Click Chemical Ligation-Initiated On-Bead DNA Polymerization for the

Article Cite This: Anal. Chem. 2018, 90, 5390−5397

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Click Chemical Ligation-Initiated On-Bead DNA Polymerization for the Sensitive Flow Cytometric Detection of 3′-Terminal 2′-OMethylated Plant MicroRNA Wenjiao Fan, Yan Qi, Liying Qiu, Pan He, Chenghui Liu,* and Zhengping Li

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Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province; Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710119, Shaanxi Province, People’s Republic of China S Supporting Information *

ABSTRACT: A versatile flow cytometric strategy is developed for the sensitive detection of plant microRNA (miRNA) by coupling the target-templated click nucleic acid ligation (CNAL) with on-bead terminal enzymatic DNA polymerization (TEP). Unlike ligase-catalyzed ligation reaction, the plant miRNA-templated enzyme-free CNAL between two single-stranded DNA (ssDNA) probes, respectively modified with Aza-dibenzocyclooctyne (Aza-DBCO) and N3, can not only simplify the operation, but also achieve a much higher ligation efficiency. More importantly, the undesirable nonspecific ligation between the Aza-DBCO- and N3-modified ssDNA, can be effectively eliminated by adding Tween-20, which allows the use of cycling CNAL (CCNAL) in a background-free manner. So each plant miRNA can template many rounds of CNAL reaction to produce numerous ligation products, forming efficient signal amplification. The ligated ssDNA can be anchored on the magnetic beads (MBs) with the 3′OH termini exposed outside. Then terminal deoxynucleotidyl transferase (TdT), a sequence-independent and template-free polymerase, would specifically catalyze the DNA polymerization along these 3′-OH termini on the MBs, forming poly(T) tails up to thousands of nucleotides long. Each poly(T) tail allows specific binding of numerous 6-carboxyfluorescein (FAM)-labeled poly(A)25 oligonucleotides to accumulate a lot of fluorophores on the MBs, leading to the second step of signal amplification. By integrating the advantages of CCNAL-TEP for highly efficient signal amplification and robust MBs signal readout with powerful flow cytometer, high sensitivity is achieved and the detection limit of plant miRNA has been pushed down to a low level of 5 fM with high specificity to well discriminate even single-base difference between miRNA targets.

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Typically, because of their low levels in biological samples, efficient nucleic acid amplification strategies are generally required for miRNA analysis to achieve high sensitivities. Up to now, various efficient amplification techniques in which miRNAs directly serve as primers, such as loop-mediated isothermal amplification (LAMP),11 rolling circle amplification (RCA), 1 2 − 1 4 isothermal exponential amplification (EXPAR), 15−19 and enzymatic repairing amplification (ERA),20 have been considered as the most powerful tools for miRNA analysis in mammals. However, totally different from the structure of animal miRNAs, plant miRNAs have a naturally occurring methyl group on the ribose of the last nucleotide at the 3′-termini (2′-O-methylated).6 Unfortunately, the methylation state can inhibit the ability of plant miRNAs to prime the polymerization reactions probably because of the steric hindrance of 2′-O-methyl group, which is next to the 3′-

icroRNAs (miRNAs) are small, nonprotein coding RNAs, which have been extensively identified in not only animals, but also plants.1−3 Numerous studies have indicated that miRNAs in mammals are closely involved in various important biological processes associated with cell growth and organ development.4 Therefore, miRNAs may become a family of promising biomarkers for both biological studies and disease diagnosis.5 It should be noted that compared with the comprehensive researches on animal miRNAs, the study on the biofunctions of plant miRNAs, which have been predicted to regulate a variety of processes in plants,2,3 is still in its infancy. More strikingly, it is recently found that exogenous plant miRNAs from food may be also present in the sera and organs of mammals, and they are also possible to regulate the target gene expression in animals including human beings.5−7 Therefore, accurate and highly sensitive plant miRNA detection is the prerequisite foundation to unveil their exact biological functions and their influence on human health.8−10 © 2018 American Chemical Society

