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Self-Replicating Catalyzed Hairpin Assembly for Rapid Signal Amplification Jianyuan Dai, Hongfei He, Zhijuan Duan, Yong Guo, and Dan Xiao Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 26 Oct 2017 Downloaded from http://pubs.acs.org on October 26, 2017

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Self-Replicating Catalyzed Hairpin Assembly for Rapid Signal Amplification Jianyuan Dai,*,†,# Hongfei He,†,# Zhijuan Duan,† Yong Guo,† and Dan Xiao*,†,‡ †

College of Chemistry, Sichuan University, Chengdu 610064, China

‡

College of Chemical Engineering, Sichuan University, Chengdu 610065, China Tel.: +86-28-85408928. E-mail: [email protected] (JD); [email protected] (DX). ABSTRACT: A rapid signal amplification system based on the self-replicating catalyzed hairpin assembly (SRCHA) was reported, in which two hairpins, H1 and H2 were well-designed that two split target/trigger DNA and two split G-quadruplex sequences were respectively integrated into H1 and H2. Target/trigger DNA can be cyclically used in this system to form the duplex DNA assemblies (H1-H2), which will bring the two G-quadruplex fragments into close-enough proximity and induce the formation of intact Gquadruplex as colorimetric signal readout. Similarly, the two split target/trigger DNA sequences will reunite into a DNA sequence which identical to the target/trigger DNA, then the obtained replica also can be cyclically used as a new activator unit to trigger the CHA reaction, leading to the rapidly and significantly enhanced formation of target/trigger DNA replicas with the concomitant generation of a higher signal. This self-replication based autocatalytic signal amplified approach has been successfully used to develop a rapid and visual assay for DNA and small molecule detection.

clinics and for emerging infectious diseases and biological warfare.19,20 However, up to now, only few enzyme-assisted rapid signal amplification detection methods have been reported,21,22 Self-replication is a process in which a system can replicate or make copies of itself, and a large number of replicas are produced and increasing exponentially over time.23,24 We speculated that the introducing of self-replication to signal amplification method will significantly reduce the reaction time and accelerate the signal amplification rate, thus a new way for the enzyme-free and rapid signal amplification detection can be realized. In the present study, a CHA signal amplified system with the capacity to self-replicate (SRCHA) was developed. Taking advantage of the self-replication and high amplification efficiency of SRCHA, we developed a rapid colorimetric DNA and small molecule assay.

The exponential amplification of nucleic acids plays an important role in genetics therapy, medical diagnostics and forensic investigations.1,2 Polymerase chain reaction (PCR) is the most widely used amplification technology for amplifying and detecting trace amounts of nucleic acids.3,4 However, PCR requires high precision thermal cycling and special DNA polymerases to achieve the successive rounds of amplification. Hence, it have limitations with respect to time-consuming, sometimes nonspecific, high costs, and dedicated instruments. Alternatively, several isothermal amplification techniques, such as rolling circle amplification,5,6 loop mediated isothermal amplification,7,8 strand displacement amplification9,10 and nucleases-assisted target recycling amplification,11,12 have been developed for bioanalytical applications. In contrast to PCR, these techniques can be performed at a constant reaction temperature, and also can provide high amplification efficiency comparable to that of PCR. However, some special experimental procedures such as the ligation of a padlock probe, the use of specific DNA polymerases and nucleases increase the experimental complexity and cost. Recently, enzyme-free target recycling amplification methods, such as hybridization chain reaction (HCR)13,14 and catalyzed hairpin assembly (CHA),15−18 have emerged as promising alternatives in which only a hybridization process is involved in the amplification reaction instead of a recycling cleavage reaction, and an efficient signal amplification can be obtained at a constant temperature without need of any protein enzyme. However, these target recycling amplification methods are limited by relatively long reaction time, thereby leading to non-specific background leakage and degrades the signal-to-noise ratio. The development of a rapid and simple detection method is still highly required in resource-limited settings, including in small

EXPERIMENTAL SECTION Materialsand Reagents. Hemin, ABTS2−, Trishydroxymethylaminomethane hydrochloride (Tris-HCl), magnesium chloride, sodium hydroxide, sodium chloride, potassium chloride, sodium citrate, adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP) and guanosinetriphosphate (GTP) were obtained from Sigma (St. Louis, MO). A hemin stock solution was prepared in DMSO and stored in the dark at −20 °C. Oligonucleotides were synthesized and purifiedby Sangon Biotech. Co., Ltd. (Shanghai, China), and their sequences were listed in Table S1. All other chemicals were used without additional purification. The water (≥ 18.2 M Ohm cm) was purified by the Millipore filtration system and used in all of the experimental processes.

