Scallop-Inspired DNA Nanomachine: A Ratiometric Nanothermometer

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Scallop-Inspired DNA Nanomachine: A Ratiometric Nanothermometer for Intracellular Temperature Sensing Nuli Xie, Jin Huang, Xiaohai Yang, Xiaoxiao He, Jianbo Liu, Jiaqi Huang, Hongmei Fang, and Kemin Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02709 • Publication Date (Web): 25 Oct 2017 Downloaded from http://pubs.acs.org on October 29, 2017

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Scallop-Inspired DNA Nanomachine: A Ratiometric Nanothermometer for Intracellular Temperature Sensing Nuli Xie, Jin Huang*, Xiaohai Yang, Xiaoxiao He, Jianbo Liu, Jiaqi Huang, Hongmei Fang and Kemin Wang* State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082, P. R. China E-mail: [email protected]; [email protected]; Fax: +86 731 88821566; Tel: + 86 731 88821566

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ABSTRACT: Accurate measurement of intracellular temperature is of great significance in biology and medicine. Using DNA nanotechnology and inspired by nature’s examples of “protective and reversible responses” exoskeletons, a scallop-inspired DNA nanomachine (SDN) is desgined as a ratiometric nanothermometer for intracellular temperature sensing. The SDN is composed of a rigid DNA tetrahedron, where a thermal-sensitive molecular beacon (MB) is embedded in one edge of the DNA tetrahedron. Relying on the thermal-sensitive MB and fluorescence resonance energy transfer (FRET) signalling mechanism, the “On” to “Off” signal is reversibly responding to “below” and “over” the melting temperature. Mimicking the functional anatomy of a scallop, the SDN exhibits high cellular permeability and resistance to enzymatic degradation, good reversibility and tunable response range. Furthermore, FRET ratiometric signal that allows the simultaneous recording of two emission intensities at different wavelengths can provide a feasible approach for precise detection, minimizing the effect of system fluctuations.

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INTRODUCTION Temperature is a greatly significant physical parameter in biology and medical science. Detection of intracellular temperature holds tremendous values to understand cell biology and promote diseases diagnosis and therapy. Firstly, it regulates most chemical and biological reactions, which is involved in various cellular biological functions, such as gene expression, enzymatic processes, and metabolism.1 Secondly, some studies reveal that cancer tissues and pathological cells have abnormal temperature variation owing to their excessive metabolic activities,2 so temperature can be regarded as an important indication of some diseases. Thirdly, cancer localized thermotherapy requires effective thermometry at nanoscale to evaluate therapeutic accuracy and efficiency.3 Thus, it is essential and significant to develop the intracellular thermometer. Fluorescent thermometers have drawn much attention while used in cellular temperature sensing. They can work remotely with high temperature resolution, spatial resolution, and are very suitable to biological samples in fluids.4, nanothermometers

have

been

reported

for

5

Over the past decades, a number of

cellular

thermometry,

including

organic

fluorophores,6-9 protein,10 nanogels,11,12 polymers,13,14 rare-earth metal complex,15,16 quantum dots,17-21 and DNA-based thermometers.22-24 Among them, DNA-based nanothermometers are promising tools for cellular temperature sensing. It not only possess high temperature resolution, high spatial resolution and excellent biocompatibility, but also have some unique advantages (e.g., programmability, fast response). However, they still suffer from some limitations: First, without extra transfection agents, simple molecular beacon (MB)-based nanothermometer cannot enter into cells, thus generating more experiment steps and additional cost; Second, bare DNA nanothermometer cannot resist nuclease attack and keep long-term detection; Last but not least,

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conventional single-dye detection system may show unreliable signal due to distinguished probe distribution, unstable light sources, temperature-fluctuating dyes and environmental fluctuations. We reasoned that if we could design a system which could protect the DNA thermometer and allow it reversibly respond to temperature and facilitate its entry into cell, we might solve the above limitations. The recent emergence of several bioinspired systems to sense biological molecules prompted us to turn to nature for inspiration.25-27 Nature has provided numerous simple yet elegant engineering solutions to this paradox as observed in some organisms such as the scallop, which utilizes a “protective and reversible responses” concept. For instance, scallop’s shells protect it from the harsh environment and respond quickly to external stimuli with the aid of central adductor muscle. Like a mechanical spring, the adductor muscle contracts, the shells close, the muscle relaxes, the shells open. Thus, we developed a scallop-inspired DNA nanomachine (SDN) for intracellular temperature sensing by DNA nanotechnology. As shown in Figure 1, The SDN is based on rigid DNA tetrahedron. In brief, one edge of DNA tetrahedron is substituted for a thermal-sensitive MB to construct a temperature-responsive DNA nanomachine. In such a way, the MB is like central adductor muscle and two faces of the DNA tetrahedron are like two shells of a scallop to protect core MB. The thermal stability of the MB is reflected in the melting temperature (Tm), defined as the temperature at which half of hairpin structure is dissociated to single-stranded DNA.28, 29 When the temperature is lower than Tm, the MB is shaped in closed stem-loop structure, making the DNA tetrahedron contractive; while temperature is higher than Tm, the MB is gradually unfolded, making the DNA tetrahedron expansive. This reversible processes of the SDN simulates the physiological behaviors of a scallop. Due to the DNA tetrahedron structure as protective shells, it can resist