Received: February 4, 2018 Accepted: March 30, 2018 Published: March 30, 2018 5390

DOI: 10.1021/acs.analchem.8b00589 Anal. Chem. 2018, 90, 5390−5397

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Analytical Chemistry

first-step signal amplification with a high signal-to-background ratio. Second, only the ligated ssDNA can expose 3′-OH termini outside the MBs, which can be specifically recognized by TdT to initiate 3′-OH terminal polymerization, producing long poly(T) tails up to thousands of nucleotides long on the surface of the MBs.38−41 Each poly(T) tail can combine with many 6-carboxyfluorescein (FAM)-labeled short poly(A) oligonucleotides to accumulate a lot of fluorophores on the MBs, leading to the second step of signal amplification. Third, by flow cytometric analysis, the optical information on each fluorophore-enriched MBs can be directly interrogated without any separation or fluorophore elution procedures, which provides a powerful and easy-to-use analytical tool for the fluorescence signal readout of MBs. Hence, by combining the high signal amplification efficiency of CCNAL-TEP with the powerful beads analyzing capability of flow cytometry, the detection limit of plant miRNA has been pushed down to a low level of 5 fM.

OH.21,22 Therefore, these efficient nucleic acid amplification methods by using target miRNAs as the primers are no longer feasible for plant miRNA analysis. In this regard, miRNA-templated rather than miRNA-primed signal amplification approaches may be good alternatives for detecting plant miRNAs. Nevertheless, the miRNAs are so short that it is difficult for them to be amplified directly as template by traditional PCR. So generally, additional miRNAtemplated reverse transcription (RT) reactions by using stemloop or locked nucleic acid (LNA) probes are required to fabricate the RT-PCR miRNA assays,23,24 where skillful/ stringent sequence design and complicated optimization are highly needed and are very challenging.25 Fascinatingly, although the short sequence of miRNAs are not suitable to act as a PCR template, we and others have proved that they are capable of acting as template to ligate two target-specific singlestranded DNA (ssDNA),4,26,27 and the ligated products can be easily amplified by PCR without requirement of the RT process. Although effective, such ligation-based assays all rely heavily on the catalysis of either DNA ligases or RNA ligases, which demand stringent buffer media and temperature28,29 to guarantee the enzyme activity. Typically, DNA ligases are mainly used to ligate DNA sequences by using DNA template, while RNA ligases mainly exhibit high specificity in ligating two RNA probes with RNA template. However, miRNA-templated ligation of two DNA probes is needed for miRNA assays, so the ligation efficiency is greatly limited either by DNA ligases or RNA ligases, which has become the main limitation for the ligation PCR-based miRNA assays. Recently, the enzyme-free and copper-free click nucleic acid ligation (CNAL) reaction have become a powerful tool for the templated ligation of two ssDNA modified respectively with Aza-dibenzocyclooctyne (Aza-DBCO) and azide (N3).30−34 The CNAL can not only lower the cost, but more fascinatingly, can achieve a more than 92% ligation efficiency35 irrespective of DNA or RNA templates. So compared with the ligase-mediated ligation, the CNAL provides a much better choice for the miRNA-templated ligation reaction. However, the click chemical ligation between the Aza-DBCO and N3 may generate a significantly more bulky junction site, which may not be easily read through by DNA polymerases.32,36 As such, the chemical ligation products may not be well amplified by traditional PCR or other polymerase-catalyzed nucleic acid amplification strategies. Inspired by the strikingly high ligation efficiency and simple operation of the miRNA-templated CNAL, we believe that the development of new strategies allowing for the specific and efficient amplification of the target-responsive CNAL products may open a new way for plant miRNA analysis. Herein, we have developed a versatile flow cytometric strategy for detecting plant miRNAs based on a two-step cascading signal amplification strategy including the targettemplated cycling CNAL (CCNAL) and the subsequent terminal deoxynucleotidyl transferase (TdT)-catalyzed terminal enzymatic DNA polymerization (TEP) on the surface of magnetic beads (MBs). This CCNAL-TEP assay shares several distinct advantages. First, for the first time we find that the undesirable nontemplated ligation between the Aza-DBCOand the N3-labeled probes, which may become significant in traditional solution reactions,31,37 can be completely suppressed in this work. Therefore, each plant miRNA target can template many rounds of enzyme-free CNAL reaction to generate numerous ligation products with thermal cycling, forming the

