DNA nanodevices have been feasibly applied for various chemo-biological applications, but their functions as precise regulators of intracellular organelles are still limited. Here, we report a synthetic DNA binder that can artificially induce mitochondrial aggregation and fusion in living cells. The rationally designed DNA binder consists of a long DNA chain, which is grafted with multiple mitochondria-targeting modules. Our results indicated that the DNA binder-induced in situ self-assembly of mitochondria can be used to successfully repair ROS-stressed neuron cells. Meanwhile, this DNA binder design is highly programmable. Customized molecular switches can be easily implanted to further achieve stimuli-triggered mitochondrial aggregation and fusion inside living cells. We believe this new type of DNA regulator system will become a powerful chemo-biological tool for subcellular manipulation and precision therapy.
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Controllable mitochondrial aggregation and fusion by a programmable DNA binder
https://doi.org/10.21203/rs.3.rs-1595440/v1
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DNA nanotechnology has been widely used to accomplish intelligent processes in many fields including synthetic soft materials, bio-computing, sensing and bioimaging, due to their outstanding programmability, high flexibility and diverse structures1-3. Recently, DNA nanodevices have become a hotspot in exploiting manipulation strategies in bio-processes, such as substrate channeling, cell migration, immune activation and cell identification4-7. The specificity and well predictability of Watson-Crick-Franklin base pairing provides the fidelity of constructing the responsively artificial DNA nano-machines by catalytic hairpin assembly, proximity-based ligation, rolling circle amplification (RCA), hybridization chain reaction (HCR)8-11. Furthermore, convenient modification on DNA strands with various functional organic molecules greatly expanded their applications to manipulate cascade enzymatic reactions, cell surface receptors assembly and cell-cell interactions4-6. For example, cholesterol-functionalized DNA nanostructures have been applied for cell surface engineering to realize adaptive cellular interactions12-14. DNA nanomachines demonstrate precise controllability and robust operation with effective biological outputs in extracellular environment15,16. However, a large number of biological processes occurs inside cells, the development of DNA-based manipulation of intracellular network still remains a big challenge.
Mitochondria are intracellular organelles playing a critical role in energy production and proliferation17. Under oxidative stress, the dynamic fusion-fission process of mitochondria can be used to regulate metabolism: fission facilitates cell apoptosis, whereas, fusion can mitigate oxidative stress via consuming the damaged mitochondria18. As an important communication process among mitochondria in the cytosol, mitochondrial fusion (mito-fusion) is regulated by membrane-anchored dynamics, of which Fzo1 heptads from two adjacent mitochondria form coiled-coil structures to tether the membrane19. Abnormal mito-fusion can cause neurodegenerative diseases, such as Charcot-Marie-Tooth 2A and dominant optic atrophy20. Traditional mito-therapeutic strategy is normally based on modulating the expression of fusion proteins, which requires either delicate gene engineering process or screening of organic molecules21-22. Recently, Wang and coworkers reported another approach for the direct induction of mitochondrial fusion based on host-guest supramolecular assembly, which coins the artificial manipulation of mito-fusion as an alternative therapeutic strategy23. However, designing molecular tools that can achieve controllable assemblies of mitochondria and precise manipulation of the fusion processes remains largely underdeveloped. In this work, a new DNA nanomachine will be introduced to fill this gap.
We designed herein a DNA binder that contains multiple mitochondrial targeting modules for the controllable manipulation of mitochondrial aggregation and fusion in living cells. Such DNA binder-mediated artificial handing of mitochondria can also result in mitigated reactive oxygen species (ROS) levels and cell repairs (Fig. 1a,b). By further taking advantages of the highly programmable DNA architectures, molecular switches could be integrated into the DNA binder to achieve precise ATP-controlled mitochondrial fusion. In addition, a variety of stimuli-triggered process could also be rationally activated using this approach. We believe the DNA binder described in this study opens a new avenue for effective and easy manipulation of intracellular organelles and related metabolism.