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Instrumentation. Concentration of DNA was measured by Qubit ® 2.0 Fluorometer (Thermo Fisher Scientific Inc.). UVvis absorption spectra were recordedby U-2900 spectrophotometer (Hitachi Co. Ltd., Japan). Photographs were obtained by Sony DSC-WX150 digital camera. Native PAGE Analysis. 10 µL of different reaction products with loading buffer were loaded into the lanes of polyacrylamidegel (10%, Acr : Bis = 29 : 1). The gel was run at 100 V constant voltage for 60 min in TBE buffer (1×) at room temperature. The gel was stained by Stains-All (SigmaAldrich) for 18 h to image the position of DNA. Finally, the polyacrylamide gel electrophoresisimage was obtained under visible light by a digital camera. DNA assay. H1 and H2 were each prepared in Tris-HCl buffer solution (50 mM, 5 mM MgCl2, 50 mM KCl, pH 7.6), annealed at 95 °C for 5 min and then slowly cooled to room temperature for 3 h. For the DNA assay, 500 nM H1 and 500 nM H2 were mixed with different concentrations of target DNA in 100 µL buffer solution. Subsequently, 2.5 µL of 50 µM hemin, 7.5 µL of 50 mM ABTS2−, and 3 µL of 50 mM H2O2 were added. The resulting solutions were incubated at room temperature for 10 min, and then followed by the nakedeye observation and UV/Vis measurement. ATP assay. ATP aptamer was annealed at 95 °C for 5 min in Tris-HCl buffer solution (20 mM, 5 mM MgCl2, 300 mM NaCl, pH 7.6). H1 and H2 were each annealed at 95 °C for 5 min in Tris-HCl buffer solution (50 mM, 5 mM MgCl2, 50 mM KCl, pH 7.6). All oligonucleotides slowly cooled to room temperature for 3 h before use. For the ATP assay, different concentrations of ATP were incubated with ATP aptamer (100 nM) in 20 µL Tris-HCl buffer solution (20 mM, 5 mM MgCl2, 300 mM NaCl, pH 7.6) at 37 °C for 10 min. Then 40 µL of the H1 and H2 (1.25 µM, respectively) in Tris-HCl buffer solution (50 mM, 5 mM MgCl2, 50 mM KCl, pH 7.6) were added into the mixed solution and then further incubated for 5 min at 37 °C. Subsequently, 2.5 µL of 50 µM hemin, 7.5 µL of 50 mM ABTS2−, and 3 µL of 50 mM H2O2 were added. The resulting solution was then incubated for 10 min at room temperature, and then followed by the naked-eye observation and UV/Vis measurement.

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Scheme 1 Principle for the rapid signal amplification based on the self-replicating catalyzed hairpin assembly (SRCHA) and its application for DNA and small molecule colorimetric detection.

(step 3), which can be used as a new catalyst to trigger additional CHA reaction, finally H1-H2 complexes are obtained (recycling I), and bring the two G-quadruplex fragments into close-enough proximity to form the intact G-quadruplex structures, which subsequently interact with hemin to form a hemin/G-quadruplex horseradish peroxidase (HRP)-mimicking DNAzyme and catalyzes the H2O2-mediated oxidation of 2,2’azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS2−) to the colored ABTS•−, thus providing the colorimetric signal readout and can be easily recognized by the naked eye.29 Meanwhile, once the H1-H2 complex is formed, the two split target/trigger DNA sequences also will come into closeenough proximity and a DNA sequence which identical to target/trigger DNA, namely target/trigger DNA replica will be formed, which can open H1 to form the H1-H2-H1 complex, and triggers the second recycling (step 4 to step 6) to form more H1-H2 complexes. The circularly using of target/trigger DNA and its replicas in CHA reaction leads to the rapidly and significantly enhanced formation of numerous target/trigger DNA replicas with the concomitant generation of a higher signal. Therefore, this SRCHA system could result in a rapid signal amplification detection system to the target molecules. To realize our design, we first examined the SRCHA system for the detection of DNA (their sequences are listed in Table S1 in supporting information). A non-self-replicating CHA (non-SRCHA) system was designed as control, in which two hairpin probes, H1C and H2C have similar sequences to H1 and H2 and only the two split target/trigger DNA sequences were replaced by thymine-rich sequences. The absorbance changes of SRCHA system and non-SRCHA system were