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EXPERIMENTAL SECTION Reagents and Materials. Dynabeads M-270 Streptavidin (STV-MBs, diameter of 2.8 μm) were acquired from Thermo Fisher Scientific (Invitrogen, Oslo, Norway). All of the oligonucleotides used in this study were synthesized by Takara Biotechnology (Dalian, China). The detailed sequences are listed in Table S1. Tween-20 was obtained from Sangon Biotech (Shanghai, China). Terminal deoxynucleotidyl transferase (TdT) and 5× TdT buffer (125 mM Tris-HCl, 1 M potassium cacodylate, 5 mM CoCl2, 0.05% (v/v) Triton X-100, pH 7.2) were supplied by Thermo Fisher Scientific. Standard Procedures of the Proposed CCNAL-TEP Assay for the Detection of Plant miRNA. The CCNAL reaction was conducted in 10 mM phosphate buffer (PBS, containing 0.3 M NaCl, 0.01% Tween-20, pH 7.4). Typically, in a total of 10 μL of reaction buffer, 40 nM Probe A (3′-AzaDBCO/5′-biotin), 40 nM Probe B (5′-N3/3′-OH), and a certain amount of ath-miR156a plant miRNA target were mixed together and then treated with the following thermal cycling conditions (denaturation at 70 °C for 30 s and hybridization at 25 °C for 150 s, 50 cycles) to perform the CCNAL assay. Afterward, ∼1.2 × 10 5 M-270 STV-MBs, which can theoretically capture ∼0.4 pmol (40 nM in a 10 μL solution) biotinylated probe, were added in and incubated with the CCNAL reaction mixture at room temperature for 30 min under shaking. After that, both the ligation products and the unreacted Probe A will be immobilized on the MBs through the STV−biotin interaction. After a thorough magnetic purification, the ligation productsanchored MBs were further dispersed in 10 μL of 1× TdT buffer containing 2 units (U) of TdT and 1 mM dTTP. The mixture was incubated at 37 °C for 1 h under gentle shaking to conduct the TdT-catalyzed TEP reaction. Afterward, 1 μM (A)25-FAM ssDNA was introduced and the mixture was further incubated at 4 °C for 1 h. Finally, the reaction mixture was subject to flow cytometry (FCM) analysis after diluted to 500 μL with PBS. The fluorescence signals (FL1 channel) of 10000 MBs for each sample were collected one-by-one on a FACS Calibur Flow Cytometer (BD Biosciences, U.S.A.). Then the target plant miRNA of each sample can be quantitatively analyzed according to the mean fluorescence intensity (MFI) of the detected MBs. Detection of ath-miR156a Level in Arabidopsis thaliana. The Arabidopsis thaliana at the stage of seedling 5391


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binding stability of poly(A) with the poly(T) tail and the high fluorophore loading capability on the MBs, (A)25-FAM is the optimal length in this study. Finally, as the MBs pass one-byone through the flow cytometer, the fluorescence signals of individual MBs can be directly and rapidly analyzed, and the amount of target ath-miR156a can be quantitatively reflected by the mean fluorescence intensity (MFI) of such MBs. It should be worth noting that only the ligation products can bring 3′OH ssDNA onto the surface of MBs, which can further lead to the TdT extension and fluorescence accumulation. On the contrary, if the ath-miR156a is absent, only Probe A exposing a 3′-Aza-DBCO group can be anchored on the MBs, which cannot be recognized and extended by TdT. This has been well supported by the polyacrylamide gel electrophoresis (PAGE) results (Figure 2). One can see from Figure 2 that with the

were acquired from College of Life Science in Shaanxi Normal University. A total of 80 mg of Arabidopsis thaliana was frozen and ground into powder with liquid nitrogen. After that, 1 mL of Trizol was added, and the mixture was kept milling until the liquid nitrogen was evaporated completely. Then total RNA was extracted following the Trizol extraction protocols. Finally, the amount of the extracted total RNA was quantified on the NANODROP 2000 equipment. The total RNA extracted from Arabidopsis thaliana was subject to the standard CCNAL-TEP procedures stated above to quantitatively evaluate the level of ath-miR156a.

