Cell-Surface-Anchored Ratiometric DNA Nanoswitch for Extracellular

Jun 5, 2019 - Very recently, we reported an aptazyme–gold nanoparticle fluorescence probe for the amplified detection of intracellular ATP.(8) Howev...
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Article Cite This: ACS Sens. 2019, 4, 1648−1653

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Cell-Surface-Anchored Ratiometric DNA Nanoswitch for Extracellular ATP Imaging Jing Yuan,† Zhiwei Deng,† Hui Liu,† Xiufang Li,† Jianbing Li,† Yao He,† Zhihe Qing,‡ Yanjing Yang,*,† and Shian Zhong*,† †

College of Chemistry and Chemical Engineering, Central South University, Changsha, 410083, PR China School of Chemistry and Food Engineering, Changsha University of Science and Technology, Changsha, 410114, PR China

‡

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S Supporting Information *

ABSTRACT: The precise detection of extracellular ATP, although a challenging task, is of great significance for understanding the related cell processes. Herein, we developed a ratiometric DNA nanoswitch by employing a DNA tweezer and split aptamer. The nanoswitch is composed of three specially designed ssDNA strands, namely, the central strands O1, O2, and O3. This nanoswitch can be anchored on the cell membrane by cholesterol labeled at the 3′ end of O3. Initially, the DNA tweezer adopts an open state, separating the dual fluorophores and giving rise to a low FRET (fluorescence resonance energy transfer) ratio. The presence of ATP induces the binding of the two split aptamers to alter the structure of the nanoswitch from the open state to the closed state, bringing the donor and the acceptor closer together and generating high FRET efficiency. The results demonstrated that the ratiometric DNA nanoswitch can be applied for quantitative analysis and real-time monitoring of the changes in extracellular ATP. We believe that the cell surface-anchored DNA nanoswitch has promising prospects for use as a powerful tool for the understanding of different ATP-related physiological activities. KEYWORDS: ratiometric, DNA nanoswitch, extracellular detection, ATP, split aptamer, cholesterol developed a fluorescent aptamer sensor for monitoring adenine. Tan15 et al. have constructed cell membraneanchored sensors based on DNAzyme for metal ions imaging. Many groups have reported ratiometric i-motif sensors for sensing extracellular pH.16−19 These cell-surface-anchored fluorescent DNA probes have been proven to be excellent extracellular analytical tools. In particular, the resonance energy transfer (FRET)-based nucleic acid probe that has been widely used for analyzing biomolecules in solution20,21 or living cells9,22,23 could provide a viable method for the precise detection of biomolecules within the cellular microenvironment. Compared to single-intensity-based sensing, FRET imaging enables the quantitative analysis of targets by using the ratio of the two fluorescence intensities. Karp24 et al. have described a FRET-based aptasensor anchored on the cell membrane for monitoring protein expression within the cellular environment. Herein, we developed a FRET-based DNA nanoswitch that combines a DNA tweezer with ATP split aptamer for extracellular ATP sensing. DNA tweezers, as a basic form of DNA nanomachine, have been demonstrated to be an ideal platform for the fabrication of biosensors due to their

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xtracellular ATP plays an important role in mediating numerous physiological activities. For example, the activities of platelet aggregation,1 muscle contraction,2 vascular tone,3 and the ion channel4,5 are related to extracellular ATP. Thus, knowledge of the localization and changes of extracellular ATP is essential for the understanding of the related cell processes. Among the various detection methods, fluorescence probes offer a highly sensitive technique for the detection of ATP. To date, several fluorescence-based probes have been explored for the intracellular detection of ATP.6−13 For example, Mirkin6 et al. have reported an aptamer-based nanoflare for ATP detection in living cells. Lin7 et al. have designed an aptamer/graphene oxide fluorescent probe for sensing ATP in live cells. Very recently, we reported an aptazyme−gold nanoparticle fluorescence probe for the amplified detection of intracellular ATP.8 However, fluorescence probes that are anchored on the cell surface to elucidate the potential regulatory effects of extracellular ATP on diverse physiological activities have been largely unexplored. Hence, the development of a facile and accurate fluorescent approach for detecting and localizing extracellular ATP on cell surfaces remains a desirable goal. Nongenetically conjugated fluorescent DNA probes, which are more convenient and versatile compared to genetically engineered sensors, have offered a new approach for studying cell-to-cell communications and cell functions within the cellular environment. For example, Sando14 et al. have © 2019 American Chemical Society

