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May 11, 2016 - T7 RNA polymerase-based rolling circle transcription (RCT) is a more powerful tool than universal runoff transcription and traditional ...
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Effect of the Concentration Difference between Magnesium Ions and Total Ribonucleotide Triphosphates in Governing the Specificity of T7 RNA Polymerase-Based Rolling Circle Transcription for Quantitative Detection Zhiyan Li, Choiwan Lau, and Jianzhong Lu* School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China S Supporting Information *

ABSTRACT: T7 RNA polymerase-based rolling circle transcription (RCT) is a more powerful tool than universal runoff transcription and traditional DNA polymerase-based rolling circle amplification (RCA). However, RCT is rarely employed in quantitative detection due to its poor specificity for small single-stranded DNA (ssDNA), which can be transcribed efficiently by T7 RNA polymerase even without a promoter. Herein we show that the concentration difference between Mg2+ and total ribonucleotide triphosphates (rNTPs) radically governs the specificity of T7 RNA polymerase. Only when the total rNTP concentration is 9 mM greater than the Mg2+ concentration can T7 RNA polymerase transcribe ssDNA specifically and efficiently. This knowledge improves our traditional understanding of T7 RNA polymerase and makes convenient application of RCT in quantitative detection possible. Subsequently, an RCT-based label-free chemiluminescence method for microRNA detection was designed to test the capability of this sensing platform. Using this simple method, microRNA as low as 20 amol could be quantitatively detected. The results reveal that the developed sensing platform holds great potential for further applications in the quantitative detection of a variety of targets.

T

RCT, T7 RNA polymerase initiates transcription at the +1 position at 3′ end of the T7 promoter, and then newly synthesizes RNA around the circular template. When encountering the 5′ end of the detection target (serves as a guide DNA, bridges 5′ end and 3′ termini of template DNA), T7 RNA polymerase releases the target sequence by strand displacement (Scheme 1). And then, the released target can anneal to new template and work repeatedly, whereas the target

he polymerase T7 RNA, a DNA-dependent RNA polymerase, can catalyze the synthesis of RNA at an average rate of 200−260nt/s. Once transcription has started, T7 RNA polymerase will synthesize RNA continuously until it encounters a termination signal or runoff the end of the template with a very long product (>10 kb).1−4 At the same time, the T7 promoter can initiate new transcription units continuously. Ascribed to these advantages, T7 RNA polymerase employed transcription has been widely applied in the analysis field. “Runoff transcription”, an in vitro transcription process using a linear double-stranded DNA (dsDNA) as the template, is a universal approach and has been used to develop a series of ultrasensitive diagnostic methods.5−7 However, a complicated dsDNA template preparing and labeling process is often required before its application, thereby limiting its practical use. As a supplementary in vitro transcription process, rolling circle transcription (RCT), using small circular single-stranded DNA (ssDNA) as the template, is more convenient and powerful than runoff transcription since these drawbacks of runoff transcription are circumvented. RCT is a type of rolling circle amplification (RCA). RCA, an efficient and versatile isothermal enzymatic amplification technique, has been widely applied in biomedical research especially in ultrasensitive DNA and RNA detection in areas of genomics and diagnostics.8−11 In © XXXX American Chemical Society