Fabrication and in-vitro performance of DNA binders. To develop mitochondria-targetable DNA binder, a long single-stranded DNA chain was first prepared by a phi29 polymerase-catalyzed RCA reaction, which consists of 48-nt-long repeated sequences containing 24-nt (Supplementary Table 1) that can be recognized by multiple triphenylphosphonium (TPP)-modified linkage DNAs (TPP-LDNAs) (Fig. 1a). Here, the TPP cation is used to target mitochondrial and induce mito-fusion24. TPP-LDNA was synthesized based on a copper-catalyzed azide/alkyne cycloaddition reaction25 between an azide-modified linkage DNA and an alkynyl-functionalized TPP cation (Supplementary Fig. 1-4). After hybridization between the DNA chain and TPP-LDNA, the successful preparation of the DNA binder was confirmed by 10% denature polyacrylamide gel electrophoresis and atomic force microscopy (Fig. 1c,d). The average length of the DNA binder was appeared to be 500±150 nm. Given that 48-nt-long repeated sequences were about 16.3 nm26, 31±9 TPP-LDNA repeating units could be counted for each DNA binder. A 93% grafting ratio of TPP-LDNA on the DNA chain was further estimated by fluorescence measurements (Supplementary Fig. 5), sufficient mito-targeting modules existed in the DNA binder.
We first wanted to test if the designed DNA binder can be used to induce mitochondrial aggregation and fusion in an extracellular experiment. As shown in Fig. 2a, the addition of either TPP-LDNA alone or a non-TPP-modified DNA chain/LDNA complex (C-binder) did not change the size or aggregation status of mitochondria. While in contrast, when mitochondria were incubated with the DNA binder (binder), particle stacking with an integrated morphology could be clearly observed, suggesting a DNA binder-induced aggregation and fusion of the mitochondria. A 15-fold enlargement in the area of mitochondrial aggregates was displayed upon the treatment of DNA binder (Fig. 2b), which was in accordance with the dynamic light scattering results (Fig. 2c). The manipulation of extracellular mitochondrial behavior can be further visualized in solution by using a mitochondria tracker red dye (MitoTracker). Under confocal fluorescence microscopy, only when the DNA binder was added, the aggregation of mitochondria can be observed (Supplementary Fig. 6). All these above results confirmed that the DNA binder could successfully induce mitochondrial aggregation in vitro. Both mito-targeting TPP moieties and the long DNA chain are essential for the function of DNA binder.
The performance and therapeutic results of DNA binder in living cells. Next, we studied if the DNA binder can be further used to regulate mitochondrial aggregations in living cells. After demonstrating the minimal cell toxicity of the DNA binder (Supplementary Fig. 7), these DNA nanodevices were transfected into 4T1 cells by means of liposomes (Supplementary Fig. 8). As shown in Fig. 3a, upon the introduction of 1 µM DNA binders, the mitochondria in the cytosol of 4T1 cells (as stained by the MitoTracker) clearly changed from scattered small spheres (~0.5 µm diameter) into elongated structures (≥2 µm length). The percentage of cells bearing these elongated mitochondria increased from 5% to 40% (Fig. 3b). As a control, the addition of either TPP-LDNA or C-binder did not change the cellular mitochondrial size or shape (Fig. 3a,b). Transmission electron microscopy images have further evidenced the DNA binder-induced formation of fused mitochondria with increased lengths (Fig. 3c,d). Thus, the DNA binder can indeed be used to effectively manipulate mito-aggregation and fusion in living cells.
We next asked whether the DNA binder could be potentially used as a therapeutic tool in neurodegenerative diseases, which are known to be closely associated with the abnormal mitochondrial fusion process. Human neuroblastoma SH-SY5Y cells were selected as a model disease cell line, since after the treatment with 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), these SH-SY5Y cells generate ROS, exhibit interrupted mitochondrial metabolism and the symptoms of Parkinson’s diseases27. Indeed, the MPTP-treated SH-SY5Y cells exhibited enhanced ROS levels (Fig. 4a,b). While after the addition of 2–8 µM DNA binders, the cellular generation of ROS was gradually decreased to that of the blank sample. Meanwhile, an obvious increase in the SH-SY5Y cell viability (from 55% to 87%) was shown after adding the DNA binders (Fig. 4c).
Confocal fluorescence microscopy was also utilized for the visual evaluation of subcellular status upon different treatment. As shown in Fig. 4d, after adding MPTP, stress-induced mito-fission resulted in obviously more mitochondrial fragmentations as compared to the blank sample23. While the introduction of DNA binders in these MPTP-pretreated cells can instead result in distinct mitochondrial aggregations and elongations. Therefore, the DNA binder could be considered as an artificial cell repair tool for mitochondria-related therapy.
