Isothermal Amplification on a Structure-Switchable Symmetric Toehold

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Isothermal Amplification on a Structure-Switchable Symmetric Toehold Dumbbell-Template: A Strategy Enabling MicroRNA Analysis at the Single-Cell Level with Ultrahigh Specificity and Accuracy Jun Chen, Taixue An, Yingjun Ma, Bo Situ, Danping Chen, Yuzhi Xu, Li Zhang, Zong Dai, and Xiaoyong Zou Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03713 • Publication Date (Web): 28 Nov 2017 Downloaded from http://pubs.acs.org on December 3, 2017

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Analytical Chemistry

Jun Chen,† Taixue An,‡ Yingjun Ma,† Bo Situ,‡,§ Danping Chen,† Yuzhi Xu,† Li Zhang,† Zong Dai*,†,§and Xiaoyong Zou†,§ †

School of Chemistry, Sun Yat–Sen University, Guangzhou 510275, PR China

‡

Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China

§

Guangdong Engineering and Technology Research Center for Rapid Diagnostic Biosensors, Guangzhou 510515, China

Supporting Information Placeholder ABSTRACT: Accurate analysis of microRNAs (miRNAs) at the single-cell level seriously requires analytical methods possessing extremely high sensitivity, specificity and precision. By rational engineering of a structure-switchable symmetric toehold dumbbell-template (STDtemplate), we propose a novel isothermal Symmetric EXPonential Amplification Reaction (SEXPAR) method. The sealed and symmetric structure of the STD-template allows exponential amplification reaction (EXPAR) to occur upon every annealing of target miRNA without loss of amplification efficiency. Besides, the rigid and compact structure of the STD-template with an appropriate standard free energy ensures SEXPAR only be activated by target miRNA. As a result, the SEXPAR method isothermally quantified let-7a down to 0.01 zmol (6.02 copies per 10 microliter) with an ultrahigh specificity which is efficient enough to discriminate one-base-mismatched miRNAs, and a remarkably high precision even for the determination of 6.02 copies let-7a (the standard deviation was reduced from >60% down to 23%). The dynamic range was also extended to 10 orders of magnitude. The method was successfully applied for the determination of let-7a in human tissues, sera and even single-cell lysate, with obviously better precision than quantitative reverse transcription polymerase chain reaction (RT-qPCR) and other EXPAR-based methods. The SEXPAR method may serve as a powerful technique for the biological research and biomedical studies of miRNAs and other short nucleic acids.

Accurate analysis of microRNAs (miRNAs) at the single-cell level is extremely important in deep understanding of the function of miRNAs as well as the progression of miRNA-related diseases.1–5 However, it still remains big challenges because of the harsh requirements for analytical methods not only to have an ultrasensitivity to determine few to thousands copies of miRNAs, but also to have a significantly high specificity to resist the more serious interference from concomitant nucleic acids, and an extremely low detection deviation to avoid mingling of results. Homogeneous amplification assays, like polymerase chain reaction (PCR)- or isothermal amplification-based strategies, may possibly provide sufficient sensitivity,6,7 while the specificity and precision usually have to compromise, because multiplexed primers and amplification steps are usually essential for achieving high amplification efficiency, and the nonspecific amplification is inevitable.8,9 Overcoming the compromise among sensitivity, specificity and precision is the key issue in accurate analysis of miRNAs at the single-cell level. Isothermal EXPonential Amplification Reaction (EXPAR), as one of the most important amplification strategies for nucleic acids, is a promising method due to its simple amplification format and relatively high amplification efficiency. Once a miRNAas primer an© XXXX American Chemical Society

neals with the 3' terminus of the template, an extension-nicking reaction repeats insistently, forming a circuiting-feedback cycle and achieving high amplification. The amplification efficiency and specificity of EXPAR greatly rely on the annealing ratio (φ) and specificity of the primer with the 3' terminus of the template. The standard EXPAR using a linear symmetric template is likely to lose huge amplification efficiency because almost half amount of the primer will anneal with the 5' terminus of the template (φ ≈ 0.5), which is inextensible.10–12 The amplification specificity ensured by the WatsonCrick base pair is also insensitive in differentiating single-base variation. We recently proposed an asymmetric EXPAR that partly solved these issues.10 On a toehold/biotin featured template, more primers are pushed to anneal with the3' terminus of the template (0.5 < φ < 1.0), resulting in great improvements in sensitivity and specificity. Although the method may still be imperfect in response to trace miRNAs, it has motivated us that through rational design of the template, sensitivity, specificity and precision can be simultaneously improved without compromise. Here, we propose a novel structure-switchable Symmetric Toehold Dumbbell-template (STD-template) that enables isothermal Symmetric EXPonential Amplification Reaction (SEXPAR) with a theoretical amplification efficiency of 2n (φ = 1.0). For the proof-of-