Scheme 1. Assay Strategy

Received: April 14, 2016 Accepted: May 11, 2016

A

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RCA reaction mixture that consisted of 2 pmol of the T7 promoter, 10 U of T7 RNA polymerase, 1× T7 RNA polymerase reaction buffer [40 mM Tris−HCl, 2 mM spermidine, and 10 mM DTT (pH 7.9), 25 μg/μL BSA], and described final concentrations of Mg2+ and rNTPs. The reactions were terminated by incubation at 95 °C for 10 min. (B) Without ligation: 10 μL of ligation reaction mixture and 10 μL of RCT reaction mixture were mixed directly and incubated at 37 °C for 3 h. The reactions were terminated by incubation at 95 °C for 10 min. Accumulation of the SA Aptamers onto SA-MB. In a typical experiment, 4 μL of the SA-MB was washed three times with 100 μL of BA buffer [20 mM Tris−HCl, 500 mM NaCl, 20 mM MgCl2 (pH 8.0)] and then diluted in 80 μL of BA buffer containing 0.4% BSA. Then 20 μL of reaction products above was added and incubated with gentle shaking for 1 h at 37 °C. Label-Free CL Measurement. Before detection, SA-MB conjugates were washed three times with wash buffer [7 mM Tris−HCl, 170 mM NaCl, 0.05% Tween 20 (pH 8.0)] and then transferred into 14 × 40 mm glass tubes with 90 μL of TMPG (30 mM in DMF). CL detection was carried out with a BPCL CL analyzer. An amount of 9 μL of tetrabutylammonium hydroxide phosphate buffer (pH 8.5) was added into the tube, and the tube was placed in the luminescence reader. The CL signal was then integrated for 10 s. Preparation of Cellular Extracts. Human lung cancer cells (A549) were cultured according to the instructions of the American Type Culture Collection. Cells were grown in DMEM (Gibco, penicillin 100 U/mL, streptomycin 100 μg/ mL) plus 10% fetal bovine serum (FBS, Gibco) and maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. The cells were collected and centrifuged at 3000 rpm for 5 min in a culture medium, washed once with PBS buffer, and then spun down at 3000 rpm for 5 min. The cell pellets were suspended in 600 μL of lysis solution. Total RNA was extracted using the miRNeasy mini kit (QIAGEN) according to the manufacturer’s procedures. The total RNA concentration was determined to be 3.2 μg/μL from the UV absorption at 260 nm with the following formula: concentration of RNA sample = (40 μg/mL)(A260)(dilution factor). The sample of let 7a in these cells was diluted and then analyzed with the proposed microRNA assays.

will irreversibly become one part of the product and thus only be used once in a typical RCA process. RCT has been widely used in the synthesis of microRNA and in the RNA nanotechnology field.12−14 However, it is rarely employed in the area of quantitative detection, due to the poor specificity of T7 RNA polymerase for small ssDNA. Small ssDNA templates can be efficiently transcribed by T7 RNA polymerase even without a promoter,1,14,15 indicating that the RCT reaction will happen without any controllable trigger once the template DNA is in the transcription environment. Thus, improving the specificity of T7 RNA polymerase is predicted to yield significant improvement of RCT. Herein we show that the concentration difference between Mg2+ and total ribonucleotide triphosphates (rNTPs) radically governs the specificity of T7 RNA polymerase. When the total rNTP concentration is 9 mM greater than the Mg 2+ concentration, T7 RNA polymerase can transcribe ssDNA specifically and efficiently. This knowledge improves our traditional understanding of T7 RNA polymerase and makes convenient application of RCT in quantitative detection possible. Thus, an RCT-based label-free chemiluminescence (CL) method for microRNA let 7a detection was designed to test the capability of this sensing platform. Using this simple method, let 7a as low as 20 amol could be quantitatively detected. What is more, it can be easily extended to a variety of targets including nucleic acids (DNA, microRNA, and mRNA), small molecules, proteins, and cells. This indicates that the developed sensing platform holds great potential for further applications in the quantitative detection.