The controlled manipulation of mitochondria aggregation and fusion by DNA binder in living cells. To further study if the DNA binder could be engineered as a molecular switch to achieve stimuli-triggered mitochondrial aggregation, we chose an ATP-recognizing aptamer as a proof-of-concept (Fig. 5a). This 27-nt-long ATP aptamer was first divided into two parts, named as SA1 and SA228,29, and labelled with a TAMRA dye and a BHQ2 quencher, respectively (Supplementary Table 1). Due to the fluorescence resonance energy transfer30, the fluorescence of TAMRA-SA1 could be efficiently quenched by the nearby BHQ2 upon the formation of an ATP/SA1/SA2 complex (Supplementary Fig. 9a). By integrating repeated SA2 sequences into the long RCA chain, a responsive DNA chain (R-DNA chain) was generated (Supplementary Table 1). Further fluorescence experiment verified that ATP could be used to selectively induce the self-assembly of R-DNA chain and TAMRA-SA1, and as a result, to develop an ATP-triggered responsive DNA binder (R-binder) (Supplementary Fig. 9b).
As shown in Fig. 5b,c and Supplementary Fig. 10, in the presence of ATP, R-binder can induce an efficient in vitro aggregation of mitochondria with increased particle sizes and integrated morphology. As a control, R-binder itself (no ATP) resulted in no morphological changes. These results have been further validated in living 4T1 cells, as the formation of elongated mitochondria can be triggered by intracellular ATP (Fig. 5d,e). To further confirm that the ATP-triggered mitochondrial aggregation and fusion, we treated the cells with oligomycin, a commonly chemical inhibitor that is known to suppress the expression of ATP in cells29. As expected, after adding oligomycin, no obviously morphological changes were observed in cells (Supplementary Fig. 11). As another control, by replacing the SA1 with a non-ATP aptamer sequence (CSA1), no mitochondrial aggregation was observed both in vitro and in living cells (Fig. 5b,c and Supplementary Fig. 11). Taken together, these results demonstrated that aggregation and fusion of mitochondria were precisely controlled by the ATP.
DNA nanodevices has been used to achieve artificial intelligent operations in material science and living systems. However, the manipulation of subcellular organelles in living cells via DNA nanodevices still remains challenging. Here, by integrating both biochemical synthesis and DNA structure design, we report a novel DNA-based nanomachine, termed DNA binder, to manipulate the aggregation and fusion of intracellular mitochondria.
The DNA binder was constructed via RCA-based one-dimensional backbone grafted with many branch DNA strands tagged with TPP as mito-targeting moiety. The DNA binder system has several advanced features for precisely controlling the mitochondrial aggregation. (1) The DNA binder is designed by DNA building blocks, providing the nanomachine with good biocompatibility. (2) Multiple mito-targeting TPPs are localized in the confined space of long RCA backbone to facilitate efficient binding of adjacent mitochondria and the resulting cell therapy. (3) The DNA binder could be deliberated programmed to achieve customized manipulation process of mitochondria aggregation. By simply changing the grafted DNA strand into a split aptamer of ATP, the DNA binder could perform the ATP triggered mitochondrial aggregation. By introducing DNAzyme31, toehold-mediated strand displacement32 or aptamer33, other important biotargets, such as metal ions, miRNAs and proteins could also be engineered as the triggers for in situ assembly of DNA binder. These controlled manipulation process could be a nano-tool for further investigating the metabolism and apoptosis of mitochondria related bioprocess. As a result, DNA binder is a universal programmable platform for manipulation of subcellular organelles in living cells.
The easy modification and hydrogen binding properties of DNA binder may also have potential application in cell imaging and drug delivery and precise therapy. Other cell therapeutic molecules could be modified on DNA binder to achieve possible synergistic effect and cell imaging, such as photodynamic34 and photoacoustic35 organic molecules. The conjugated planer structure of double stranded structure and oligonucleotides on DNA binder could be the suitable docking sites for drug delivery, endowing DNA binder as the multi-functional therapeutic machine.
In sum, we report here a programmable DNA-based molecular tool for the manipulation of mitochondrial aggregation and fusion in living cells. The designed DNA binder could efficiently gather mitochondria together based on multivalent mito-targeting modules. The DNA binder may potentially even be used as an alternative strategy for cell therapy. By redesigning a stimuli-responsive DNA binder, we demonstrated that ATP could be used as a switch to achieve regulatable mitochondrial aggregation. The highly editable and predictable nature of Watson-Crick-Franklin base pairing endows the DNA binder as a new versatile platform for regulating intracellular metabolism, which also further extended the functions of existing DNA nanodevices.