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concept study, the let-7a miRNA, which is closely related to several tumors and diseases and has a large family with numerous analogues,13 was employed as the target model. A series of STDtemplates were designed and the STD-template was rationally engineered to have an appropriate standard free energy (G), which allowed the STD-template to keep stable in dumbbell-shape while only to be “activated” by target miRNAs for SEXPAR through toehold-mediated strand displacement. An efficient amplification was achieved on the optimized STD-template, and an extremely low amplification bias was obtained from the rigid and compact structure of the STD-template. The feasibility of the SEXPAR method was demonstrated on the determination of the let-7a expressions in complicated samples of tissues, sera and even single-cell lysate, which showed significant improvements in sensitivity, specificity and precision comparing to RT-qPCR and other EXPAR-based methods.

DNAs were purchased from Shanghai Sangon Bio. Eng. Tech.& Services Co., Ltd (Shanghai, China). The precursors of STD-templates were modified with 5’-phosphate group. miRNAs were purchased from TaKaRa Biotech. Co. (Dalian, China) and purified by high-performance liquid chromatography. The sequences of the used oligonucleotides are listed in detail in Table S1. T4 DNA ligase, Exonuclease I, Exonuclease III, Vent (exo-) DNA polymerase, Nt.BstNBI NEase, RNase inhibitor, 10 × T4 DNA ligase reaction buffer, 10 × Vent (exo-) DNA polymerase reaction buffer, and 10 × Nt.BstNBI NEase reaction buffer were purchased from New England Biolabs (Beijing, China). Sybr Green I, diethyl oxydiformate (DEPC), deoxyribonucleotides mixture (dNTPs) were purchased from Shanghai Sangon Bio. Eng. Tech. & Services Co., Ltd. (Shanghai, China). The used solutions and deionized water were all treated with DEPC and autoclaved to protect from RNase degradation. The buffers used in the work are as follows: 10 × T4 DNA ligase reaction buffer (400 mM Tris-HCl, 100 mM MgCl2, 100 mM dithiothreitol, 5 mM ATP, pH 7.8); Nt.BstNBI buffer (25 mM Tris-HCl, pH 7.9, 50 mM NaCl, 5 mM MgCl2, 0.5 mM dithiothreitol; Tris = 2amino-2-hydroxymethylpropane-1,3-diol); ThermoPol buffer (20 mM Tris-HCl, pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100); 5 × TBE buffer (445 mM Tris-Boric Acid; 10 mM EDTA); D-PBS buffer (10 mM sodium phosphate buffer, 0.1 M NaCl, pH 7.4). The activation process of SEXPAR consisted of two reactions, which were decomposed into four separate reactions. The ΔG of each reaction was calculated on the ViennaRNA website14,15 under conditions as follows: temperature: 55 oC, salt concentration: 10 mM Na+, 10 mM Mg2+, STD-template: 150 nM, miRNA: 1 nM. The “dangles” parameter was set to “on both sides of a helix in any case”. The bending energies of STD-templates were omitted as single stranded DNA, because it is very flexible and the ratio of duplex length of miRNA-STD-template is less than 40% of the full length. ΔG22 was calculated approximately as the formation of hybridization of two linear strands, miRNA and linear probe, omitting the bending energies of STD-template. According to the Hess's law, the ΔG of the whole activation process was calculated byΔG11 + ΔG12 + ΔG21 + ΔG22.