RESULTS AND DISCUSSION Scheme 1 exhibits a schematic representation of this new assumption for rapid signal amplification and its application for DNA and small molecule colorimetric detection. In this self-replicating catalyzed hairpin assembly (SRCHA) system, a pair of DNA hairpins (H1 and H2) were well-designed and can partially hybridize to each other, the two split target/trigger DNA and two split G-quadruplex sequences were respectively integrated into H1 and H2. The spontaneous hybridization between these two hairpins were kinetically blocked since the complementary sequences were placed in the stems.25−28 In the presence of target or trigger DNA (trigger DNA and aptamer sequences were respectively integrated into H3, and trigger DNA can be released once H3 is opened by the target through the formation of the target-aptamer complex), the hairpin structure of H1 is opened (step 1) and the newly exposed sticky end of H1 will hybridize with the stickyend of H2 to form the short-life intermediate Target-H1-H2 (step 2), then the target/triggerDNA will be released by H2

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Figure 2. (A) Photograph of the colorimetric detection of different concentrations of target DNA. Concentration of H1 and H2: 500 nM. (B) Absorption spectra of the detection system in the presence of target DNA (from bottom to top: 0, 10 pM, 50 pM, 100 pM, 200 pM, 300 pM, 500 pM, 1 nM, 5 nM, 10 nM). (C) The relationship between the intensity change of absorbance and target DNA concentrations. Inset shows the absorbance response to the target DNA from 10 pM to 500 pM.

the formation of target DNA replicas, which lead to the rapid and continuous generation of more H1-H2 complexes at the same reaction time. This SRCHA system was also examined by gel electrophoresis. Consistent with the absorbance and visual assay, there is more H1-H2 products obtained from the SRCHA system relative to the non-SRCHA system (Figure 1B). To further achieve the highest sensing performance, several experimental conditions, such as reaction time, concentration of hairpins and Mg2+ were optimized (Figure S1). In accordance with the results, these optimum conditions were used in the subsequential experiments. The corresponding photograph of the target-induced color change was demonstrated in Figure 2A, the solution color gradually turned to green as target DNA concentration increases, and a visible color change could be distinguished when the target DNA concentration as low as 50 pM. The UV-vis data showed in Figure 2B depicts that the absorbance at 420 nm gradually increased with the increase of the target DNA concentration. Accordingly, we use the absorbance intensity at 420 nm to quantitatively scale the target concentration. From Figure 2C, a linear relationship between the absorbance and the target concentration from 10 pM to 500 pM was obtained, and the detection limit was determined to be 5 pM (S/N=3), which is comparable to the previously reported signal amplified DNA assays. However, the reaction time needed, 10 min, was much shorter than previous assays, especially compared to the other CHA-based signal amplification assays (Table S2). To investigate the selectivity of the proposed DNA detection system, four different 500 pM target DNAs, deleted, inserted, mismatched and perfectly matched target DNA were examined. As shown in Figure S2, only a significant absorbance increase was observed for perfectly matched target DNA, indicating that the rapid signal amplification was triggered. This result can be confirmed by the difference of the solution color in the presence of different target DNAs (Figure S2).

Figure 1. (A) Time-dependent absorbance changes of SRCHA system (a) and non-SRCHA CHA system (b) in the presence of 10 nM target DNA, with corresponding backgrounds (c and d), respectively. The inset photographs show the colorimetric changes, the images a–d correspond to curves a–d, respectively. (B) The gel electrophoresis image for the SRCHA system and nonSRCHA CHA. Lane 1: H1 only; Lane 2: H2 only; Lane 3: H1C+H2C; Lane 4: H1+H2; Lane 5: H1C+H2C+target; Lane 6: H1+H2+target; Lane 7: DNA ladder. Concentration of H1, H2, H1C and H2C: 500 nM; target concentration: 10 nM. All measurements were performed in Tris-HCl buffer solution (50 mM, 5 mM MgCl2, 50 mM KCl, pH 7.6).