Received: March 11, 2019 Accepted: June 5, 2019 Published: June 5, 2019 1648

DOI: 10.1021/acssensors.9b00482 ACS Sens. 2019, 4, 1648−1653

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ACS Sensors

Figure 1. Mechanism of the cell-surface anchored ratiometric DNA nanoswitch for the imaging of extracellular ATP.

straightforward response mechanisms.25−28 Split aptamers that consist of two fragments of the intact nucleic acid aptamer and can specifically fold into the right secondary structure in the presence of the target have been widely used in biosensors by taking advantage of their low background.29−33 Because of the advantages of the DNA tweezer, the split aptamers and the signal readout mode of FRET, this nanoswitch can be applied for reporting and quantifying extracellular ATP in real time. Therefore, we have, for the first time, applied a FRET-based DNA nanoprobe for the analysis of extracellular ATP. As illustrated in Figure 1, the nanoswitch is composed of three specially designed ssDNA stands: central strand (O1) labeled with two-fluorophores (Cy3 and Cy5), O2 containing split aptamers (blue), and O3 that is composed of split aptamer (blue) and is labeled with cholesterol to modify the DNA tweezer on the cell surface. In the absence of the target ATP, the DNA tweezer assumes an unbound state, separating the two fluorophores and leading to low FRET value. The presence of ATP can bind the two split aptamers to alter the structure of the nanoswitches from the open to the closed state, bringing the donor and acceptor closer together and inducing a high FRET ratio. The FRET ratio (FA/FD) can be utilized as a readout signal for the quantification of ATP on the cell surface. This cell surface-anchored ratiometric DNA nanoswitch offers a flexible and facile method for imaging ATP on the cell surface.

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potential of 110 V. The electrophoresis time was determined by the location of the two indicators in the loading buffer and was approximately 40 min. A PBS buffer was used for electrophoresis to dilute the DNA oligonucleotides (2 μM). After electrophoresis, the gel was visualized using a BioDoc-It2 315 imaging system. Fluorescence Measurement. An F-4600 fluorescence spectrophotometer produced by Shimadzu Corporation of Japan was used for the fluorescence measurements. Fluorescence spectra were recorded from 550 to 750 nm with a 532 nm excitation wavelength and a 5 nm slit for both excitation and emission. The 10 μL DNA nanoswitch (2 μM) and ATP samples with different volumes were added to PBS containing 50 mM magnesium ions. Different ATP concentrations of 0 μM, 5 μM, 10 μM, 20 μM, 30 μM, 60 μM, 100 μM, 200 μM, 500 μM, and 800 μM were obtained in the total volume of 120 μL. Then, the samples were heated to 37 °C, incubated for an hour, and then the fluorescence was measured. To prove the specificity of the DNA nanoswitches, the DNA probes were dissolved in PBS buffer, and then ATP, CTP, UTP, and GTP were added separately (500 μM). In the end, the samples were incubated for an hour at 37 °C and fluorescence was measured. Cell Culture. A549 cells and HEK 293T cells were cultivated in a prepared culture medium containing 90% DMEM medium (Thermo Fisher Scientific Co., Ltd.) and 10% fetal bovine serum (Thermo Fisher Scientific Co., Ltd.). SMMC-7721 cells were incubated in RPMI 1640 medium containing 12% gibco. SK-BR-3 cells were incubated in the McCoy’s 5A medium containing 10% FBS. CO2 Incubators (Thermo Scientific, USA) were used to incubate the cells. All of the cells were incubated in moist air with 5% CO2 at 37 °C. Cell Membrane Imaging. The cells were cultured (100 μL substrate per well) in a 96-well plate for 24 h, and two wells were selected in which the cells were in good condition under the microscope. The cells in the two selected wells were washed with PBS, followed by incubation with Hoechst 33342 at 10 μg/mL for 15 min. After 15 min, the Hoechst 33342 was washed off with PBS. The cells were immersed with 40 μL DNA nanoswitches (one group used DNA nanoswitches without cholesterol; the other group used DNA nanoswitches modified by cholesterol groups, 2 μM) at 4 °C for 10 min. After washing off the material, cells were resuspended in a PBS buffer containing 20 mM magnesium ions for measurements using an Operetta High-Content Imaging System. Imaging of ATP Cells at Different Concentrations. The cells were cultured in a 96-well plate for 24 h. A column of cells in the 96well plate was selected. The cells were washed with a PBS buffer, immersed with 40 μL material (2 μM), and placed at 4 °C for 10 min. Then, a total of 100 μL of PBS buffer (8 mM magnesium ions) with different concentrations of ATP were ready for fluorescence imaging (compared to standard ATP buffers, we ignored the extracellular ATP). All of the fluorescence image results were obtained using an Operetta High-Content Imaging System. Flow Cytometry Assays. (1) A549 cells were cultivated overnight in a 6-well plate (approximately 1 × 106 cells). The cells were first washed with PBS, and then the probe was added (2 μM, 300 μL), and the mixture was incubated at 4 °C for 10 min. Then, the cells were washed with PBS to remove the unbound probe. The cells