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The STD-templates with different toehold length from 9 to 14-nt (termed as STD-template-9 to 14, respectively) were prepared from 5′-phosphorylated STDtemplate precursors by ligation with T4 DNA ligase.16,17 The STDtemplate-0 (toehold = 0) was prepared from circular probe with the aid of ligation template. The ligation reaction was performed in a 100 μL of reaction mixture which composed of 20 μL of STD-template precursor (10 μM), 10 μL of 10 × T4 DNA ligase reaction buffer, 65 μL of DEPC-treated H2O and 5 μL of T4 DNA ligase (200 U μL–1). After incubation at 16 °C for 4 h, the reaction mixture was heated at 65 °C for 15 min to terminate the reaction, followed by the addition of Exonuclease I (20 U μL–1) and Exonuclease III (100 U μL–1) to digest all the non-ligated DNAs. The digestion was terminated by heating the reaction mixture at 85 °C for 30 min. The STD-templates in 0.5 × gel loading buffer (TBE buffer) were characterized by native 15% polyacrylamide gel electrophoresis (PAGE) at 200 V for 30 min. As comparison, the non-ligated and non-digested STD-templates were tested on the prepared gel for electrophoresis. After electrophoresis, the gel was stained with SYBR Gold and visualized via DigiGenius gel system (Syngene, UK ). The concentration and the purity of prepared STD-templates were evaluated from the absorbance at 230, 260 and 280 nm with a NanoDrop-2000c spectrophotometer (NanoDrop Technologies, USA). The STD-templates were stored at –20 °C until use. The melting temperature (Tm) values of STD-templates were measured on a CFX ConnectTM Real-Time System (Bio-Rad, USA) to determine the fluorescence signal changes with the increase in the temperature from 45 to 95°C at a rate of 6 °C min–1. The solution consisted of 0.15 µM STD-template in 10 µL buffer solution same as that used in SEXPAR but without Vent (exo-) DNA polymerase and let-7a miRNA (250 µM dNTPs, 0.15 U μL–1 Nt.BstNBI, 0.8 U μL–1 RNase inhibitor, 0.4 × SYBR Green I, 1 × ThermoPol buffer, and 0.5 × Nt.BstNBI buffer). Two reaction solutions of A and B were prepared on ice separately. The solution A consisted of Nt.BstNBI buffer, STD-template, dNTPs, RNase inhibitor and 1 μL total RNA samples. The solution B was composed of ThermoPol buffer, Nt.BstNBI NEase, Vent (exo-) DNA polymerase and SYBR Green I. After preparation, the solutions A and B were mixed immediately and adjusted with DEPC-treated deionized water to a final volume of 10 μL containing 0.15 μM STD-template, 250 μM dNTPs, 0.15 U ml–1 Nt.BstNBI, 0.1 U ml–1 Vent (exo-) DNA polymerase, 0.8 U ml– 1 RNase inhibitor, 0.4 mg ml–1 SYBR Green I, 1 × ThermoPol buffer, and 0.5 × Nt.BstNBI buffer. The mixture was placed in a CFX ConnectTM Real-Time System (Bio-Rad, USA) and reacted at 55 °C. The real-time ﬂuorescence intensity was monitored using an isothermal protocol at 60 s intervals. Human lung cancer tissues and cervical cancer tissues were obtained from Guangzhou Huayin Medical laboratory Center of Southern Medical University. Fresh tissue was added with 0.8 mL of trizol and vortex vigorously to a homogenous lysate. The lysate was added with 0.2 mL of chloroform and vortex for 15 s, and then incubated at 25 – 30 oC for 2 – 3 min. The resultant mixture was centrifuged at 4 oC for 15 min at 12,000 r min–1 to separate the aqueous and organic phase. The aqueous phase was transferred into a fresh tube, and added with equal volume of isopropyl alcohol. After incubation at 15 – 30 oC for 10 min and centrifugation