measured before and after the addition of 10 nM target DNA (Figure 1A). In the ideal case, there should be no increase in absorbance intensity in the absence of target DNA, however, slight increases of absorbance intensity were observed in both SRCHA system and non-SRCHA system, this can be attributed to the uncatalyzed reaction between H1 and H2 because of the “breathing” of hairpin helix.16 After the addition of target DNA, the absorbance intensity of the SRCHA system increased much faster than the non-SRCHA system and only took around 10 min to reach a plateau, however, non-SRCHA system didn’t reach a plateau even after 1 h (data not shown). The ratio of the absolute absorbance intensity between SRCHA system and non-SRCHA system ((ASRCHA−A0SRCHA)/(Anon-SRCHA−A0non-SRCHA), A0SRCHA and ASRCHA are the absorbance intensities of the SRCHA system detected in the absence and presence of target DNA, respectively. A0nonSRCHA and Anon-SRCHA are the absorbance intensities of the nonSRCHA system detected in the absence and presence of target DNA, respectively) at 10 min is around 3.0. Meanwhile, only the solution corresponding to SRCHA system turned into green (Figure 1A inset). These results indicated that the rapid signal amplification has been truly realized in the SRCHA system. Clearly, this rapid signal amplification was caused by

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previously reported ATP assays but has a significant short detection time (Table S3). The selectivity test was performed by measuring and comparing the colorimetric response of ATP to its analogous molecules, such as UTP, GTP, and CTP. As shown in Figure S3, only ATP exhibited a significantly higher absorption signal compared to its analogous molecules, indicating that this detection system was able to recognize ATP as the target with high selectivity. Such high selectivity can be attributed to the high specificity between ATP and its aptamer, and the sequence-specific discrimination of the SRCHA system.30 In addition, five repetitive experiments for ATP at 0.2 µM and 1 µM were performed, and the RSD of 2.1% and 5.2% were obtained, respectively, demonstrating that the proposed method was coupled with good reproducibility.

CONCLUSION

Figure 3. UV-vis absorption spectra of the assay with and without of 2 µM ATP. H1 and H2 concentration: 500 nM. Inset: Photographs of the reaction solutions.

In summary, a well-designed catalyzed hairpin assembly system with the capacity to self-replicate was proposed, in which numerous target/trigger DNA replicas can be rapidly formed and rapid signal amplification was then realized. Benefiting from the target/trigger self-replication, our detection system has been successfully applied for the rapid and visual detection of DNA and small molecule, which can be extended to the detection of other targets by simply modifying the detection system with corresponding recognition domains. More importantly, this self-replication-based approach opens a new way for the enzyme-free and rapid signal amplification instead of the conventional enzyme-assisted or target recycling amplification methods, provides a potential technology for the realtime rapid analysis.

Reproducibility of the proposed method was evaluated by relative standard deviation (RSD). Under the optimal conditions, five repetitive measurements for target DNA at 50 pM and 300 pM showed the RSDs were 2.2 % and 3.4 %, respectively, suggesting this method had good reproducibility. In order to prove the versatility of this method, we analyzed the platform for the detection of small molecule (adenosine triphosphate (ATP)). As shown in Figure 3, after the addition of ATP, an obviously color change and significantly increase in UV–vis absorption intensity are observed, indicating that the trigger DNA has been released from hairpin probe through the formation of the ATP-aptamer complex and triggered the SRCHA reaction. From Figure 4 we can see that avisible color change can be observed for ATP concentration as low as 0.2 µM, and the absorbance versus the ATP concentration showed a linear range from 0.1 µM to 2 µM. The detection limit was estimated to be about 48 nM (S/N=3). This SRCHA-based ATP detection system exhibits a similar sensitivity to the

ASSOCIATED CONTENT AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; [email protected].

Author Contributions #

J.D. and H.H. contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Financial supports from the National Natural Science Foundation of China (No. 21377089, 21407109 and 21475091) are gratefully acknowledged.

Supporting Information Sequences of DNA used in the experiments, comparison of DNA and ATP detection in sensitivity and detection time, conditions optimization for the DNA detection and the selectivity of the SRCHA system for DNA and ATP detection. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure 4. (A) Photograph of the colorimetric detection of different concentrations of ATP. H1 and H2 concentration: 500 nM. (B) Plot of ATP concentration vs. absorbance for the ATP assay. ATP concentrations: 0, 0.1 µM, 0.2 µM, 0.5 µM, 1 µM, 1.5 µM, 2 µM.

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