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RESULTS AND DISCUSSION Principle of the CCNAL-TEP Flow Cytometric Assay for Plant miRNA Analysis. The principle of the proposed CCNAL-TEP strategy is illustrated in Figure 1. In this work,

Figure 1. Schematic illustration of the CCNAL-TEP flow cytometric assay for plant miRNA analysis.

ath-miR156a, which regulates the timing of the youth-to-adult transition in Arabidopsis thaliana,42 is selected as a proof-ofconcept target. Accordingly, Probe A (5′-biotin/3′-Aza-DBCO) and Probe B (5′-N3/3′-OH) are, respectively, designed to be complementary to the half sequence of ath-miR156a and the Aza-DBCO, and N3 should be labeled on the neighboring nucleotides after Probes A and B have hybridized with the miRNA target. So in the presence of ath-miR156a, the targettemplated formation of sandwich-type hybrid will lead to efficient click chemical ligation between the Aza-DBCO and N3 groups because Probes A and B are brought into close proximity. With the help of thermal cycling, the ath-miR156a target will be released from the ligated ssDNA and become the template again for next round of CNAL reaction between other nonligated Probes A and B to generate a large number of ligated ssDNA, resulting in the first step of signal amplification. Afterward, the ligation products bearing both 5′-biotin and 3′-OH can be anchored on the STV-MBs. In this regard, numerous 3′-OH terminal ssDNA, which can be specifically recognized by TdT, will be introduced on the MBs as a reflection of the target ath-miR156a level. Then the templatefree polymerase TdT, would catalyze the repetitive incorporation of dTTPs at each 3′-OH terminal to form long poly(T) ssDNA tails up to thousands of nucleotides long.38 Each poly(T) tail can capture a lot of poly(A)25-FAM ssDNA to accumulate numerous fluorophores on the surface of MBs, leading to another step of signal amplification. It is worth noting that the length of poly(A) probe should not be too short or too long (Figure S1 in SI). In consideration of both high

Figure 2. Polyacrylamide gel electrophoresis (PAGE) analysis of TdTcatalyzed terminal polymerization of Probes A and B: lane 1, doublestranded DNA ladder; lane 2, pure Probe B (400 nM); lane 3, pure Probe A (400 nM); lane 4, Probe A (400 nM) + 2 U TdT + 5 mM dTTP; lane 5, Probe B (400 nM) + 2 U TdT + 5 mM dTTP.

catalysis of TdT, only Probe B with 3′-OH terminus can produce a long poly(T) tail with the band located at the very top of the gel, while Probe A with 3′-Aza-DBCO terminus cannot be extended by TdT. Investigation of the Critical Effect of Tween-20 To Suppress Nonspecific Chemical Ligation. In conventional homogeneous solutions, the nonspecific ligation between N3and Aza-DBCO-labeled DNA probes without template may occur inevitably.31 This may become more serious in this study because their random collision will be accelerated during the thermal cycling. As shown in Figure 3a, with 50 thermal cycles in the CCNAL-TEP miRNA assay, the nonspecific signal of blank control (in the absence of plant miRNA) is so significant that 50 pM ath-miR156a-generated fluorescence response cannot be distinguished. As a contrast, the fluorescence signal aroused by pure Probe A-labeled MBs (in the absence of Probe B) is very low. These results clearly suggest that the significant blank signal is originated from nonspecific ligation between Probe A and Probe B, even in the absence of ath-miR156a, 5392


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concentration in the CCNAL-TEP were studied and optimized by using ath-miR156a as the model target. The effect of thermal cycle number on the proposed CCNAL-TEP assay was first investigated, and the results were shown in Figures S2 and 4. One can see that the

Figure 4. Effect of the thermal cycle number on the proposed CCNAL-TEP strategy for ath-miR156a analysis. 50 pM of athmiR156a was used for this optimization in comparison with blank control without adding ath-miR156a. Other conditions: TdT, 2 U; dTTP, 1 mM. FL1 voltage for the FCM measurement, 330 V.

