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Reversible Activation of pH-Responsive Cell-Penetrating Peptides in Model Cell Membrane Relies on the Nature of Lipid Xia Hu, Junjun Tan, and Shuji Ye J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b03092 • Publication Date (Web): 26 Jun 2017 Downloaded from http://pubs.acs.org on June 26, 2017

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The Journal of Physical Chemistry

Reversible Activation of pH-Responsive Cell-Penetrating Peptides in Model Cell Membrane Relies on the Nature of Lipid

Xia Hu,1,2 Junjun Tan,1,2 Shuji Ye,1,2,*

1

Hefei National Laboratory for Physical Sciences at the Microscale, and Department of

Chemical Physics, & 2Synergetic Innovation Center of Quantum Information & Quantum Physics, University of Science and Technology of China, Hefei, Anhui 230026, China

1

ACS Paragon Plus Environment


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Abstract The pH response of pH- responsive cell-penetrating peptides in cell membrane is directly associated with many potential applications and cell activities such as drug delivery, membrane fusion, and protein folding. But it is still poorly understood. In this study, we used GALA as a model, and applied sum frequency generation vibrational spectroscopy (SFG-VS) to systematically investigate the pH response of GALA in lipid bilayers with different hydrophobic length and lipid head groups. We determined the GALA structures in lipid bilayers by combining second-ordered amide I and amide III spectral signals, which can accurately differentiate the loop and α-helical structures at the interface. It is found that GALA can insert into fluid-phase lipid bilayers even at neutral pH, while lies down on the gel-phase lipid bilayer surface. In acidic conditions, GALA inserts into both fluid-phase and gel-phase lipid bilayers. GALA adopts a mixed loop and α-helical structures in lipid bilayers. Besides, the reversible activation of GALA in lipid bilayers depends on the nature of lipid. After membrane insertion, GALA exits from the negative phosphoglycerol and positive ethylphosphocholine lipid bilayers at neutral pH while does not move out from the zwitterionic phosphocholine lipid bilayers. These findings will help to understand how to enhance the efficacy of drug/gene delivery in cell membrane.

2


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1. Introduction The pH- responsive cell-penetrating peptides (CPPs) are known to be bound to the membrane surface or soluble in aqueous solution with disordered structures at neutral pH while they insert into and penetrate the membrane with α-helical structure at acidic pH conditions.1-3 Such structural transition dynamics has led to several important potential applications of these peptides: 1) working as imaging probes and drug targeting carriers for the diagnostics and treatment of diseases such as cancer;4-8 and 2) serving as a well-defined model system to get insights into the mechanisms of cell penetration, membrane fusion, and protein folding.9-12 Because of the promising biomedical applications and the importance in protein folding, the pH response in structural reconfiguration of such peptides in solution has been extensively studied.12-15 In contrast, the detailed structural information of pH modulation on the CPPmembrane interactions has not been clearly described. However, it is crucial to shed light on the mechanisms of peptide folding/ insertion into membrane, and enhance the efficacy of drug/gene delivery systems as well. In this study, we used GALA as a model, and applied sum frequency generation vibrational spectroscopy (SFG-VS) to investigate the pH response of GALA in model cell membrane. GALA is a 30 amino acid synthetic peptide with a repeating sequence of Glu-Ala-Leu-Ala,6 which is assumed to adopt a random coil structure at pH~7 in solution and at membrane surface due to the repulsive force caused by the negative charge of Glu residues, but form an α-helical structure upon insertion into lipid membranes at acidic pH.1,16-20 GALA targets the membrane with several characters of specific selectivity, efficient endosomolytic 3



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activity, and low cytotoxicity.18-21 It thus provides a ‘benchmark’ model for the site-specific delivery of diagnostic and therapeutic agents. Even though the interfacial behaviors of GALA at the air/water and water/lipid monolayer interface have been investigated by SFG-VS recently,1,16 little is known about the pH response of GALA in lipid bilayers that are the most suitable model for mimicking cell membranes.22,23 A recent study on the conformation of GALA in its fully protonated and deprotonated state in the bulk solution and at the interface indicated that GALA shows different behavior in the bulk and at interface.16 It was found that the fully reversible structural transition occurs in solution while a large fraction of the GALA population remains helical at high pH at the water-air interface.16 Therefore, it is critical to examine the behavior of GALA at the lipid bilayer interface. In addition, to improve the efficiency of GALA-assisted drug delivery, systematic studies are required to examine the dependence of GALA activity on the membrane nature and probe whether the activity of membrane-embedded GALA is reversible when the pH is switched to neutral value.1,24 Furthermore, only amide I ssp spectra were used to determine the interfacial structure of GALA in previous SFG studies.