Reagents. All the chemicals were purchased from Sigma unless otherwise noted. Commercial reagents were used without further purification. 4',6-Diamidino-2-Phenylindole (DAPI) and SYBR Gold were purchased from Invitrogen (Carlsbad, CA, USA). Mitochondrial extraction kit was obtained from Abbkine Scientific Co., Ltd (USA). Mitochondrial tracker red (MitoRed) purchased from KeyGEN Biotech Co., Ltd (Nanjing, China). Phi29 DNA polymerase, T4 DNA ligase, exonuclease I, exonuclease III and dNTPs were purchased from New England Biolabs Ltd (USA). DNA oligonucleotides were synthesized and purified by Sangon Biotech Co., Ltd (Shanghai, China). The detailed sequences have been listed in Table S1. All aqueous solutions were prepared using ultrapure water (Z18 MO, Milli-Q, Millipore).
Apparatus. The concentrations of nucleic acids were all measured using a NanoDrop One UV-Vis spectrophotometer. The gel electrophoresis was performed on a Tanon EPS-300 Electrophoresis Analyser (Tanon Science & Technology Company, China) and imaged on Bio-rad ChemDoc XRS (Bio-Rad, USA). All the in vitro fluorescence measurements were measured on an F-7000 spectrometer (HITACHI, Japan). All the intracellular images were taken by a Nikon A1& SIM-S&STORM super resolution microscope (Tokyo, Japan). ROS levels and cell viabilities were measured with a Safire microplate Analyzer (Molecular Devices, America). AFM imaging was performed under ambient conditions with a Dimension Icon Atomic Force Microscopy (Bruker, Germany). Dynamic lights scattering (DLS) was observed on a 90 Plus/BI-MAS equipment (Brookhaven, USA). Scanning electron microscopy (SEM) images were recorded using a JEOL JSM-7800F field emission scanning electron microscope. MALDI-TOF MS was carried out by ABSCIEX MALDI TOF-TOF 4800 plus.
Synthesis of alkynyl TPP cation (but-3-yn-1-yltriphenylphosphonium). Triphenylphosphine (520 mg, 2mmol) and 4-Bromobutyne (400 mg, 3 mmol) was dissolved in acetonitrile (20 mL). The reaction mixture was heated to 80 oC for 72 h under nitrogen. The solvent was removed at room temperature followed by addition of benzene (80 mL). The resulting mixture was cooled to -20 oC for 0.5 h and the product was filtered off as white solid (512 mg, 82% yield). The spectroscopic data was in agreement with previous reports36.
Synthesis of TPP-LDNA. The TPP-LDNA was synthesized by adding 5 mM (5 μL) alkynyl TPP cation to 100 μM (200 μL) of linkage DNA (or SA1). The mixture was diluted with 250 μL DMSO before addition of 40 μL (50 mM) ascorbic acid. The cycloaddition reaction between azide group of linkage DNA (or SA1) and alkynyl group of TPP cation was initiated by adding 10 mM (50 μL) CuSO4 stock solution. The reaction was incubated for 24 hours and the resulting TPP-LDNA was then purified by gel filtration37. MALDI-TOF MS results confirmed the successful synthesis of TPP-LDNA (Supplementary Fig. 4).
Preparation of circular DNA (circ-DNA). For the synthesis of circ-DNA1, 4.2 μL of 100 μM phosphorylated linear DNA1 and ligation DNA1 were mixed at a ratio of 1:1. After the annealing procedure, 1 μL of T4 DNA ligase (400 U/μL), 2 μL of 10×T4 DNA buffer and 8.6 μL of ultrapure water were added for 16 h-incubation at 25 °C. Then, the mixture was heated at 65 °C for 10 min to inactivate T4 DNA ligase. After reaction, 4 μL of exonuclease I (20 U/μL) and 4 μL of exonuclease III (100 U/μL) were added, and the mixture was incubated at 37 oC for 8 h to degrade ligation DNA1. Afterward, the mixture was incubated at 80 oC for 15 min to denature the exonuclease I and exonuclease III. The circ-DNA1 was generated and stored at 4 oC before use. The circ-DNA2 was obtained by using phosphorylated linear DNA2 and ligation DNA2 following the same approach.