Figure 3. Effect of Tween-20 on the suppression of nonspecific ligation in the proposed CCNAL-TEP ath-miR156a assay. 50 pM of ath-miR156a was used in this study (green lines) in comparison with blank control (red lines) by using Tween-20 dosage of (a) 0; (b) 0.005%; (c) 0.01%; (d) 0.05%; (e) 0.2%. All of the CCNAL-TEP was performed identically according to the standard procedures stated in the Experimental Section, except for the different amounts of Tween20. The pink line in image a (indicated by arrow) represents the fluorescence signal of Probe A-functionalized MBs after the TdTcatalyzed TEP. Other conditions: thermal cycle number, 50; TdT, 2 U; dTTP, 1 mM. FL1 voltage for the FCM measurement, 330 V.

fluorescence signal produced by 50 pM ath-miR156a increases gradually as the number of thermal cycles increases from 0 to 100. Meanwhile, the fluorescence signals of the blank control without ath-miR156a almost keep constant when the cycle number is in the range of 0−50. However, when the cycle number reaches 60 or higher, the blank signal also increases obviously (Figure S2), which may be unfavorable for the detection of low concentrations of target miRNA. What is more, if 100 cycles are used, the cycling click reaction time will need more than 6 h, which may be too long for practical applications. In consideration of both appropriate assaying time and high sensitivity, 50 cycles are selected as the optimum for the CCNAL reaction in this work. The influence of the amount of TdT enzyme was further investigated by varying the TdT dosage from 0.1 U to 20 U after the CCNAL reaction. As shown in Figure 5a, the athmiR156a-produced fluorescence signals of the MBs increase sharply with the increase of TdT dosage from 0.1 U to 20 U. Meanwhile, the fluorescence signals of the blank (without athmiR156a) also display an increasing tendency with the increase of TdT. As displayed in Figure 5b, the highest value of S/B ratio is obtained when the amount of TdT enzyme is 2 U. Taking into consideration both high target-induced signal and relative low background to achieve better discrimination between the target and the blank, 2 U of TdT is selected in this study for plant miRNA analysis. Analytical Performance of the CCNAL-TEP Flow Cytometric Assay for ath-miR156a Analysis. To investigate the analytical performance of the proposed method, series dilutions of synthetic ath-miR156a with varying concentrations were detected by the proposed assay under the optimized conditions. It can be seen from the fluorescence histograms (Figure 6a) that the fluorescence signal of the CCNAL-TEP system rises gradually with the ath-miR156a concentration increasing from 10 fM to 200 pM. The ath-

which may greatly compromise the sensitivity of the CCNALTEP assay. Fortunately, for the first time, we find that the nonspecific ligation can be almost completely eliminated by adding Tween20 in the ligation reaction. As displayed in Figure 3b−e, even in the presence of a small quantity (0.005%, v/v) of Tween-20 in the CCNAL reaction, the signal of blank control can be sharply reduced, while the 50 pM target-produced signal still remains high. What is more, the response of the blank control in the presence of Tween-20 may keep nearly identical to that produced by Probe A-labeled MBs, indicating that the nonspecific ligation is suppressed almost totally with the introduction of Tween-20. We suppose that the random collision rate of Probe A and Probe B may be significantly decreased due to their restricted diffusion with the help of Tween-20. Although the exact mechanism as to why Tween-20 can suppress the nonspecific chemical ligation is still unclear and needs further investigation, the effectively reduced background by Tween-20 may eventually lead to high signalto-noise ratio and high detection sensitivity. So 0.01% Tween20 is used for the CCNAL-TEP throughput of this study. This important finding may be also quite useful for the fabrication of CNAL-based biosensing systems and biomaterials.37,43,44 Optimization of Thermal Cycle Number and TdT Concentration in the CCNAL-TEP Assay. In order to achieve the best assay performance, the experimental conditions, such as the number of thermal cycles and TdT 5393