EXPERIMENTAL SECTION Chemicals and Materials. All chemicals were analytical grade and used as received. All of the solutions were prepared with ultrapure water (Millipore purification system, 18.2 MΩ cm), treated with DEPC, and sterilized with high-pressure steam. All oligonucleotides were HPLC-purified and synthesized by Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. (Shanghai, China). Deoxynucleotides (dNTPs), microRNA, and T4 DNA ligase were obtained from TaKaRa Biotechnology Co. Ltd. (Dalian, China). T7 RNA polymerase (50 U/μL) and rNTPs (100 mM) were obtained from both New England Biolabs (Ipswich, MA) and Fermentas (U.S.A.). Repli phi29 DNA polymerase (100 U/μL) was obtained from Epicenter (Madison, WI). SYBR green II dye were purchased from Invitrogen. Streptavidin-coated magnetic beads (SA-MB) were purchased from Polysciences (Warrington, PA). 3,4,5-Trimethoxylphenylglyoxal (TMPG) was synthesized as described previously.16 Bovine serum albumin (BSA) was bought from TaKaRa Biotechnology Co. Ltd. (Dalian, China). Other reagents were bought from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Ligation Reaction. The ligation reaction was carried out with a 10 μL of reaction mixture containing 1× ligation buffer [66 mM Tris−HCl, 6 mM MgCl2, 10 mM dithiothreitol (DTT), 0.1 mM ATP (pH 7.6)], 20 U of T4 DNA ligase, 2 pmol of the padlock A, and different concentrations of let 7a. Before adding T4 DNA ligase, the oligonucleotide mixture was denatured at 95 °C for 1.5 min and cooled slowly to room temperature over a 30 min period. After annealing, T4 DNA ligase was added to the mixture and incubated at 37 °C for 2 h. RCT Reaction. (A) With a ligation step: RCT reaction was conducted at 37 °C for 3 h in a 20 μL reaction mixture containing 10 μL of ligation reaction products and 10 μL of



RESULTS AND DISCUSSION Principle of Quantitative Detection Assay. The designed circular DNA template (padlock A, Table S1) has three functional regions (T, SA, and P). Region T (red), located at the 5′- and 3′-end of the template, was designed to be complementary to the microRNA let 7a target. Region SA (green) was complementary to the SA RNA aptamer.17 Region P (black) was the T7 RNA polymerase promoter site for initiating transcription. The T7 promoter initiates transcription at its 3′ end; however, the products cannot elongate due to the truncation of the template in the absence of let 7a. Upon hybridization of region T with let 7a, the padlock probe can be ligated specifically and circularized in the presence of T4 DNA ligase. The T7 promoter subsequently initiates an isothermal rolling circle transcription in the presence of T7 RNA polymerase, releasing let 7a and resulting in a product composed of many repeated copies of the circle. The released let 7a can now hybridize to a new padlock probe and initiate multiple reaction cycles. The transcription products containing B

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Analytical Chemistry multiple SA RNA aptamers are then accumulated by SA-MB for the generation of a CL signal, with the employment of an instantaneous derivatization reaction between TMPG and G bases on the SA aptamer backbone. SA aptamers were accumulated and employed as both a specific separation medium and signaling molecules, whereas other G bases from free padlock probes, targets, and those remaining rNTP substrates did not interfere with let 7a detection since they can be simply washed away through magnetic separation before detection. Therefore, a series of complicated labeling and releasing processes are avoided and the matrix effects are minimized. Specificity of T7 RNA Polymerase in the Universal Reaction Buffer. T7 RNA polymerase has been wellinvestigated over the past 2 decades. Prior studies and most T7 RNA polymerase suppliers (such as NEB, Fermentas, Invitrogen, TAKARA, etc.) suggest that, to obtain higher yields of RNA, the Mg2+ concentration should be raised to 4 mM above the total rNTP concentration, and a universally adopted reaction buffer contains 6 mM Mg2+ and 2 mM total rNTPs.18−20 According to the above suggestions, we first established a simple test under the universal reaction conditions. Briefly, 2 pmol of template DNA (padlock A) as described in Scheme 1 was ligated for 1 h by DNA ligase in the presence of 5 fmol or 0 fmol of let 7a. The template was then transcribed in the presence of 2 pmol or 0 pmol of T7 promoter ssDNA in the universal reaction buffer. After a 3 h incubation at 37 °C, the amount of products was quantified by 1× SYBR II dye and fluorescence spectrophotometry rather than chemiluminescence (CL) approaches mentioned in Scheme 1. Figure 1A shows that no obvious difference was observed between the sample (5 fmol let 7a) and blank (no let 7a) groups. Both of them showed very high fluorescence intensity,