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cellular nuclease degradation and enter into cells by receptor-mediated pathways without any transfection agents.27, 30-36

Furthermore, in view of the drawbacks of conventional single-dye systems, we introduce FRET mechanism into the SDN to construct a ratiometric thermometer. Two dyes (FAM and TAMRA) are respectively labeled at the two ends of P4 strand, which partially hybridized with P3 strand containing MB hairpin structure. Thus, once the MB opens or closes, the distance between donor (FAM) and acceptor (TAMRA) changes synchronously. According to the ratio of Acceptor-to-Donor fluorescence intensity (A/D) derived from FRET, we can realize fast and reliable temperature sensing in living cells. Compared with single-dye system, FRET-based ratiometric thermometer can distinctly avoid inaccurate measurement arising from changes in probe concentration, ion strength, pH and complex biochemical milieu. EXPERIMENTAL SECTION Materials and Instruments. All DNA oligonucleotides were synthesized and HPLC purified by Sangon Biotechnology Co., Ltd (Shanghai, China), and the sequences are shown in Table S1. All aqueous solutions were prepared using ultrapure water (≥18MΩ, Milli-Q water purification system, Millipore). Cell medium RPMI 1640 was obtained from GIBICO (USA). Human cervical cancer cell line HeLa were obtained from our lab. All fluorescent spectra were measured using F-7000 fluorescence spectrometer (Hitachi, Japan). Cells were incubated by using a Thermo FORMA 3111 CO2 incubator (ThermoFisher, USA). The confocal fluorescence imaging studies were performed using an Olympus IX-70 inverted microscope with an Olympus FV 500 confocal scanning system (Japan) with an oil objective lens (100×), which was equipped with temperature controller (TOKAI HIT). The

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transmission electron microscopic (TEM) images were obtained on a JEM-2100 transmission electron microscope (JEOL Ltd., Japan). The UV-vis absorption spectra were obtained with a Biospec-nano UV-vis spectrophotometer (Japan). DNase I was purchased from Takara Biotechnology Co. Ltd. (Dalian, China). Preparation and Characterization of the SDN nanothermometer. The SDNs were selfassembled according to a well-understood protocol.30, 37 Four customized oligonucleotide strands (P1, P2, P3 and P4) were respectively diluted with TM buffer (20 mM Tris, 50 mM MgCl2, pH=8.0) to stock solutions which have a final concentration of 10 µM. The four strands were then mixed in equimolar in TM buffer, heated to 95oC for 5 minutes (min) and then immediately cooled on ice in 1 min, finally stored at 4oC for at least 4h. The MB stem-loop structure was embedded in P3 strand. To assemble the SDN with different Tm, it was necessary to change different P3 strands containing designed MB structure. The characterization of synthesized SDN was performed by agarose gel electrophoresis and dynamic light scattering (DLS). Gel analysis of the nanostructure was conducted using a 2% agarose gel in 1×TBE (Tris borate-EDTA) buffer, respectively. After adding the samples, the gels were run at a constant voltage of 100 V for 2h. The DLS characterization of the SDN was performed using diluted sample. Fluorescence Analysis. The fluorescence spectrum was determined using F-7000 fluorescence spectrometer equipped with a thermostat controlled cell holder. A slit width of 5 nm was used in both excitation and emission light path, while the photomultiplier tube voltage by the detector was set to 700 V. Fluorescence emission intensity of FAM (522 nm) and TAMRA (580 nm) was recorded under an excitation wavelength of 484 nm. For melting curves, the concentration of SDN in Tris buffer (20 mM Tris, 20 mM MgCl2, pH=7.4) was 50 nM, and the temperature