EXPERIMENTAL SECTION

Materials and Reagents. The sequence of O1 is taken from the literature.28 DNA strands and ATP analogues were obtained from Sangon Biotech Co., Ltd. (Shanghai) and were purified by highperformance liquid chromatography. The DNA sequences are listed in Table S1. The phosphate buffered saline (0.01 M, pH 7.2−7.4) used in vivo and in vitro was purchased from Beijing Solarbio Science & Technology Co., Ltd. The gibco used in the SMMC-7721 cells was obtained from Zhejiang Tianhang Biotechnology Co., Ltd. (Hangzhou, China). Hoechst 33342 was purchased from Beyotime Biotechnology Co., Ltd. (Hunan, China). Trypsin-EDTA (0.25%, 1X) was obtained from New Cell & Molecular Biotech Co., Ltd. CD spectra were measured using a circular dichroism chiroptical spectrometer manufactured by JASCO Electric Co., Ltd. All of the reagents were commercially available and were used without further purification. Construction of the DNA Nanoswitch. DNA oligonucleotides (2 μL of O1, O2, or O3) were separately placed in 98 μL of PBS buffer to achieve a concentration of 2 μM and were heated at 95 °C for 5 min in a metal bath. Then, the heater was turned off and the mixture was cooled naturally. The prepared DNA nanoswitch was stored at 4 °C for further use in the following experiments. Electrophoresis Experiments in Vitro. Native polyacrylamide gel (8%) electrophoresis (native PAGE) was run at a constant 1649

DOI: 10.1021/acssensors.9b00482 ACS Sens. 2019, 4, 1648−1653

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ACS Sensors were digested with trypsin and centrifuged at the speed of 1500 rpm for 5 min, and then the liquid supernatant was removed, followed by washing, and finally the sample was suspended in 250 μL PBS. (2) Another well in the 6-well plate was selected, washed, and incubated with PBS at 4 °C for 10 min to generate the blank sample. All of the cells data were obtained by flow cytometry in the APC channel. Flow cytometric analysis was performed using a Beckman CytoFLEXFCM instrument. Time Imaging. The cells were cultured in a 96-well plate for 24 h. Then, the cells were washed, and DNA nanoswitches (2 μM) were added to the 96-well plate and incubated for 10 min at 4 °C. Then, the cells were washed and immersed in a 0.1 M CaCl2 solution that was cooled to at 4 °C in advance. The 96-well plate was incubated at 4 °C for 30 min. Finally, the cells were immersed in a PBS buffer containing 20 mM magnesium ion for analysis with an Operetta HighContent Imaging System.