Preparation of DNA chain. The DNA chain was synthesized by rolling circle amplification (RCA)38. 10 μL of 3 μM circ-DNA1 (or circ-DNA2) and 0.5 μL of 100 μM DNA primer1 (or DNA primer2) were mixed and annealed at 95 oC for 4 min, then gradually cooled to 25 oC at a rate of 1 oC/min. The mixture was incubated with phi29 DNA polymerase (0.2 U/μL), BSA (0.4 μg/μL) and dNTPs (0.1 mM) at 37 oC for 5 h in 150 μL of 1×Phi29 reaction buffer. The RCA reaction was terminated through heated at 65 oC for 10 min, then purified by 30 kDa MWCO ultrafiltration device (Millipore) to obtain the long DNA chain (R-DNA chain). DNA chain has repeating strand segments with sequences complementary to circ-DNA. The concentration of the repeat unit in DNA chain was obtained by measuring ultraviolet absorbance at 260 nm and used as the concentration of DNA chain39.
Preparation of DNA binder. 25 μL of 10 μM TPP modified linkage DNA (TPP-LDNA) and 300 μL of DNA chain were mixed together for 2 h at 37 oC to synthesize the DNA binder. The prepared DNA binder was purified by 30 kDa MWCO ultrafiltration device (Millipore) for three times. The concentration of TPP-LDNA was used as DNA binder. As controls, the C-binder was obtained by using linkage DNA and DNA chain following the same approach.
To measure the amounts of linkage DNA in each DNA binder, 5-Carboxyfluorescein (FAM)-labelled linkage DNA (FAM-LDNA) was used as components to synthesize a fluorescent DNA binder (F-binder). From the fluorescence intensities of FAM in DNA binder and the standard calibration curves for FAM-LDNA, its amounts could be obtained.
The responsive DNA binder (R-binder) that was able to activate with ATP was constructed by using TPP modified SA1 (TPP-SA1) and R-DNA chain. As controls, no responsive DNA binder (NR-binder) that was unable to activate with ATP was constructed by using TPP modified negative control SA1 (TPP-CSA1) and R-DNA chain.
To demonstrate the response of the split ATP aptamer to ATP, 2 μM tetramethylrhodamine labeled SA1 (TAMRA-SA1) or tetramethylrhodamine labeled CSA1 (TAMRA-CSA1), and 2 μM black hole quencher (BHQ2) labeled SA2 (BHQ-SA2) with various concentrations of ATP (CTP, GTP or UTP) were used and incubated for 1 hour at 37 oC. Fluorescence measurements were performed with excitation at 550 nm and emission spectra were collected in the range of 570–650 nm. The slit width was set to be 2 nm for both excitation and emission.
To characterize the ATP activated R-binder response, 2 μM TAMRA-SA1 and 2 μM R-DNA chain with 4 mM ATP were used and incubated at 37 oC for 1 hour. Afterward, the solution was purified by ultrafiltration (30,000 MW cut off membrane, Millipore) for three times. The fluorescence measurement of concentrate and filtrate at the third ultrafiltration were performed with excitation at 550 nm and emission spectra were collected in the range of 570–650 nm. All the data were plotted using the Origin software.
Polyacrylamide gel electrophoresis Analysis. 10% native polyacrylamide gel was prepared using 1×TBE buffer. The loading samples were prepared by mixing 7.5 μL DNA samples and 1.5 μL 6×loading buffer, and placed for 3 min before injected into polyacrylamide gel. The gel electrophoresis was run at 100 V for 60 min in 1×TBE buffer, stained with 1×SYBR Gold, and scanned with a Molecular Imager Gel Doc XR.
AFM imaging. 6 µL DNA binder was deposited on freshly cleaved mica surface and incubated for 10 min. After the solvent water was absorbed with filter paper, 20 µL ultrapure water was added to wash the mica for three times. The mica was then dried with a nitrogen flow and scanned in tapping mode.
Cell culture. The SH-SY5Y and 4T1 cell lines (Procell Life Science & Technology, Wuhan, China) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 μg/mL streptomycin and 100 U/mL penicillin-streptomycin at 37 oC in a humidified incubator containing 5% CO2 and 95% air. Short tandem repeats (STR) profiling and mycoplasma testing were conducted for each cell line before use. Cell numbers were determined with a Petroff-Hausser cell counter (USA).