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response test was performed at a series of temperature points with an interval of 3oC. The samples were equilibrated at each temperature for 5 min. The fluorescence A/D ratios at different temperature points (the value of TAMRA fluorescence intensity at 580 nm / FAM fluorescence intensity at 522 nm) were normalized by taking the A/D ratio at 54oC as 1.0. Then, two different temperatures with 27oC interval were set to study the reversibility of this SDN-1 nanothermometer. The temperature shifted up and down between 27oC and 54oC over 50 cycles. Each point was equilibrated for 5 min. The fluorescence A/D ratios were normalized by taking the A/D ratio at 54oC as 1.0. For signal stability experiments, the samples was added into the buffer with different pH or K+ concentrations, respectively. In cell lysate experiments, the fluorescence intensity of all samples was monitored using time-scan model at a constant temperature of 25oC or 37oC. All fluorescence experiments were repeated three times. The thermal sensitivity of the fluorescent thermometer can be calculated by equation (1).38


 =

 

∆ 

(1)

Where  /  and ∆ represent the derivative value in the fluorescence A/D ratio– temperature diagram and the variation of normalized fluorescence A/D intensity, respectively. The temperature resolution of the fluorescent thermometer can be generally evaluated by wellunderstood method39 using equation (2). 

δT =  

(2)

Where / and  represent the inverse of the slope in the fluorescence A/D ratio– temperature diagram and the standard deviation of the fluorescence A/D intensity, respectively. / was obtained by differentiating the fitting polynomial FI (T) according to the melting

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curves and  was calculated with the following equation (3) as the averaged difference between FI (T) and the fluorescence A/D intensity experimentally acquired.  =

∑ |   | 

(3)

Where n and   imply the experiment number and the fluorescence A/D intensity of each measurement at T °C, respectively. Nuclease-Resistance Studies of the SDN. To evaluate the nuclease resistance ability of the SDN, 0.05 U/µL Dnase I, which was significantly higher than what could exist in cellular milieu, was used to digest probes. The simple MBs were used as the control sample. The samples were incubated with Dnase I for different times (0 min, 10 min, 20 min, 40 min and 60 min). Then, the samples were treated with EDTA solutions of high concentration to stop nuclease digestion. Then, all of the samples were collected to perform gel electrophoresis analysis. Gel analysis was conducted using a 2% agarose gel in 1×TBE (Tris borate-EDTA) buffer, respectively. After adding the samples, the gels were run at a constant voltage of 100 V. Cell Viability Assay. To investigate the cytotoxicity, a standard MTT assay was operated. HeLa cells were dispersed with replicate 96-well microplates at a density of 1×106 cells/well. Plates were then maintained at 37°C in 5% CO2 atmosphere for 24 h. Afterwards, the cells were treated with three concentrations of SDN-1 (0, 50, 100nM) for 0h, 12h, 24h, 36 h and 48 h, respectively, and 100 µL MTT solutions were then added to each well for 4 h. After removing the remaining MTT solution, 150 µL DMSO was added to each well to dissolve the formazan crystals. The absorbance was measured at 490 nm with a RT 6000 microplate reader. Photothermal Effect of Au NRs in Buffer. 1 mL buffer containing approximately 4.4× 1010 particles of the Au nanorods was irradiated by a 980 nm NIR laser irradiation (650 mW). The temperatures of the solutions were monitored by a thermocouple microprobe submerged in the

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solution. The temperatures of 1 mL buffer without Au nanorods with laser treatment was also monitored. Cell Culture and Confocal Fluorescence Imaging. HeLa cells (Human cervical cancer cell line) were grown in RPMI 1640 medium supplemented with 10% inactivated fetal bovine serum, 100 U/ml 1% penicillin and streptomycin solution. All cells were cultured in a humidified CO2 incubator containing 5% CO2 at 37°C. For confocal imaging studies, HeLa cells were cultured on 35-mm confocal laser culture dishes with the same medium for 24 h. Then the SDN (the final concentration is 100 nM) were incubated with HeLa cells in culture medium at 37 °C for 3 h. after removing the probes by PBS buffer three times, confocal fluorescence imaging was performed with oil immersion objective 100X and a heating stage (TOKAI HIT) for temperature control. The FAM and TAMRA fluorescence emission channels were collected under an exictation laser at 488 nm. For data analysis by Image-Pro plus 6.0, fluorescence intensity of the cells at different temperature was normalized by taking the fluorescence A/D ratio at 40oC as 1.0. Each point in the calibration curve was calculated by averaging the normalized A/D ratio of more than 3 cells. At each temperature, cells were incubated for 10 min to equilibrate the temperature of the medium and cells. For the photothermal study of Au NRs in cells, HeLa cells were first incubated with the SDN in culture medium at 37 °C for 3 h. Next, SDN-treated cells were incubated with Au NRs (2.2× 1010 particles per cm3) for 3 h. The cells dishes without AuNRs served as control samples. After cleaned 3 times with PBS and immediately immersed in a mixture of serum-free medium, the dishes were treated with a peripheral NIR laser (650 mW) for 0, 4, 8, 12, 15 min, respectively. Finally, confocal fluorescence imaging was performed with oil immersion objective 100X under the excitation wavelength at 488 nm.