slowly than the bands in lanes 2−4. These results proved that the DNA nanoswitch did indeed form as designed. To demonstrate the structure change upon the addition of ATP, the circular dichroism (CD) spectra of the DNA nanoswitch in the absence or presence of ATP were obtained (Figure S1). Upon changing the concentration of ATP from 0 to 5.0 mM, the positive band of the DNA nanoswitch near 270 nm was red-shifted to 280 nm, while a negligible shift was found in the negative band near 245 nm. This result is in agreement with a previous report.19 Moreover, Figure 2B shows that the FRET nanoswitch incubated with ATP had a similar FRET efficiency to that of the nanoswitch treated with complementary DNA, which is complementary to the ATP split aptamer. These results suggest that ATP can induce a change in the DNA nanoswitch conformation from the open state to the closed state. We first investigated the ATP sensing behavior of the designed FRET nanoswitch. The FRET signal was monitored upon changing the ATP concentrations from 5 μM to 800 μM. Figure 3A shows that the FRET spectra of the acceptor/donor

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RESULTS AND DISCUSSION First, we used gel electrophoresis to verify the formation of the DNA nanoswitch. In Figure 2A, lanes 5, 6, and 7 represent the O3′, O2, and O1 strands, respectively. Incubation of O1, O2, and O3′ generated a new band that migrated much more

Figure 3. (A) Fluorescence spectra of the DNA nanoswitch response to different ATP targets in vitro at 37 °C. (B) FRET ratio of FA/FD as a function of ATP concentrations. Inset shows the calibration curve for the concentrations ranging from 5 to 60 μM.

depend on the ATP concentration. Figure 3B shows that the FRET ratio changed as a function of ATP. The nanoswitches respond with an 8.5-fold increase in the FA/FD values upon ATP binding. Meanwhile, there was a linear relationship between the FRET ratio and the ATP concentration in the range of 5−60 μM, and the limit of detection (LOD) was 1.02 μM. Control experiments revealed that the presence of ATP led to an increase in the fluorescence ratio, while only a minute

Figure 2. (A) Image of 8% Native polyacrylamide gel electroctrophoresis. Lane 1: O1+O2+O3′; Lane 2: O2+O3′; Lane 3: O1+O2; Lane 4: O1+O3′; Lane 5: O3′; Lane 6: O2; Lane 7: O1. (B) Fluorescent spectra of DNA nanoswitches treated with blank, 2 μM complementary DNA, and 800 μM ATP. 1650

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ACS Sensors FRET signal change was observed in the presence of CTP, UTP, and GTP (Figure S2). This result suggested the high selectivity of the FRET nanoswitch. To anchor the nanoswitch on the cell surface, we used cholesterol as the anchoring unit. We chose cholesterol because lipid or Chol-modified DNA probes were reported to be efficiently attached to the membrane.34 Fluorescent imaging and flow cytometry experiments were carried out to verify the modification of the DNA nanoswitch on the cell surface. A549 cells were first treated with nanoswitches for 10 min, followed by treatment with Hoechst 33342 for 15 min. Figure 4 shows that the fluorescence signal of the probe was

Figure 5. Fluorescence imaging of A549 cells incubated with the nanoswitches and with different ATP concentrations. The scale bar is 20 μm.

To test the selectivity of the FRET nanoswitch on the cell surface, we added ATP, CTP, UTP, or GTP to four different groups of A549 cells. Figure S6 shows that the FRET signal was stronger in the cells treated with ATP relative to those in the other three groups. Thus, the probe on the cell membrane retained its selectivity for ATP. Next, we carried out the timedependent imaging experiment to examine the duration of the effect of ATP. In Figure S7, little change in the FRET ratio was observed in the cells until 15 min, suggesting that the whole detection process could be sustained for at least 15 min. Finally, different types of cells including HEK 293T, SMMC7721, and SK-BR-3 cells were incubated with the DNA nanoswitches to investigate the generality of the DNA probe. The fluorescent imaging results (Figure S8) suggested that this DNA nanoswitch was successfully anchored to the cell membranes of all three cell lines, thereby demonstrating the generality of this sensing protocol. We next investigated the ability of the nanoswitch to sense the changes in the ATP concentrations at the cell surface. It was reported that a low-temperature CaCl2 solution induced the formation of competent cells for which the membrane permeability was increased compared to that of normal cells, resulting in the release of ATP from the cytoplasm to the cell surface.35−38 Figure 6 shows that the FRET ratio was higher in the competent cells (A) than in the control cells (B). This result indicates that the fluorescence ratio correlates well with the concentration of ATP in the extracellular environment. Moreover, to test the ability of our cell surface-anchored