MTT assay. MTT assays were performed to investigate DNA binder biocompatibility. SH-SY5Y and 4T1 cell lines were respectively seeded within replicate 96-well plates at a density of 1×106 cells/well. Plates were maintained at 37 °C for 24 h. After discarding the medium, the cells were washed twice with PBS and incubated with serial concentrations of the DNA binder transfected with Lipofectamine® 2000 transfection reagent. Cells incubated with only the PBS was served as control. After 24 hour-incubation, the cells were washed twice with PBS buffer and 100 μL of a fresh medium containing 10 μL of MTT (5 mg/mL) was added and incubated for 4 h. After removing the remaining MTT solution, 100 μL of dimethylsulphoxide was added to dissolve the formazan crystals precipitates. After shaking the plate for 10 min, the optical density at a wavelength of 490 nm was measured with a Safire microplate Analyzer.
Measurement of the ROS generation. Human neuroblastoma SH-SY5Y cells were seeded in a 6-well plate at a density of 50000 cells per well for 24 h at 37 oC. Then, incubated with 1 mM MPTP in the absence and in the presence of 8 μM C-binder or 2 μM, 4 μM or 8 μM of DNA binders transfected with Lipofectamine® 2000 transfection reagent (Thermo Fisher Scientific) for another 24 h. After washing with PBS for three times, the cells were stained with DCFH-DA and measured by a Safire microplate Analyzer.
Cellular imaging and data analysis. 1×104 4T1 cells were seeded in a confocal dish for 24 h at 37 oC, then transfected, respectively, with 1 μM binder, TPP-LDNA, C-binder, or F-binder using a Lipofectamine® 2000 transfection reagent (Thermo Fisher Scientific). 24 h after transfection, the cells were washed twice with Dulbecco’s phosphate-buffered saline (DPBS) and stained with 200 nM of mitochondrial tracker red and 5 mg/mL of DAPI for 15 min for imaging.
To demonstrate the potential capability of DNA binder for molecular mitochondrial aggregation/fusion, 1×104 human neuroblastoma SH-SY5Ycells were seeded in a confocal dish for 24 h at 37 oC, then incubated with 1 mM MPTP in the absence and in the presence of 1 μM DNA binder transfected with Lipofectamine® 2000 transfection reagent (Thermo Fisher Scientific) for another 24 h. Afterward, the cells were washed twice with DPBS and stained with 200 nM of mitochondrial tracker red and 5 mg/mL of DAPI for 15 min for imaging.
For the ATP controlled mitochondrial aggregation/fusion experiment, 1×104 4T1 cells were seeded in a confocal dish for 24 h at 37 oC, then transfected, respectively, with 1 μM TPP-SA1 and R-DNA chain, TPP-CSA1 and R-DNA chain, or TPP-SA1, R-DNA chain and 10 μM oligomycin using a Lipofectamine® 2000 transfection reagent (Thermo Fisher Scientific). After incubation for 24 h, the cells were washed with DPBS and stained with 200 nM of mitochondrial tracker red and 5 mg/mL of DAPI for imaging.
All the fluorescence images were collected with Nikon A1& SIM-S&STORM super resolution microscope. FAM, mitochondrial tracker red and DAPI were excited with 488 nm, 640 nm and 405 nm lasers, respectively. A 100× oil immersion objective was used for imaging cells. Image analysis was performed with a NiS-Elements AR Analysis software. Data analysis was done using the Origin and GraphPad Prism software.
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Acknowledgements
This work was supported by the National Key Research and Development Program of China for International Science & Innovation Cooperation Major Project between Governments (Grant No. 2018YFE0113200), National Natural Science Foundation of China (21775072, 21874071, 22104058, 22174066), and the Natural Science Foundation of Jiangsu Province (BK20200459).
Author contributions
K.W.R. and Y.W. initiated the projects. L.Y.Z., S.Y.D., Y.W. and K.W.R. conceived the experiments. L.Y.Z. and Y.T.S. performed the experiments. Y. S and J.L. provided experimental advice. L.Y.Z., and K.W.R. analyzed the data and co-wrote the manuscript. K.W.R., Y.W., M.X.Y. and C.H.F. supervised and coordinated all investigators for this project. All authors discussed the results and commented on the manuscript.
Additional information
Supplementary information are available in the online version of the paper. Correspondence and requests for materials should be addressed to K.W.R.
Competing financial interests
The authors declare no competing financial interests.
There is NO Competing Interest.
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Supplementary information