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RESULTS AND DISCUSSION To confirm the feasibility of our SDN, we first designed a SDN-1 with four base-pair stem in the MB by mixing four strands (P1, P2, P3-1 and P4, Table S1) in equimolar. The assembly protocol has been well built according to previous papers.30, 37As shown in Figure 2, each strand runs round one of the four faces and is hybridized to other three strands running round the adjacent three faces. Because each face is an entire triangle, this DNA tetrahedron is one of the simplest DNA polyhedron with powerful structural rigidity. Here, P3-1 is a 76-nt oligonucleotide strand containing MB structure with four base-pair stem. The SDN-1 is evaluated by gel electrophoresis (Figure S1) and dynamic light scattering (Figure S2). An average hydraulic diameter of 13.7 nm conformed to the theoretical value of DNA tetrahedron, making it suitable for temperature sensing with high spatial resolution. In fluorescence experiment, with temperature increasing, the fluorescence A/D ratios gradually declined (Figure 3(a)). It is consistent with the behavior of SDN. When the temperature is lower than Tm (calculated value is 40.0oC), the MB is shaped in closed stem-loop structure, making the SDN contractive and showing strong FRET signal; while temperature is higher than Tm, the MB is gradually unfolded, making the SDN expansive. Two dyes are separated, FRET signal declining. Nevertheless, when the temperature is excessively higher than 55oC, the nanomachine would be melt, resulting in the changeless signal of this thermometer. The fast temperature response range (30 to 50oC) of our SDN-1 is slightly sharper than that of single MB (27 to 50oC) (Figure S3). It could be attributed to the stabilizer function of peripheral tetrahedron structure, 24 displaying high cooperative behaviors of the SDN. Then, two different temperatures with a big interval were set to study the reversibility of the SDN-1. Responding to the temperature fluctuation between 27oC and 54oC, the fluorescent A/D ratio shifted up and down with little

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variation over 50 cycles (Figure 3(b)). It demonstrated that the SDN thermometer had good reversibility and reusability, and was capable of long-time observation. The thermal sensitivity, is commonly used to reflect the relative variation of the fluorescence intensity with temperature.38, 40 Our SDN-1 showed an excellent thermal sensitivity of more than 5.2% /oC in the fast response range (30 to 50oC), which is greater than a minimum signal variation (0.5% /oC) required to resolve a temperature difference. The temperature resolution, evaluated as the product of the inverse of the slope of intensity versus temperature and the standard deviation of the averaged fluorescent intensity,39 is used to indicate the minimal temperature distinction. As shown in Figure 3(a), the temperature resolution in each point were calculated by the well-understood equations (shown in the experiment section). The temperature resolutions ranging from 30 to 50oC were determined to be smaller than 0.5oC, which is comparable to or better than the resolution of current fluorescent thermometers,39,41 enabling precise detection with high temperature resolution, especially while detecting small temperature variations. In order to take whole comparison with other representative cellular thermometry systems reported previously, a table was introduced (Table 1).39 A series of detailed parameters were indicated in this table, including response range, temperature resolution, thermal sensitivity and fluorescent spectrum information. The SDN thermometers not only had a proper response range for cellular temperature sensing, but also showed good performance in temperature resolution and thermal sensitivity. One virtue of DNA nanothermometers is its programmability with tunable response ranges to apply in various practical applications. It can be tuned by different MB sequences, either different longer base-pair stem or GC base pairs. In order to investigate the tunability of our SDN, P3-1 was respectively replaced by another four well-designed P3 strands (P3-2, P3-3, P3-