Figure 4. Fluorescent imaging of A549 cells after incubation with the nanoswitch, with and without cholesterol. Signals from the green channel (570−620 nm) and the red channel (655−760 nm) were collected with a 530 nm excitation wavelength. The excitation wavelength of Hoechst 33342 for staining nuclei was collected in the range of 430−500 nm by using a 355 nm excitation wavelength (the Cy3 and Cy5 channels are artificially colored). The scale bar is 20 μm.

mainly distributed on the cell membrane, with negligible signal inside the cells. The flow cytometry results indicated that the cells incubated with nanoswitches were more fluorescent than the control cells (Figure S3). Additionally, we studied the distribution of the sensor on the membrane. Cu2+ was selected to quench the fluorescence of Cy3 and Cy5. The fluorescent experiments in vitro suggest that the fluorescence of Cy3 was dramatically decreased in the presence of 5 mM Cu2+ (Figure S4A). As shown in Figure S4B, the fluorescence of the cell surface disappeared completely upon addition of Cu2+, indicating that the probe was anchored on the outside of the cell surface. These data clearly indicated that the nanoswitches were located on the outside of the cell surface. Then, we evaluated the performance of the cell membraneanchored DNA nanoswitch for extracellular ATP imaging using living A549 cells. As shown in Figure 5, with increasing extracellular ATP concentrations, a gradual decrease in the fluorescence intensity of Cy3 and a significant increase in the fluorescence intensity of Cy5 on the A549 cell membranes were observed. The relationship between the FA/FD ratios and the concentrations of extracellular ATP was analyzed using ImageJ software, and quantitative fluorescent intensities data were obtained (Figure S5). It was demonstrated that the FRET value was enhanced as the extracellular ATP increased, corresponding to the results obtained in the buffer solution. These results indicated that the FRET nanoswitch retained its performance as a fluorescent probe when anchored on the cell membrane.

Figure 6. Fluorescence images of ATP using the nanoswitch on A549 cells treated with (A) and without (B) CaCl2 solution. The scale bar is 20 μm. 1651

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ACS Sensors Notes

nanoswitch to monitor the cellular ATP extrusion process, the cells were first treated with a cold CaCl2 solution to generate competent cells and then analyzed via high content imaging at certain intervals. As shown in Figure 7, FRET signals gradually

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was financially supported by the National Natural Science Foundation of China (21804143, 21576295) and the Open-End Fund for the Valuable, Precision Instruments of Central South University (CSUZC201830).

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Figure 7. Time-dependent imaging with the addition of Ca2+ at 4 °C to stimulate the cells. The Operatta high-content imaging system was used to perform measurements at 2 min intervals for a total of 10 images for the merge channel. The scale bar is 20 μm.

decreased until 12 min, indicating the gradual decrease in the extracellular ATP concentration. This phenomenon may be due to the recovery of the competent cells and the consumption of the ATP energy molecules.

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CONCLUSIONS In summary, we have developed a cell membrane-anchored FRET nanoswitch by employing a DNA tweezer and split aptamer for extracellular ATP analysis. The nanoswitch can be directly anchored to the cell membrane and retain its performance. It was demonstrated that this cell membraneanchored FRET nanoswitch exhibited high sensitivity and selectivity. Moreover, due to the use of the DNA nanoswitch, the probe possesses several advantages including quick response kinetics and easy application to other targets. Furthermore, our FRET-based nanoswitch can be applied for the quantitative analysis and real-time analysis of extracellular ATP by using FRET signals, which has not been reported in previous work. This nanoswitch provides a potentially useful tool for the investigation of the extracellular ATP-related cell physiological processes.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.9b00482. Experimental data, including sequences of DNA strands, circular dichroism of the DNA nanoswitch, specificity experiments in buffers and A549 cells, the FA/FD ratios of cell imaging for different concentrations of ATP, different types of cell imaging, fluorescent and cell imaging experiments with a quencher, and cell real-time monitoring (PDF)

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Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Shian Zhong: 0000-0002-0729-2304 1652

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DOI: 10.1021/acssensors.9b00482 ACS Sens. 2019, 4, 1648−1653