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precision of the proposed method, the relative standard deviation (RSD) values of each data point are calculated from three parallel measurements, which are all less than 5%. It is worth noting that previously, Zhao group have proposed some pioneering methods to detect plant miRNAs by integrating sophisticatedly designed three-way junction structures (3WJ) with nicking endonuclease and polymerase-assisted synergetic amplification mechanisms, where the detection limits of ath-miR156a typically fall within the range of 1−5 pM.8,9 So with much simpler probe design and assaying conditions, the detection limit of our CCNAL-TEP assay is about 2 orders of magnitude lower than that of such previous plant miRNA assays. The sensitivity of the CCNAL-TEP assay is also slightly higer than that of the 3WJ-EXPAR method with an exponential amplification mechanism.22 Furthermore, to prove that the high sensitivity of CCNALTEP for ath-miR156a analysis is indeed because of the combination of cascading CCNAL and TEP signal amplification, three further verification experiments (VE1∼VE3) were conducted (Figure 7). For the VE1 illustrated in Figure 7a, the ath-miR156atemplated CNAL was performed between Probe A and Probe C (the same sequence as Probe B but with 5′-N3/3′-FAM modifications, see Table S1) without thermal cycles or TdTcatalyzed extension. As such, each of the target miRNA can only capture one Probe C with one FAM on the MBs. As can be seen from the FCM results in Figure 7a, the lowest athmiR156a concentration that can be distinguished from the blank control is 1 nM in VE1, indicating that the integration of CCNAL with TEP has led to a 105-fold signal amplification. In VE2, the ath-miR156a-templated CNAL was performed between Probe A and Probe C with 50 thermal cycles but without TdT-catalyzed signal amplification. Under this condition, the detection limit of ath-miR156a is approximately 10 pM (Figure 7b), which is 3 orders of magnitude higher than the CCNAL-TEP approach. For the VE3, the TdT-catalyzed signal amplification was performed directly after the athmiR156a-templated isothermal CNAL between Probe A and Probe B without thermal cycling. In such conditions, the detection limit of ath-miR156a is approximately 1 pM (Figure 7c). From the results shown in Figure 7, it can be clearly concluded that the ligation cycling and the TEP can, respectively, contribute to ∼102 and ∼103 enhancement of the detection sensitivity. So the coupling of CCNAL with TEP is critical to obtain the excellent analytical performance for athmiR156 analysis. Specificity Evaluation of the CCNAL-TEP Approach for ath-miR156a Analysis. To investigate the specificity of the proposed miRNA assay, different miRNA sequences were randomly selected and detected by the CCNAL-TEP system with Probes A and B specific to ath-miR156a. As shown in Figure 8a, only the fluorescence signal aroused by ath-miR156a can be distinctly distinguished from the blank control. While for other noncomplementary miRNA sequences, all of their fluorescence responses are almost the same as the blank control. Furthermore, we have also investigated whether the proposed CCNAL-TEP assay can discriminate similar miRNA sequences which are only 1−3 nucleotides different from athmiR156a. It can be seen from Figure 8b that all the responses of synthetic miR156a-1 (1 nucleotide difference), miR156a-2 (2 nucleotides difference), and miR156a-3 (3 nucleotides difference) can be clearly discriminated from that of ath-miR156a. The interference for detection of ath-miR156a arisen from the

Figure 5. (a) Effect of TdT dosage on the proposed CCNAL-TEP strategy for ath-miR156a analysis. 50 pM of ath-miR156a was used for this optimization (green lines) in comparison with blank control (red lines) in the presence of different amount of TdT: (I) 0.1 U; (II) 0.5 U; (III) 2 U; (IV) 5 U; (V) 10 U; (VI) 20 U. (b) Corresponding S/B ratios under different dosage of TdT. S/B ratio refers to the ratio of the 50 pM ath-miR156a-produced MFI value to that of blank control. Other conditions: cycle number, 50 cycles; dTTP, 1 mM; FL1 voltage for the FCM measurement, 440 V.