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at 4 oC for 10 min at 12,000 r min–1, the resultant aqueous phase was discarded, and the precipitation was washed with 800 μL of 75% alcohol. The resultant precipitation was further centrifuged at 4 oC for 5 min at 7,500 r min–1. Abandon of the alcohol, the obtained total RNA were eluted with DEPC-treated water and kept in refrigerator at –80 oC for further use. The reverse transcription of the obtained total RNA was carried out in a mixture containing 12.5 μL of RNase free doubly-distilled water, 2 μL of 5 × PrimeScriptTM Buffer (for real time), 1 μL of Prime ScriptTM RT Enzyme Mix I, 0.5 μL of let-7a RT Primer (10 pmol μL–1) and 4 μL of the extracted total RNA. The mixture was incubated at 37 oC for 40 min, and then at 85 oC for 2 min. Quantitative real-time fluorescence analysis was performed on a 7300 Real-Time PCR System (Applied Biosystems, USA) based on the protocol of literature.18 Forty cycles of amplification were performed. Each cycle comprised an initial denaturation step at 95 oC for 2 min, a denaturation step at 93 oC for 15 s, and annealing and extension steps at 55 oC for 25 s. The detection system was 25 μL of a mixture containing 18.5 μL of doubly distilled water, 2.5 μL of 10 × PCR buffer (with SYBR Green I and ROX), 0.5 μL of dNTPs, 0.5 μL of TAQ, 2 μL of cDNA, 0.5 μL of forward primer and 0.5 μL of reverse primer (Table S1). The Ct values were converted into absolute let-7a copy numbers using a standard curve obtained from synthetic let-7a miRNA. Human serum samples were supplied by Nanfang Hospital. miRNAs in serum samples were extracted using the miRNeasy RNA isolation kit from Qiagen, according to the recommended procedure with minor modifications. Briefly, 5 mL of Qiazol solution was added to 0.50 mL of serum. The mixture was vortexed briefly and incubated for 10 min at room temperature to dissociate nucleoprotein complexes. After adding 1.0 mL of chloroform, the mixture was vigorously vortexed for 60 s, followed by a centrifugation at 14,000 g for 15 min at 4 oC. Precipitation and purification of RNA in the aqueous phase (upper phase) was performed according to the recommended protocol. The total RNA was stored at –80 °C for further use. The A549 cell lines and BEAS-2B human bronchial epithelial cells were obtained from the Cell Resource Center, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences. The cells were cultured in 5 mL of DMEM Medium (GBICO, Cat. 12100-046) containing 10% (v/v) fetal calf serum (GBICO, Cat. 1600036), 1% NaHCO3, 100 U mL–1 penicillin, 100 μg mL–1 streptomycin and 3 mM L-glutamine at 37 oC in a humidified atmosphere of 5% CO2. The A549 cell lines or BEAS-2B human bronchial epithelial cells were centrifuged at 700 rpm for 5 min to remove the culture medium after trypsinization (0.2% trypsin, 1 mM EDTA, Invitrogen), washed three times with cold D-PBS buffer, and then

re-suspended in cold D-PBS buffer at a concentration of ~100 cells μL–1. The cell suspension was serially diluted to ~50, 10 and 1 cell μL–1 by cold D-PBS buffer. 1 μL of these diluted cell suspensions were transferred to the microslide, respectively. The cells were counted on a Cell CelectorTM (Automated Lab Solutions GmbH's, Germany), and 1, 10, 50 and 100 cells were collected by a pipettor to 0.2 mL DEPC treated PCR tubes containing 1 μL of 0.9% NaCl solution and 10 U RNase inhibitor (TAKARA, China) and then incubated on ice. Approximately 15 individual cells can be picked and placed in individual tube in 30 min. Every 30 min, the single-cell samples were frozen rapidly in liquid nitrogen for more than 2 min. Frozen cells were heat-denatured at 98 C for 3 min to break the cell membranes and were then immersed in liquid nitrogen as quickly as possible. The frozen samples were then treated with 1 U proteinase K and 10 U RNase inhibitor at 53 o C for 1 h to digest the proteins binding on miRNAs. o

For the proof-of-concept study, the let7a miRNA was employed as a target model. The SEXPAR method is based on a novel STD-template. The STD-template is a closed and symmetric circular DNA, consisting of antisense sequences of the target miRNA and endonuclease (ENase) recognition sites. The antisense sequence contains a toehold part and an incumbent toehold part (Figure 1a). The STD-template is rationally engineered to have an appropriate G. When nontarget nucleic acids hybridize with the STD-template, the ΔG of the reaction is positive and the STDtemplate remains stable in the dumbbell-shape, which is "inactive" for SEXPAR. When target miRNA binds to the toehold part, spontaneous branch migration activates the dumbbell-shaped template to a circular form (Figure 1b). After activation, the target miRNA hybrid with the STD-template is then extended from its 3' terminus by DNA polymerase, and the formed dsDNA is nicked by ENase. After releasing from the template, the amplification product as a new primer will initiate another round of SEXPAR, and the regenerated duplex is ready for the next extension-nicking reaction (Figure 1c). Because of the sealed and symmetric structure, extension-nicking reaction can be triggered by the primer hybrid with either side of the STD-template, which suggests that a low concentration of target miRNA even down to few copies can still initiate the SEXPAR without loss of amplification efficiency. In addition, as toehold-mediated strand displacement can remarkably boost the specificity of nucleicacid recognition compared to simple Watson-Crick base pairing, the STD-template is hardly activated by mismatched nucleic acids, resulting in high specificity. Furthermore, the rigid and compact structure of the STD-template efficiently avoids the formation of secondary structure and chimeras, which greatly suppress the amplification bias. The SEXPAR is expected to show ultrahigh sensitivity, specificity and accuracy, even in response to copies of target miRNAs in complex samples like tissues, body fluids and cell lysate.