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4 and P3-5, Table S1). All the alteration presented at the MB stem structure. To be specific, P32 and P3-3 reduced one base-pair than P3-1. Contrarily, P3-4 and P3-5 increased one base-pair than P3-1. Notably, P3-3, P3-5 respectively increased one GC base-pairs than P3-2 and P3-4. Then, four congeneric SDN thermometers (SDN-2, SDN-3, SDN-4 and SDN-5) were assembled, respectively. Similarly, fluorescence analysis was used to evaluate their performance. As a result, with the longer stem or higher GC content from SDN-2 to SDN-5, the Tm shifted to higher point (Figure 4, Figure S4). Compared to SDN-1, the Tm of SDN-2 decreased nearly 6oC due to the deletion of one stem base-pair and the substitution of one GC base pair to AT base pair. On the contrary, The Tm of SDN-5 increased 6oC due to the longer stem and higher GC content. Interestingly, although the response ranges regularly changed as different MB sequences, but all of the highest detectable temperature were at the same point (54oC). This unique phenomenon could be explained by that the two dyes weren’t directly labeled at the MB strand. The temperature above 54oC might affect the stability of this nanostructure, then destroying hybridization between MB and dye-labeled strand. So, all of them have the same highest temperature limitation. This property can be regarded as the “fuse wire” function. Furthermore, the change of stem sequences did not affect their temperature resolutions. All the temperature resolutions located in fast response range are almost lower than 0.5oC (Figure S4). So, it possesses sequence programmability and temperature tunability to adapt different practical applications. To be an eligible intracellular thermometer, it must possess reliable signal output, noncytotoxicity and the nuclease-resistant stability. Firstly, SDN-1 was employed to investigate the influences of pH and K+ ion concentrations. It hardly displayed correlation under both pH (6.0, 7.4, and 8.0) and K+ ion concentrations (100mM, 150mM and 200mM) (Figure S5), suggesting

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its independence of environmental pH and ion strengths. Furthermore, we used diluted cell lysate to examine whether this SDN-1 could be disturbed by the complicated biological environment (Figure S6). The fluorescence signal exhibited very little change at a fixed temperature (25oC and 37oC) respectively, proving that it can keep stable and normally function in cellular environment. Furthermore, MTT assays showed that our SDNs had no cytotoxicity, and were suitable to cellular applications (Figure S7). In addition, excellent thermometer must be resistant to nuclease degradation while functioning in living cells. As shown in electrophoresis results of Figure S8, the SDN can greatly delay nuclease degradation under a very high concentration of Dnase I (0.05 U/µL, significantly higher than what could exist in cellular milieu). In contrast, single MBs were degraded in several minutes. The results were coincident with our previous fluorescence experiments, proving that the DNA tetrahedron showed high resistance to enzymatic degradation.35, 36 Obviously, the DNA tetrahedron structure played like the shells of a scallop to protect MB from nuclease attacking. Next, SDN-1 was chosen to test its temperature sensing performance in HeLa cells because of its proper response range. The SDN can be delivered into cytoplasm through receptor-mediated endocytosis, without any transfection agents.34,

42

After cellular uptake, we used confocal

microscopy to observe the FRET fluorescence signal of the thermometers under diverse temperatures (Figure 5(a)). At 25oC, bright red fluorescence and green fluorescence in HeLa cells was detected under the laser excitation at 488 nm wavelength. While temperature rose to 40oC, green fluorescence gradually showed brighter, but red fluorescence intensity declined greatly. The variation tendency of fluorescence intensity confirmed the occurrence of good FRET. For further analysis, the average fluorescence A/D intensity at five temperature points (25oC, 29oC, 33oC, 37oC and 40oC) were investigated to plot a cellular temperature calibration

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curve. Each point in the calibration curve was calculated by averaging the normalized A/D ratio of more than 3 cells. As temperature increasing, the relative values of A/D ratio become smaller (Figure 5(b)), which was consistent with the calibration curve of SDN-1 in buffer. Then, the thermal sensitivity of SDN-1 in both buffer and HeLa cells were calculated. Compared with the thermal sensitivity in buffer, the value of that in living cells do not suffer great losses (Figure 5(c)). In addition, the SDN-1 was demonstrated to function well in different cell lines. This result can be shown in the confocal imaging and their cellular temperature calibration curves (Figure S9). Thus, the results demonstrated that this nanomachine can be well applied as a thermometer for temperature sensing in living cells. In order to clarify the practicability of the SDN-1, we utilized it to assess the photothermal effect on living cells. Photothermal treatment can generating hyperthermia induced by internalized therapeutic agents under laser irradiation.43,

44

Significantly, the use of cellular

thermometers can in-depth improve the Photothermal applications because it can realize synchronous visualization of temperature variation in heating area, as well as imaging-guided comprehensive evaluation of photothermal killing efficiency. Some nanothermometers have been already used for intracellular temperature sensing to control and assess photothermal treatment.22, 45