indicating the formation of large amounts of RNA products. Similar results were also seen in both the presence and absence of T7 promoter ssDNA, which is complementary to part of the circular template and upon hybridizing forms a complete double-stranded promoter. Polyacrylamide gel electrophoresis was employed to investigate the products. As shown in Figure 1B, the length of the main products was the same as that of the template ssDNA, suggesting that T7 RNA polymerase transcribed the whole template DNA, even when the promoter was located on the middle of the template. This result is consistent with the recognized understanding that T7 RNA polymerase can transcribe small ssDNA nonspecifically and efficiently, and RCT cannot be directly applied for quantitative detection, as is the case with RCA. It is imperative to improve the specificity of T7 RNA polymerase for quantitative detection, making ssDNA transcription under the absolute control of the T7 promoter possible. Specificity of T7 RNA Polymerase in Our Reaction Buffer. To obtain good specificity of T7 RNA polymerase, a series of experimental parameters were tested. We hypothesized that, to obtain higher yields of RNA, early researchers focused their attention on the activity of the T7 RNA polymerase, such that current T7 RNA polymerase reaction conditions are optimal for high activity. Consequently, the activity of T7 RNA polymerase under these conditions is so high that promoterless small ssDNA can be transcribed efficiently. To regulate the activity of T7 RNA polymerase, various Mg2+ concentrations were tested and the total rNTP concentration was fixed at 20 mM rather than the classical 2 mM to allow more flexibility in regulation. The same experiments described at the beginning were tested with 2 pmol of T7 promoter under these conditions. The data show that T7 RNA polymerase could synthesize RNAs efficiently and specifically when 12 mM Mg2+ and 20 mM rNTPs were used (Figure 2B, lanes 4 and 4B and Figure S1A). Most RNA products were specifically synthesized by T7 RNA polymerase, where only about 12% nonspecific products still remained (lane 4B and lane 5/5B on Figure 2B and Figure S1B). However, as seen in Figure 2B, almost all of the products were synthesized nonspecifically when the Mg2+ concentration

Figure 1. (A) Fluorescence intensity of T7 RNA polymerase transcription products. (B) Polyacrylamide gel electrophoresis of products. Each lane of the gel corresponds to the column in panel A above it. Black bands at the top of each lane (red arrow) are the bottom of the loading well and do not correspond to any specific products. Control 1 (lane 5) refers to the product of the reaction, which is identical to that in lane 1 (containing padlock, let 7a, T7 promoter sequence, and other reagents) but without T7 RNA polymerase. Control 2 (lane 6) contains only 2 pmol padlock A loaded for electrophoresis.

Figure 2. (A) Fluorescence intensity of T7 RNA polymerase transcription products with 2 pmol padlock A and 2 pmol T7 promoter sequence under 20 mM total rNTPs and various Mg2+ concentrations. (B) Polyacrylamide gel electrophoresis of the products. Each lane corresponds to the column in panel A above it. Red boxes indicate the specific products of rolling circle transcription. C

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which is consistent with that of prior studies. It may also explain why most T7 RNA polymerase suppliers often claim that, to obtain higher yields of RNA, the Mg2+ concentration should be raised to 4 mM above the total rNTPs concentration, due to the complexion between Mg2+ ions and nucleotides and thus decreasing the concentration of free Mg2+ ions; (2) in our reaction buffer, the concentration of Mg2+ was actually lower than that of total rNTPs, and thus the activity of T7 RNA polymerase is partly suppressed, leading to that it is almost impossible to initiate a promoterless transcription. Only in the presence of a specific promoter, can transcription be initiated and thus a concentration-dependent specificity of T7 RNA polymerase is greatly improved; (3) as Daube and von Hippel suggested, to initiate transcription, T7 RNA polymerase needs bind to the “bubble” structures of the ssDNA, possibly mimicking the open transcription complex formed by the polymerase.1,25,26 Mg2+ ions typically play more than just a backbone charge compensation role and are important in the stabilization of the bubble structures.27 Thus, low Mg2+ ions in our reaction buffer may destabilize the bubble structures, and further contribute to the concentration-dependent specificity of T7 RNA polymerase. Optimization of Reaction Parameters. A series of experiments were established to optimize the assay reaction parameters. We found that the ligation time has little effect on the enhancement of sensing performance (Figure 4). It means