Figure 6. (a) Fluorescence histograms of the CCNAL-TEP system in the presence of different concentrations of ath-miR156a. Ath-miR156a from left to the right: 0 (blank), 10 fM, 50 fM, 100 fM, 500 fM, 1 pM, 5 pM, 10 pM, 20 pM, 50 pM, 100 pM, and 200 pM, respectively; (b) the relationship between the MFI values and ath-miR156a concentrations. The inset shows the plot between the MFI values and ath-miR156a concentrations in the range of 0−1 pM. The error bar represents standard deviation of three replicates for each data point. FL1 voltage for the FCM measurement, 330 V.

miR156a level can be quantitatively reflected by the MFI value of each sample. As can be seen from the plot in Figure 6b, the MFI values rises linearly with the increasing concentrations of ath-miR156a. The linear regression equation is MFI = 1.25 Cath‑miR156a (pM) + 3.36 with the correlation coefficient R2 = 0.9953. Accordingly, the detection limit of ath-miR156 is estimated to be ∼5 fM (3σ, n = 11). Furthermore, to test the 5394


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Figure 7. (a) Schematic illustration (top panel) and the corresponding fluorescence histograms (bottom panel) of the MBs in the presence of different concentrations of ath-miR156a under the condition of the target-templated 1:1 ligation mechanism between Probe A and Probe C (5′-N3/ 3′-FAM) without either thermal cycling or TdT-catalyzed DNA extension; (b) Schematic illustration and the corresponding fluorescence histograms of the MBs with different concentrations of ath-miR156a by target-templated click chemical ligation between Probe A and Probe C (5′-N3/3′-FAM) with thermal cycling but without TdT-catalyzed signal amplification; (c) Schematic illustration and the corresponding fluorescence histograms of the MBs treated with different concentrations of ath-miR156a by coupling the target-templated 1:1 ligation between Probe A and Probe B (no thermal cycling) with TdT-catalyzed signal amplification.

Probes A and B need to be changed according to the let-7a sequence, while other reagents and reaction procedures are all the same as those for ath-miR156a analysis. As can be seen from Figure S4, if Tween-20 is absent, the nonspecific signal of blank control is so high that the 50 pM let-7a-aroused fluorescence response cannot be distinguished from the blank control. As a contrast, when Tween-20 is introduced, the nonspecific signal of blank control can be sharply reduced while the 50 pM target-produced signal still remains high enough. Such results clearly suggest that the fascinating effect of Tween20 on suppressing nonspecific chemical ligation is general and not sequence-dependent. Figure 9 displays the results for let-7a analysis. As low as 10 fM let-7a can be unequivocally distinguished from the blank, and the target dosage-responsive increase of fluorescence signals can be clearly identified with let-7a concentration increasing from 10 fM to 200 pM, which is in good agreement with the detection of ath-miR156a. Therefore, since the TdT-catalyzed TEP is template-free and sequence-independent, the CCNAL-TEP strategy should have good generality and specificity for the detection of different miRNA targets just by simply changing the two target-specific ssDNA probes.

signals produced by the same concentration of miR156a-1, miR156a-2, and miR156a-3 are calculated to be 10.2%, 6.4%, and 1.4%, respectively. All of these results suggest that the specificity of the CCNAL-TEP flow cytometric assay is satisfactory to well discriminate quite similar miRNAs even with single base difference. Detection of ath-miR156a in Real Complex Samples. Furthermore, the ath-miR156a level in total RNA extraction of Arabidopsis thaliana is assessed by the sensitive and specific CCNAL-TEP strategy. According to a calibration curve constructed by synthetic ath-miR156a standard, the amount of ath-miR156a in 50 ng total RNA sample is calculated to be 4.5 pM (in a 10 μL reaction). To prove the reliability of this method, 10 pM synthetic ath-miR156a was spiked into 50 ng total RNA, and 14.8 pM ath-miR156a is determined in the spiked sample with the recovery of 102.6%. Meanwhile, the same batch of total Arabidopsis thaliana RNA sample was also analyzed by a modified stem-loop RT-PCR method (see details in Supporting Information).8,45 By using the RT-PCR assay, the amount of ath-miR156a in 50 ng total RNA is determined to be 5.6 pM, which is in good consistence with the result of our CCNAL-TEP strategy. These results suggest that the CCNALTEP strategy is practicable to detect plant miRNAs in real biosamples. Generality of the CCNAL-TEP miRNA Assay. Actually, according to the work principle illustrated in Figure 1, the CCNAL-TEP strategy is not only suitable for the detection of plant miRNA, but also generally applicable for animal miRNA analysis. Human let-7a is thus selected as another miRNA target to test the generality of the CCNAL-TEP assay. Only