Herein, SDN-1 was employed to sense temperature change after a photothermal process

caused by gold nanorods (AuNRs). AuNRs were synthesized by the reported method46 and prepared for intracellular applications. Both of the UV-Vis spectrum (Figure S10) and TEM image (Figure 6(a)) showed a proper size (32.5 ± 4.5 nm) with good length-width ratio. Then, we studied the photothermal conversion efficiency by using 1mL PBS buffer containing approximately 4.4× 1010 particles under a 980 nm NIR laser irradiation (650 mW). As a result, the temperature increased nearly 15oC during 30 min, while that of control samples without any

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AuNRs or without laser irradiation increased only a little, exhibiting good photothermal performance of the AuNRs (Figure 6(b)). Next, our SDNs were used to examine the photothermal effects in HeLa cells. A number of AuNRs (2.2× 1010 particles per cm3) were incubated with HeLa cells dishes in culture medium. TEM images indicated that AuNRs were present inside the cells (Figure S11). Then, different irradiation time of 0 min, 4 min, 8 min, 12 min, 15 min were investigated by using a peripheral 980 nm NIR laser (650 mW) respectively. HeLa cells without AuNRs served as the control samples. Finally, confocal fluorescence imaging was quickly collected under the excitation wavelength at 488 nm. As shown in Figure 6(c), the red fluorescence intensity of experiment sample with 15 min irradiation largely declined, suggesting weak FRET proficiency, while that of the control sample still kept bright. Furthermore, the correlation curve between average fluorescence A/D intensity and different irradiation time was calculated, indicating that temperature change lower after longer irradiation time (Figure S12). It demonstrated the temperature variation of the cells treated with AuNRs after laser irradiation, can be well sensed by our SDN-1. Therefore, the SDNs can be a helpful tool for intracellular temperature detection. CONCLUSIONS In summary, we developed a scallop-inspired DNA nanomachine as a ratiometric nanothermometer for intracellular temperature sensing. By introducing thermal-sensitive MB hairpin structure into DNA tetrahedron, this SDN can simulate the scallop to perform open and close behaviors due to “below” and “over” the melting temperature. This nanotherometer possesses many advantages. First, the SDN has good structural rigidity, high cellular permeability, excellent biocompatibility and reversibility. Second, the SDN not only assists thermal-responsive MB to function well, but also play an important role in resisting cellular

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nuclease attack, showing high resistance to enzymatic degradation. Third, benefit from our design, it has good programmability with tunable temperature response range, which could be used for multiplex applications. Forth, it can enter into cells without any transfection agents, which can reduce experiment steps and additional cost. Fifth, compared to conventional singledye labeled systems, FRET-based system allows ratiometric measurement with high temperature resolution, and eliminate signal fluctuation induced by complicated surroundings. Therefore, our SDN nanothermometer can be as a promising tools for temperature sensing in living cells, and promote the development of cell biology and disease studies.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. All DNA sequences, the gel and DLS characterization of SDN, the studies of pH, ion strength and complicated biological environment, the nuclease resistance study of SDN, MTT, fluorescence analysis of different SDN, the characterization of gold rods (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; *E-mail: [email protected] Notes

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The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21675046, 21675047 and 21735002), the National Natural Science Foundation of Hunan Province (2017JJ2039), the Foundation for Innovative Research Groups of NSFC (21521063) and the Fundamental Research Funds for the Central University. REFERENCES (1) Lowell, B. B.; Spiegelman, B. M. Nature 2000, 404, 652-660. (2) DeBerardinis, R. J.; Lum, J. J.; Hatzivassiliou, G.; Thompson, C. B. Cell Metab. 2008, 7, 11-20. (3) Lal, S.; Clare, S. E.; Halas, N. J. Acc. Chem. Res. 2008, 41, 1842-1851. (4) Bai, T.; Gu, N. Small 2016, 12, 4590-4610. (5) Jaque, D.; Vetrone, F. Nanoscal 2012, 4, 4301-4326. (6) Chen, Y.; Wood, A. W. Bioelectromagnetics 2009, 30, 583-590. (7) Zohar, O.; Ikeda, M.; Shinagawa, H.; Inoue, H.; Nakamura, H.; Elbaum, D.; Alkon, D. L.; Yoshioka, T. Biophys. J. 1998, 74, 82-89. (8) Huang, H.; Delikanli, S.; Zeng, H.; Ferkey, D. M.; Pralle, A. Nat. Nanotechnol. 2010, 5, 602-606. (9) Homma, M.; Takei, Y.; Murata, A.; Inoue, T.; Takeoka, S. Chem. Commun. 2015, 51, 6194-6197. (10) Donner, J. S.; Thompson, S. A.; Kreuzer, M. P.; Baffou, G.; Quidant, R. Nano Lett. 2012, 12, 2107-2111.