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Figure 1. (a) Structure of STD-template-11. (b) Activation of STD-template. (c) Scheme of SEXPAR. The reaction involves five steps: hybridization of target miRNA (primer) with STD-template; activation of STD-template from dumbbell-shape to circular-shape; extension of primer from its 3' terminus along template by DNA polymerase; cleavage of the upper strand DNA by NEase; and release of the amplification product from template for the next SEXPAR..

The SEXPAR starts with a toehold-driving process whose kinetics and specificity critically rely on the structure of the STDtemplate. The STD-template should be stable enough in its selfcomplementary state (dumbbell-shape) so that it can sufficiently resist the hybridization of nontargets and flexible enough to be activated by targets through toehold-mediated strand displacement. To precisely design the structure of the STD-template, the ΔG of the activation reaction was evaluated by theoretical calculation. The activation process consists of two reactions: (1) transformation of STD-template from dumbbell-shape to circular-shape, and (2) hybridization of target miRNA with the circular-shaped STD-template. The two reactions were further decomposed into four separate reactions of (1-1), (1-2), (2-1) and (2-2) (Figure 2a). We designed several STD-templates with different toehold lengths from 9 to 14-nt (termed as STD-template-9 to 14, respectively). As revealed, the ΔG1 values (ΔG11 + ΔG12) of these STD-templates are all positive and decrease with the increase in the toehold length (Figure 2b, Table S2), indicating that these STD-templates initially maintain the dumbbell-shape, and the dumbbell structure would be less stable with the increase of the toehold length. Upon the reaction with miRNAs, the STD-template remain inactive when the toehold length is shorter than 11-nt, while some other miRNAs, aside from let-7a, can initiate the SEXPAR when the toehold length is longer than 11-nt. The STD-template-11 has the appropriate structure that is activated only by let-7a (ΔG = –0.79 kJ mol–1) (Figure 2c, Table S3).

Figure 2. (a) Scheme of the activation reaction process. Histogram of ΔG1 (b) and ΔG (c) versus the toehold length of STD-template. The STD-templates reacted with let-7a (red), let-7b to 7g and 7i miRNAs (gray). In order to further verify the results of calculation, the STD-templates-9 to 14 were prepared from 5’-phosphorylated STD-template precursors (Table S1). The prepared STD-templates were tested by native PAGE. Figure S1 shows the characterization of the preparation of STD-template-11. The STD-template-11 precursor initially self-complemented to form dumbbell-shape and showed one bright band (lane 1). After the ligation of the STD-template-11 precursor by T4 DNA ligase,

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the resultant mixture showed two bands (lane 2). The mixture was further digested by Exonuclease I and Exonuclease III, and the resultant product showed only a band (lane 3), whose position is identical to that in lane 1. The results suggest that pure sealed STDtemplate-11 was obtained. Other STD-templates were all characterized by the same method.

Figure 3. (a) Determination of the Tm of STD-templates. (b) POI for SEXPAR on different STD-templates against toehold length. Error bars were obtained from three parallel experiments. (c) Normalized ΔPOI for SEXPAR on different STD-templates. (d) Real-time fluorescence curves on standard-template, biotin-template-2, toehold/biotintemplate-2 and STD-template-11 at 55 oC. The concentrations of miRNAs in (b), (c) and (d) were 10 fmol. Biotin-template-2 is a standard-template labeled with a biotin at the second base from its 5’ terminus. Toehold/biotintemplate-2 is a standardtemplate with a biotin at the second base from its 5’ terminus and a hairpin structure at its 3’ terminus. The effect of toehold length on the stability and reactivity of STD-template was investigated. As shown in Figure 3a, with the increase in the toehold length, the Tm values decreased from 86.05 to 79.74 oC (Table S4), proving the reduction of the stability of the dumbbell structure. On different STD-templates, the SEXPAR in response to 10 fmol let-7a family miRNAs showed clearly different real-time fluorescence curves (Figure S2). The point of inflection values (POI, defined as the time corresponding to the maximum slope of the real-time fluorescence curve) for let-7a to 7g and 7i reduced with the increase in the toehold length. The POI for blank experiments also showed the same trend (Figure 3b). As the dumbbellsharped STD template trends to be less stable and is more likely to be “activated” to circular-sharp with the increase in the toehold length, amplification reaction will be initiated more easier. Even in blank experiments, the collision of dNTPs with template has more chances to initiate amplification reaction. The results further proved the theoretical conclusions. The difference between POIlet-7a and POIlet-7x represents the amplification specificity. When the toehold length was longer than 11-nt, the POI values for let-7d to 7g were close to that of let-7a (Figure 3b). When the toehold length was shorter than 11-nt, all the POI values increased (Figure 3b), indicating that the STD-templates are too stable. To precisely estimate the