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CONCLUSIONS In summary, a versatile CCNAL-TEP flow cytometric assay, which is very suitable for detecting plant miRNAs with known sequences, is developed by integrating the cascading signal amplification of cycling click chemical ligation and TdTcatalyzed template-free on-bead DNA polymerization. We find that by adding Tween-20 in the CCNAL reaction, the 5395


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Figure 9. (a) Fluorescence histograms of the CCNAL-TEP system in the presence of different concentrations of let-7a. Let-7a concentration from left to the right: 0 (blank), 10 fM, 50 fM, 100 fM, 500 fM, 1 pM, 5 pM, 10 pM, 20 pM, 50 pM, 100 pM, and 200 pM, respectively; (b) the relationship between the MFI values and let-7a concentrations. The inset shows the plot between the MFI values and let-7a concentrations in the range of 0−1 pM. The error bar represents standard deviation of three replicates for each data point. FL1 voltage for the FCM measurement, 330 V.

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ASSOCIATED CONTENT

S Supporting Information *

Figure 8. (a) Specificity evaluation of the proposed miRNA assay by using ath-miR156a-specific probes. The fluorescence response of the ath-miR156a is normalized to be 1. The inset displays the fluorescence histograms aroused by different miRNA sequences; (b) Specificity evaluation of the proposed assay toward different synthetic miRNA sequences with 1−3 nucleotides difference to ath-miR156a. The inset displays the corresponding fluorescence histograms. The concentrations of all of these miRNAs are 50 pM. FL1 voltage for the FCM measurement, 330 V.

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b00589. The sequences of oligonucleotides, effect of the length of poly(A)-FAM probe, the effect of the thermal cycle number on the proposed CCNAL-TEP assay, the quantification of ath-miR156a in total Arabidopsis thaliana RNA by using stem-loop RT-PCR method, generality of Tween-20 on suppressing the nonspecific chemical ligation, and the discussion about the bimodal phenomena of the fluorescence histograms (PDF).

nontemplated ligation between N3- and Aza-DBCO-labeled probes is completely restricted so that the enzyme-free CCNAL can achieve a high signal-to-noise ratio with quite simple operation. Furthermore, the TdT-based on-bead TEP amplification is template-free and sequence-independent, which may greatly simplify the probe design. Benefiting from the highly efficient two-step amplification and FCM analysis for powerful beads signal readout, a low detection limit of 5 fM target plant miRNA is obtained by using the proposed CCNAL-TEP strategy. This method can also discriminate quite similar miRNA with high specificity and can be used to determine miRNA target in real samples reliably. Actually, besides for plant miRNA analysis, this robust method can be generally extended to the detection of all miRNA species without considering whether the miRNA is 2′-O-methylated or not. Considering the high sensitivity, specificity and universal applicability for different miRNAs, we believe this method may have great potential for the applications in miRNA-related biological studies and disease diagnostics.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone/Fax: +86 29 81530859. ORCID

Chenghui Liu: 0000-0001-8375-0242 Zhengping Li: 0000-0002-7573-8822 Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21622507, 21575086, 21335005), the 5396


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Natural Science Basic Research Plan of Shaanxi Province (2015KJXX-22), the Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R43), the Program for Innovative Research Team in Shaanxi Province (No. 2014KCT-28), and the Fundamental Research Funds for the Central Universities (GK201802016).

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