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(11) Zhu, H.; Li, Y.; Qiu, R.; Shi, L.; Wu, W.; Zhou, S. Biomaterials 2012, 33, 3058-3069. (12) Fischer, L. H.; Harms, G. S.; Wolfbeis, O. S. Angew. Chem. Int. Ed. 2011, 50, 45464551. (13) Okabe, K.; Inada, N.; Gota, C.; Harada, Y.; Funatsu, T.; Uchiyama, S. Nat. Commun. 2012, 3, 705-712. (14) Tsuji, T.; Yoshida, S.; Yoshida, A.; Uchiyama, S. Anal. Chem. 2013, 85, 9815-9823. (15) Suzuki, M.; Tseeb, V.; Oyama, K.; Ishiwata, S. Biophys. J. 2007, 92, L46- L48. (16) Van Houten, J.; Watts, R. J. J. Am. Chem. Soc. 1976, 98, 4853-4858. (17) Li, S.; Zhang, K.; Yang, J. M.; Lin, L.; Yang, H. Nano Lett. 2007, 7, 3102-3105. (18) Hsia, C. H.; Wuttig, A.; Yang, H. ACS Nano 2011, 5, 9511-9522. (19) Albers, A. E.; Chan, E. M.; McBride, P. M.; Ajo-Franklin, C. M.; Cohen, B. E.; Helms, B. A. J. Am. Chem. Soc. 2012, 134, 9565-9568. (20) Ye, F.; Wu, C; Jin, Y.; Chan, Y.; Zhang, X.; Chiu, D. J. Am. Chem. Soc. 2011, 133, 8146-8149. (21) Xu, C.; Zipfel, W.; Shear, J. B.; Williams, R. M.; Webb, W. W. Proc. Natl. Acad. Sci. USA 1996, 93, 10763-10768. (22) Ke, G.; Wang, C.; Ge, Y.; Zheng, N.; Zhu, Z.; Yang, C. J. J. Am. Chem. Soc. 2012, 134, 18908-18911. (23) Ebrahimi, S.; Akhlaghi, Y.; Kompany-Zareh, M.; Rinnan, A. ACS Nano 2014, 8, 10372-10382. (24) Gareau, D.; Desrosiers, A.; Vallee-Belisle, A. Nano Lett. 2016, 16, 3976-3981. (25) Mao, C.; Liu, A.; Cao, B. Angew. Chem. Int. Ed. 2009, 48, 6790-6810.

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(26) Wang, Y.; Ju, Z.; Cao, B.; Gao, X.; Zhu, Y.; Qiu, P.; Xu, H.; Pan, P.; Bao, H.; Wang, L. ACS nano 2015, 9, 4475-4483. (27) Tay, Y. Chor.; Yuan, L.; Leong, D. T. ACS nano 2015, 9, 5609-5617. (28) Song, S.; Liang, Z.; Zhang, J.; Wang, L.; Li, G.; Fan, C. Angew. Chem. Int. Ed. 2009, 48, 8670-8674. (29) Barilero, T.; Saux, T. Le; Gosse, C.; Jullien, L. Anal. Chem. 2009, 81, 7988-8000. (30) Goodman, R. P.; Schaap, I. A. T.; Tardin, C. F.; Erben, C. M.; Berry, R. M.; Schmidt, C. F.; Turberfield, A. J. Science 2005, 310, 1661-1665. (31) Giovanni, M.; Setyawati, M. I.; Tay, C. Y.; Qian, H.; Kuan, W. S.; Leong, D. T. Adv. Funct. Mater. 2015, 26, 3840-3846. (32) Li, J.; Pei, H.; Zhu, B.; Liang, L.; Wei, M.; He, Y.; Chen, N.; Li, D.; Huang, Q.; Fan, C. ACS Nano, 2011 5, 8783-8789. (33) Zhu, D.; Pei, H.; Yao, G.; Wang, L.; Su, S.; Chao, J.; Wang, L.; Aldalbahi, A.; Song, S.; Shi, J.; Hu, J.; Fan, C.; Zuo, X. Adv. Mater. 2016, 28, 6860-6865. (34) Pei, H.; Liang, L.; Yao, G.; Li, J.; Huang, Q.; Fan, C. Angew. Chem. Int. Ed. 2012, 51, 9020-9024. (35) Xie, N.; Huang, J.; Yang, X.; Yang, Y.; Quan, K.; Wang, H.; Ying, L.; Ou, M.; Wang, K. Chem. Commun. 2016, 52, 2346-2349. (36) Xie, N.; Huang, J.; Yang, X.; Yang, Y.; Quan, K.; Ou, M.; Fang, H.; Wang, K. ACS Sens. 2016, 1, 1445-1452. (37)