effect of toehold length on the specificity of SEXPAR, the ΔPOI values of mismatched miRNAs were normalized by that of let-7a (ΔPOIlet-7x/ΔPOIlet-7a), in which the ΔPOI is the difference between the POI values of miRNAs and that of the blank. It is clear that SEXPAR on the STD-template-11 gave the best specificity (Figure 3c). The employment of the STD-template is supposed to greatly enhance the annealing ratio φ of EXPAR, thus responding faster to target miRNAs. As proven in Figure 3d, in response to 10 fmol let-7a, the EXPAR on the standard-template started at ~23 min, which means that many let-7a were invalid at the beginning. Because more let-7a were pushed to hybridize on the 3’ terminus of the templates, the starting times of the EXPAR on biotin-template-2 or toehold/biotin-template-2 were greatly reduced. The SEXPAR showed a further shortening of starting time, indicating that most of let-7a were valid for initiating the amplification reaction, which is very important for trace analysis. Under the optimal conditions of 150 nM STD-template-11 in the presence of 0.15 U μL–1 Nt.BstNBI and 0.1 U μL–1 Vent (exo-) DNA polymerase (Figures S3 – S5), well-defined real-time fluorescence curves were obtained for let-7a measurement by SEXPAR at 55 °C (Figure 4a). In a logarithmic scale, the POI values decreased linearly with an increase in the amount of let-7a between 0.01 zmol (1 aM, 6.02 copies per 10 μL) and 0.01 nmol (1 nM), with a correlation equation of POI = –18.9 – 2.01lgAlet-7a(mol) (R = 0.997) (Figure 4b). In comparison with other techniques (Table S5), the SEXPAR is the most sensitive method, in consideration that the detection of 0.001 zmol (0.602 copies) let7a is actually unreliable. Besides, the method has a wider dynamic range of 10 orders of magnitude, and the simplest amplification format that only needs one amplification template. In addition, the SEXPAR method also shows ultrahigh specificity to discriminate concomitant miRNAs. As shown in Figure 4c, the real-time fluorescence curve of SEXPAR in response to let-7a was far ahead of those to each individual let-7b to 7g and 7i, which were close to that of the blank control. The interference (I) on the detection of let-7a arising from its family members was evaluated (Figure 4d, Table S6). The standard EXPAR provided interference in the range of 7.8 × 10–5% ~ 13.2%.10 The EXPAR on biotin-template-2 reduced most of the interference; however, the interference from let7e was still significant (9.93%). The EXPAR on toehold/biotin-template-2 suppressed the interference from let-7e to 1.2 × 10–2% and further reduced other interferences.10 SEXPAR showed the highest specificity in that all the interferences were controlled less than 6.5 × 10–4%. Besides, admixture experiments that target miRNA in the presence of mismatched miRNAs were performed. As shown in Figure S6, the POI for individual let-7a is very close to those for the let7a in the presence of 1000-fold let-7b to 7g and 7i. The specificity (S%) was evaluated to be in the range of 8.3 × 10–3 ~ 7.7 × 10–2 (Table S7). The results clearly indicate that the proposed SEXPAR method is efficiently enough to discriminate all mismatched miRNAs. The expected low detection variation was also achieved on SEXPAR. The detection variation usually becomes more serious in response to a low concentration of the target. As shown in Table S8, in response to 0.1 zmol to 10 zmol let-7a, the standard deviations (SDs) of strand EXPAR were 62% ~ 82%, while those of the asymmetric EXPAR were 27% ~57% and 24% ~ 49%, respectively. The SDs from SEXPAR was much lower (13% ~ 18%). Even in response

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to 0.01 zmol let-7a, in comparison with the high SDs from the asymmetric EXPAR (66% and 61%), the SD from SEXPAR was only 23%, which greatly ensures the precision of detection even with trace amount of target.