was higher than 16 mM. A small number of specific products (red box of lane 3) emerged when the Mg2+ concentration was reduced to 16 mM. There were no products, specific or nonspecific, synthesized when the Mg2+ concentration was further reduced to 6 mM, indicating that T7 RNA polymerase loses its transcriptional activity under this condition. Additionally, the middle band of lane 4 is much lighter than those of lanes 4B and 5 (Figure S1B), although only 5 fmol of let 7a was added, which is much less than the amount of template (2 pmol). This result suggests that the released target can anneal to new template and work repeatedly. Note also that RCT products are too large to be resolved by 8% polyacrylamide gel (red box of Figure 2B) and therefore remain at the top of the lane, i.e., bottom of the loading well, and RCT products were further confirmed by another 1% agarose gel (Figure S1A). Concentration Difference between Mg2+ and rNTPs versus the Specificity of T7 RNA Polymerase. Various concentrations of rNTPs and Mg2+ were further tested to reveal how their concentrations affect the specificity of T7 RNA polymerase. As shown in Figure 3, the optimal Mg 2+

Figure 3. Performance of T7 RNA polymerase with various concentrations of Mg2+ ions under different total rNTPs (10, 20, 30, and 40 mM). F and F0 represent the fluorescence intensity in the presence (5 fmol) and absence of microRNA let 7a, respectively. Mg2+ concentrations were 6, 8.5, 11, and 13.5 mM when 10 mM rNTPs was used; 8.5, 11, 13.5, 16, and 21 mM when 20 mM rNTPs was used; 11, 16, 18.5, 21, 24.5, 26, and 31 mM when 30 mM rNTPs was used; 21, 26, 28.5, 31, 34.5, 36, and 41 mM when 40 mM rNTPs was used.

Figure 4. CL intensity vs the ligation time. Experimental conditions: 1× T4 buffer, 5 fmol let 7a, 2 pmol padlock A, and 20 U of T4 DNA ligase in 10 μL; after different ligation times in 25 °C, 10 μL of RCT reaction mixture containing 1× T7 buffer, 2 pmol T7 promoter, 20 mM rNTPs (5 mM for each rNTP), 11 mM MgCl2 and 10 U of T7 RNA polymerase were added for transcription. After 3 h of incubation, CL was determined as described in the Experimental Section.

concentration was different, depending on the rNTP concentration. This result suggests that it is the concentration difference between Mg2+ and total rNTPs rather than a single parameter that radically affects the specificity of T7 RNA polymerase. T7 RNA polymerase showed the best performance when the total rNTP concentration was about 9 mM greater than the Mg2+ concentration. In addition, we further confirm our conclusion by using one fixed rNTPs concentration of 20 mM and more precise Mg2+ concentration examination (Figure S2). Under these conditions, both specificity and activity are suitable for applications of RCT in quantitative detection. To the best of our knowledge, these characteristics of T7 RNA polymerase that we have described here have not been previously reported. For such concentration-dependent specificity of T7 RNA polymerase, we speculated the following three reasons: (1) it is well-known that the RNA synthesis activity of T7 RNA polymerase is Mg2+-dependent; however, Mg2+ ions also bind to the phosphate of nucleotide (rNTP or dNTP) due to electrostatic interaction,21−24 and thus it is the relative concentration between Mg2+ and rNTPs rather than single parameter of them affects the behavior of T7 RNA polymerase,