Goodman, R. P.; Heilemann, M.; Doose, S.; Erben, C. M.; Kapanidis, A. N.;

Turberfield, A. J. Nat. Nanotechnol. 2008, 3, 93-96.

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(38) Baleizão, C.; Nagl, S.; Borisov, S. M.; Schäferling, M.; Wolfbeis, O. S.; BerberanSantos, M. N. Chem. Eur. J. 2007, 13, 3643 –3651. (39) Gota, C.; Okabe, K.; Funatsu, T.; Harada, Y; Uchiyama, S. J. Am. Chem. Soc. 2009, 131, 2766-2767. (40) Jaque, D.; Rosal, D. B.; Rodriguez, M. E.; Maestro, M. L.; Haro-Gonzalez, P.; Sole, G. J. Nanomedicine 2014, 9, 1047-1062. (41) Yang, J.; Yang, H.; Lin, L. ACS Nano 2011, 5, 5067-5071. (42) Liang, L.; Li, J.; Li, Q.; Huang, Q.; Shi, J.; Yan, H.; Fan, C. Angew. Chem. Int. Edit. 2014, 53, 7745-7750. (43) Jain, P. K.; Huang, X.; EI-Sayed, I. H.; EI-Sayed, M. A. Acc. Chem. Res. 2008, 41, 1578-1586. (44) Skrablak, S. E.; Chen, J.; Sun, Y.; Lu, X.; Au, L.; Cobley, C. M.; Xia, Y. Acc. Chem. Res. 2008, 41, 1587-1595. (45) Maestro, M. L.; Haro-González, P.; Iglesias-de la Cruz, M. C.; SanzRodríguez, F.; Juarranz, Ángeles; Solé, J. G.; Jaque, D. Nanomedicine 2013, 8, 379-388. (46) Tapan, K. S.; Catherine, J. M. J. Am. Chem. Soc. 2004, 126, 8648-8649.

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For TOC only:

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Figure 1. Design and working principle of the SDN. (a) Illustration of the scallop, where the shell can provide essential protection from the harsh environment and respond quickly to external stimuli with the aid of central adductor muscle. Like a mechanical spring, the adductor muscle contracts, the shell closes; the muscle relaxes, the shell opens. (b) The dual-labeled thermal-responsive MB is embedded in one edge of six edges of the DNA tetrahedron. Relying on the temperature-responsive hairpin structure and the FRET signal mechanism, the FRET “On” to “Off” signal is reversibly responding to “below” and “over” the melting temperature. The donor dye is 6’-Carboxyfluorescein (FAM) and the acceptor dye is tetramethyl rhodamine (TAMRA).

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Figure 2. The principle illustration of self-assembled DNA tetrahedron. The DNA tetrahedron is composed of four strands (P1, P2, P3 and P4), P3 is a 74-78 nt strand containing temperatureresponsive MB hairpin structure. P4 is a 53 nt strand which is labelled with FAM at 5’ terminal and TAMRA at 3’ terminal, respectively.

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Figure 3. (a) Blue line is the melting curves of the SDN-1, as a function of temperature. The fluorescence emissions were collected under the laser excitation at 484 nm. The normalized curve was plotted by using the A/D ratios as Y axis. The A/D intensities were normalized to the value at 54oC. Red dotted line is relative temperature resolution. (b) The normalized fluorescence A/D ratio reversibly changes between 27oC and 54oC over 50 cycles, showing good reversibility of the SDN. The intensity of Y axis is normalized to the value at 54oC.

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Table 1. A comparison table of different fluorescent thermometry systems.

Name

Response range/oC

Resolution/oC

Sensitivity

λexc/nm

λem/nm

Polymer plus CdSeCdS

20-40

0.2

2.4%, K-1

400

650

Nanogel

25-40

0.5

NA

456

560

Fluorescent polymer

20-50

0.2-0.6

NA

456

565

Genetically encoded GFP

20-50