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Table 1 Levels for Let-7a (×106 copies µL–1) in RNA Samples from Cancer Tissues and Sera Quantified by RT-qPCR and SEXPAR (n = 3). RT-qPCR Samples

NSCLC tissue 1 NSCLC tissue 2 NSCLC tissue 3

2.52 1.22 2.04

SD a % 12.70 10.40 15.13

Cervical cancer tissue 1

1.88


The level of let-7a is known to be related to non-small cell lung cancer (NSCLC) and cervical cancer.20,21 The let-7a expressions in total RNA samples from three NSCLC tissues and two cervical cancer tissues were determined by SEXPAR and standard RT-qPCR method. As shown in Table 1, the results from SEXPAR are well consistent with those from the standard RT-qPCR method, and obviously better precision (SDs = 1.3% ~ 5.6%), in comparison with those of RT-qPCR (SDs = 11 ~ 16%). In comparison with tissues, body fluids like blood, sputum and stool are easily sampled with less destruction. The analysis of the miRNAs in body fluids is more grateful but more challenging due to the relatively low abundance of diseased cells in these samples.22–26 The SEXPAR was also utilized for the analysis of the let-7a expressions in total RNAs extract from three normal human sera and three lung cancer patient sera. As expected, the results from SEXPAR were in good agreements with those from RT-qPCR, with obviously better precision (Table 1). Since many diseases often begin with cellular abnormalities, the investigation of cell-to-cell variations of miRNA levels is highly demanded. After verifying that the environment of cell lysate did not affect the analysis of miRNAs by SEXPAR (Figure S7), we determined the let-7a miRNA in the lysate of 1, 10, 50 and 100 human lung cancer cells (A549). The cells were collected by ALS Automated Lab Solutions GmbH's Cell CelectorTM (Figure S8). As shown in Figure 5a, even in response to the lysate from 1 cell,

a

RE b %

2.88 1.02 1.78

SD a % 1.28 3.35 2.39

15.10

2.02

5.54

7.45

2.34

14.58

2.03

4.98

–13.25


1.25

15.84

1.40

1.30

12.01

Normal serum 1

3.85

9.14

4.14

2.80

7.53

Normal serum 2

4.34

13.02

3.96

2.73

–8.76

Normal serum 3

3.67

14.81

3.15

4.21

–14.17

Lung cancer serum 1

0.37

13.81

0.40

4.07

8.11

Lung cancer serum 2

0.24

18.32

0.28

5.67

16.67

0.43

15.15

0.39

4.15

–9.31

Alet-7a

Lung cancer serum 3

Figure 4. (a) Real-time fluorescence curves of SEXPAR in response to different amounts of let-7a at 55 oC. (b) Plot for the POI of SEXPAR versus logarithmic amount of let-7a. Error bars indicate the standard deviation of three replicate tests. (c) Real-time fluorescence curves of SEXPAR in response to 100 amol of let-7a to 7g and 7i at 55 oC. (d) Histogram of let-7a detection interference raised from let-7b to 7g and 7i.

SEXPAR

Alet-7a

14.28 –16.39 –12.74

b

SD: standard deviation; RE: relative error

the amplification curve was still clearly different from that of the blank control. An excellent linear relationship in the range of 1 to 100 cells was obtained between POI value and logarithm of cell number (R = 0.999) (Figure 5a, inset). In addition, the let-7a levels in 20 individual A549 lungcancer cells and 20 individual BEAS-2B healthy cells were determined. The copies of let-7a in 20 individual A549 lung cancer cells varied from 1183 to 4572, while those in 20 individual BEAS-2B healthy cells varied from 8202 to 24917 (Figure 5b). Notably, the mean value of let-7a from 20 individual A549 lung cancer cells (2992 copies) was much lower than that from 20 individual BEAS-2B healthy cells (14128 copies) (Figure 5b, inset), which is consistent with previous studies.27–29 These results suggest that the proposed method can directly distinguish the expression of let-7a among lung cancer cells and healthy cells with excellent accuracy, holding a great potential for application in clinical diagnosis.

Figure 5. (a) Real-time fluorescence curves for detecting diluted extracts equivalent to 1, 10, 50 and 100 cells. (b) The copies of let-7a in individual A549 cells and BEAS-2B cells. The numbers 1-20 are A549 cells; the numbers 21-40 are BEAS-2B cells. Insets: (a) The linear relationship between the POI values of the corresponding amplification curves and logarithm of the A549 cell numbers. Error bars indicate the standard deviation of three replicate tests. (b) Box chart of the mean content of let-7a from 20 individual A549 lung cancer cells and 20 individual BEAS-2B healthy cells.