that the ligation and transcription can be conducted synchronously. This is further proof that the released target can anneal to a new template and work repeatedly, since the 1 h ligation step before amplification is often required in traditional RCA, in which the target cannot be innately reused. In all optimization experiments, the default conditions were 2 pmol of padlock probe, 2 pmol of T7 promoter sequence, 20 U of T4 DNA ligase, 10 U of T7 RNA polymerase, 20 mM total rNTPs, and 11 mM Mg2+. After 3 h of incubation in 37 °C, signal intensity was measured as described in Experimental Section in the presence of 5 fmol let 7a. The rest of the optimization results are shown in Figures S3−S7. Selectivity of the Let 7a Assay. We investigated the selectivity of the let 7a assay which is very important due to the high sequence homology among family members and small size of microRNA. Note that members of the let 7 family are too D

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Real Sample Analysis. We demonstrated the performance of our assay by measuring the absolute amounts of let 7a in a cancerous cell line. The total RNA extracted from A549 human lung cancer cells was determined to be 0.17 μg/μL from the UV absorption at 260 nm. It is calculated that the concentration of let 7a in human lung cancer cell is 4.33 ± 0.57 nM in 1 μg/μL of extracted total RNA. This result is consistent with previous observations.35

similar that they differ only by one or two nucleotides in sequence with the same length. In this assay, to initiate the amplification, the target let 7a must coincidently hybridize to the complementary segments at the opposite ends of a linear DNA probe molecule. Due to the strict requirement for coincident hybridization to two target segments, let 7a was recognized with high specificity; single-nucleotide variants of sequences could be distinguished using the padlock probe.28−30 This mechanism is the same as the traditional RCA so that the selectivity of RCT also is similar to traditional RCA. As shown in the Figure 5, CL signals produced by let 7f, let 7g, and let 7i



CONCLUSION In summary, our work has improved the specificity of T7 RNA polymerase such that small ssDNA can be transcribed under the control of the T7 promoter. An RCT-based, highly sensitive and convenient sensing platform was developed that is adaptable to a variety of targets including nucleic acids (DNA, microRNA, and mRNA), small molecules, proteins, and cells. Compared to traditional RCA, RCT is more powerful because it can innately utilize targets circularly without the help of other endonucleases and is more convenient because an additional ligation step is not required. Additionally, it facilitates the detection of mRNA, which is difficult to realize by traditional RCA, in which an exposed 3′ end is required. Free from the complicated steps in template preparation and labeling, RCT is promising as a feasible and robust signalamplification method to replace runoff transcription.

Figure 5. CL intensity vs let 7a/mismatch microRNA. Experimental conditions were the same as in Figure 6, and let 7a, 7b, 7c, 7d, 7e, 7f, 7g, and 7i were 5 fmol, respectively. After 3 h of incubation, CL was determined as described in the Experimental Section.



ASSOCIATED CONTENT

S Supporting Information *

are very low due to the one base mismatch located at the ligation site which significantly affects the ligation efficiency. Meanwhile, a good selectivity was also achieved with other members of the family. Sensitivity of the Let 7a Assay. Under the optimized reaction conditions, the sensitivity and dynamic range of the proposed method was evaluated with different concentrations of let 7a. As shown in Figure 6, a good linear response was

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b01460. Supplementary gel analysis, performance of T7 RNA polymerase with one fixed rNTPs and more precise Mg concentration examination, optimization of reaction parameters, and calibration curve by a similarly designed traditional RCA-based let 7a (PDF)



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 0086-21-51980058. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of China (nos. 21375025, 21575029).

Figure 6. Calibration plots. Experimental conditions: 1× T7 buffer, various concentrations of let 7a, 2 pmol padlock A, padlock/promoter ratio is 1:0.5, 30 mM rNTPs, 21 mM MgCl2, 20 U of T4 DNA ligase, and 10 U of T7 RNA polymerase in 20 μL. After 6 h of incubation, CL was determined as described in the Experimental Section.

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