA, BRCA1 mRNA, and beta-actin mRNA and blank were simultaneously detected according to the protocol in the experimental section. As shown in Fig. 6C, the fluorescence curves produced by 1 pM BRCA 1 mRNA and beta-action mRNA were same as that of blank, which suggested that the proposed assay exhibited high specificity for target mRNA detection. 3.6. Determination of p53 mRNA in total RNA sample In order to investigate the practicality of the cleavage-mediated IEXPAR-based mRNA assay, we applied the proposed method to detect p53 mRNA in the total RNA sample extracted from MCF-

7 breast cancer cells. Results were shown in Table S2. With the calibration curve, the amount of the p53 mRNA in the total RNA sample (5 ng) was estimated to be 0.46 amol. To verify whether the actual p53 mRNA in total RNA sample have the same response as the synthesized p53 mRNA fragment in our method, we added 1.20 amol synthesized p53 mRNA fragment to 5 ng total RNA sample. According to the calibration curve, the average amount of all p53 mRNA target (synthesized fragment and mRNA) from three measurements was 1.77 amol, and the average recovery was estimated to be 109.2% (n = 7). Moreover, the p53 mRNA amount in the same batch of total RNA sample was also detected by using RT-PCR method, as can be seen in Table S2, the p53 mRNA content in 5 ng total RNA is calculated to be 0.51 amol and the average recovery was 105.8%, which agrees well with the result obtained by our cleavage-mediated IEXPAR method. Therefore, this cleavage-

Fig. 6. Evaluation of the performance of cleavage-mediated IEXPAR for mRNA detection. (A): Real-time fluorescence curves produced by p53 mRNA with different concentration. The concentration of p53 mRNA was 10 nM, 1 nM, 100 pM, 10 pM, 1 pM, 100 fM, 10 fM, and blank, respectively. (B): The linear relationship of the POI and the logarithm (lg) of the p53 mRNA concentrations. Error bars were estimated from the standard deviations of three repetitive tests. (C): Evaluation of the specificity of the cleavage-mediated IEXPAR for mRNA assay.

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mediated IEXPAR-based mRNA assay is a practical and reliable tool for the quantitative detection of mRNA in the complex biological sample at amol level. 4. Conclusion In summary, we have established a highly specific, sensitive, and reverse transcription-free approach for the quantitative detection of mRNA based on cleavage-mediated IEXPAR. The DNA probe recognized the mRNA target in the digestion reaction. Meanwhile, the template I in IEXPAR reaction recognized the mRNA trigger in the amplification reaction. These recognition steps with three specifically recognized sequences made the proposed method to have high specificity for mRNA detection. The IEXPAR reaction was monitored in real time without any labeled probes under isothermal condition that achieved high sensitivity. As low as 100 zmol target mRNA was accurately determined and the dynamic range covered six orders of magnitude. Moreover, the results indicated that the proposed method can be well applied to accurate detection mRNA in small amounts of total RNA sample. The proposed mRNA assay presented here is suitable for any RNA targets (such as long noncoding RNA, lnRNA), as long as the RNA targets can form a loop structure through hybridization with flexibly designed DNA probe, making the RNA assay a versatile method. Furthermore, the comparison between the proposed method and previously reported methods are listed in Table S3 which suggested that the proposed mRNA assay will provide an alternative avenue for simple, rapid and sensitive detection of mRNAs. 5. Acknowledgment We thank the financially supporting from National Natural Science Foundation of China (21335005 and 21472120), Program for Changjiang Scholars and Innovative Research Team in University (IRT 15R43), and the Excellent Doctor Innovation Project of Shaanxi Normal University (X2013YB05). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2017.06.008. References [1] F. Crick, Central dogma of molecular biology, Nature 227 (1970) 561–563. [2] Z. Wang, M. Gerstein, M. Snyder, RNA-Seq: a revolutionary tool for transcriptomics, Nat. Rev. Genet. 10 (2009) 57–63. [3] F. OZsolak, P.M. Milos, RNA sequencing: advances, challenges and opportunities, Nat. Rev. Genet. 12 (2010) 87–98. [4] M. Mandal, R.R. Breaker, Gene regulation by riboswitches, Nat. Rev. Mol. Cell Biol. 5 (2004) 451–463. [5] H. Tanabe, A. Yagihashi, N. Tsuji, Y. Shijubo, S. Abe, N. Watanabe, Expression of survivin mRNA and livin mRNA in non-small-cell lung cancer, Lung Cancer 46 (2004) 299–304. [6] S. Morishita, H. Tani, S. Kurata, K. Nakamura, S. Tsuneda, Y. Sekiguchi, et al., Real-time reverse transcription loop-mediated isothermal amplification for rapid and simple quantification of WT1 mRNA, Clin. Biochem. 42 (2009) 515–520. [7] D.A. Relman, The search for unrecognized pathogens, Science 284 (1999) 1308–1310. [8] Y.Y. Zhao, L. Zhou, Z. Tang, Cleavage-based signal amplification of RNA, Nat. Commun. 4 (2013) 1493. [9] A. Mader, U. Riehle, T. Brandstetter, E. Stickeler, A. zur Hausen, J. Ruhe, Microarray-based amplification and detection of RNA by nucleic acid sequence based amplification, Anal. Bioanal. Chem. 397 (2010) 3533–3541. [10] K.A. Curtis, D.L. Rudolph, S.M. Owen, Rapid detection of HIV-1 by reverse-transcription, loop-mediated isothermal amplification (RT-LAMP), J. Virol. Methods 151 (2008) 264–270.

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Biographies Hui Wang is a Ph.D. candidate in School of Chemistry and Chemical Engineering, Shaanxi Normal University. His current research is focused on the detection of nucleic acids and single cell analysis. Honghong Wang is a Ph.D. candidate in School of Chemistry and Chemical Engineering, Shaanxi Normal University. Her current research is focused on the development of isothermal amplification methods for DNA and RNA analysis. Xinrui Duan is a professor in School of Chemistry and Chemical Engineering, Shaanxi Normal University. His current research is focused on bioanalytical chemistry and cytobiology. Xiangdong Wang is a Ph.D. candidate in School of Chemistry and Chemical Engineering, Shaanxi Normal University. His current research is focused on the quantitative detection of plant miRNAs. Yuanyuan Sun is a Ph.D. candidate in School of Chemistry and Chemical Engineering, Shaanxi Normal University. Her current research is focused on the determination of SNPs. Zhengping Li is a professor in School of Chemistry and Chemical Engineering, Shaanxi Normal University. His current research mainly focused on biochemical and molecular diagnosis.