Cdx.doi.org/10.1021/acXXXXXXX | Anal. Chem. XXXX, XXX, XXX−XXX ACS Paragon Plus Environment

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By employing the well-engineered STD-template, we have developed a novel SEXPAR that shows clear improvements in sensitivity, specificity, and dynamic range over standard methods and enables the sensitive and reliable analysis of miRNA with better precision than RT-qPCR and other EXPAR-based methods. This method allows for miRNA analysis in complicated samples of tissues, sera and even single-cell lysate. Furthermore, the strategy does not require expensive fluorescence imaging and thermal cycling instruments. We believe that the SEXPAR method may serve as a powerful technique for the fundamental biological research and biomedical studies of miRNAs and other short nucleic acids, and has great potential for application in diseases diagnostics, environmental analysis, food security and other areas.

This material is available free of charge via the Internet at http://pubs.acs.org.”

[email protected] The authors declare no competing financial interests.

This work was supported by the National Natural Science Foundations of China (21375154, 21422510, 21675170 and 21775169), the Scientific Technology Project of Guangdong Province (2016B010108007, 2014A040401022, 2015A030401033, 2017B020221001), and the Scientific Technology Project of Guangzhou City (201604020145).

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Ddx.doi.org/10.1021/acXXXXXXX | Anal. Chem. XXXX, XXX, XXX−XXX ACS Paragon Plus Environment


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For TOC only 84x47mm (96 x 96 DPI)


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Figure 1. (a) Structure of STD-template-11. (b) Activation of STD-template. (c) Scheme of SEXPAR. The reaction involves five steps: hybridization of target miRNA (primer) with STD-template; activation of STDtemplate from dumbbell-shape to circular-shape; extension of primer from its 3' terminus along template by DNA polymerase; cleavage of the upper strand DNA by NEase; and release of the amplification product from template for the next SEXPAR. 250x148mm (300 x 300 DPI)



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Figure 2. (a) Scheme of the activation reaction process. Histogram of ∆G1 (b) and ∆G (c) versus the toehold length of STD-template. The STD-templates reacted with let-7a (red), let-7b to 7g and 7i miRNAs (gray). 237x177mm (300 x 300 DPI)


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Figure 3. (a) Determination of the Tm of STD-templates. (b) POI for SEXPAR on different STD-templates against toehold length. Error bars were obtained from three parallel experiments. (c) Normalized ∆POI for SEXPAR on different STD-templates. (d) Real-time fluorescence curves on standard-template, biotintemplate-2, toehold/biotintemplate-2 and STD-template-11 at 55 oC. The concentrations of miRNAs in (b), (c) and (d) were 10 fmol. Biotin-template-2 is a standard-template labeled with a biotin at the second base from its 5’ terminus. Toehold/biotintemplate-2 is a standard-template with a biotin at the second base from its 5’ terminus and a hairpin structure at its 3’ terminus. 219x179mm (300 x 300 DPI)



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Figure 4. (a) Real-time fluorescence curves of SEXPAR in response to different amounts of let-7a at 55 oC. (b) Plot for the POI of SEXPAR versus logarithmic amount of let-7a. Error bars indicate the standard deviation of three replicate tests. (c) Real-time fluorescence curves of SEXPAR in response to 100 amol of let-7a to 7g and 7i at 55 oC. (d) Histogram of let-7a detection interference raised from let-7b to 7g and 7i. 228x171mm (300 x 300 DPI)


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Figure 5. (a) Real-time fluorescence curves for detecting diluted extracts equivalent to 1, 10, 50 and 100 cells. (b) The copies of let-7a in individual A549 cells and BEAS-2B cells. The numbers 1-20 are A549 cells; the numbers 21-40 are BEAS-2B cells. Insets: (a) The linear relationship between the POI values of the corresponding amplification curves and logarithm of the A549 cell numbers. Error bars indicate the standard deviation of three replicate tests. (b) Box chart of the mean content of let-7a from 20 individual A549 lung cancer cells and 20 individual BEAS-2B healthy cells. 220x90mm (300 x 300 DPI)


Isothermal Amplification on a Structure-Switchable Symmetric Toehold

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