1,16,17

However, the amide I spectra alone do not permit to

unambiguously distinguish α-helical and loop structures because their vibrations overlap in the frequency at 1655-1660 cm-1.25,

26

The loop-helix transition is a critical step in the

transmembrane process. Herein, we performed a systematic study on investigating the interaction between GALA and lipid bilayers with different hydrophobic length and lipid head groups at different pH conditions. We determined the GALA structures in lipid bilayer by combining second-ordered amide I and amide III spectral signals, which have been demonstrated to be a powerful tool to accurately differentiate the loop and α-helical structures 4


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at the interface. 25, 26 It is found that the association dynamics and pH-driven reversibility of GALA activity depend on the nature of lipid bilayers.

2. Experimental Section 2.1 Materials and Sample Preparations The peptide GALA (sequence: WEAALAEALAEALAEHLAEALAEALEALAA, purity > 98%) was purchased from Shanghai Apeptide Co., Ltd. The phospholipids of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine(DLPC),

phosphocholine

(DPPC),

1,2-dilauroyl-sn-glycero-3

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

(POPC),

1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine(chloride salt) (DPEPC), 1,2-dipalmitoyl -sn-glycero-3-phospho-(1'-rac-glycerol)(sodium

salt)

(DPPG),

1,2-dipalmitoyl-d62-sn-

glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (d-DPPG), 1,2-dilauroyl-sn-glycero-3 -phospho-(1'-rac-glycerol) (sodium salt) (DLPG) were purchased from Avanti Polar Lipids (Alabaster, AL). GALA was dissolved in methanol (purchased from Sinopharm Chemical Reagent Co., Ltd.) at a concentration of 2 mg/ml and kept at -20°C. DPPC, DLPC, DPEPC and POPC were dissolved in chloroform (purchased from Sinopharm Chemical Reagent Co., Ltd.) at a concentration of 1.0 mg/mL. DPPG, d-DPPG and DLPG were dissolved in mixed solvents of chloroform and methanol (with a volume ratio of 65:35) (purchased from Sinopharm Chemical Reagent Co., Ltd.) at a concentration of 1.0 mg/mL. All of the phospholipids solutions were kept at -20°C. The molecular structures of the used phospholipids are shown in Figure S1. The solutions with targeted pH or pD value were prepared by diluting the hydrochloric acid (0.1M) into ultrapure water or deuterated water (D2O). The ultrapure water was produced by Millipore system (Millipore, Bedford, MA). D2O 5



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was ordered from Aldrich (Milwaukee, WI) with a purity of >99.0% and hydrochloric acid was purchased from Sinopharm Chemical Reagent Co., Ltd. All of the chemicals were used as received. Right-angle prisms (CaF2) were purchased from Chengdu Ya Si Optoelectronics Co., Ltd (Cheng Du, China). We cleaned the prisms and prepared lipid bilayers using a standard procedure given in our previous reports.27, 28 pH value of the solution was measured using Testo 206-pH1 (Testo, Germany). 2.2 SFG-VS Experiments SFG-VS is a second-ordered nonlinear optical technique that measures the vibrational spectra of the molecules at the surface and interface in situ and in real time.29-33 It thus permits to determine the interfacial molecular structures and interactions.1,

17, 25-39

It has been

employed to identify the structures and orientation of peptides and proteins in different surfaces and interfaces.1, 17, 25,26, 39-44 The SFG setup in this study is similar to that described in our earlier publications.45 In this research, all SFG experiments were carried out at room temperature (25 °C). The SFG spectra were collected with different polarization combinations of ssp (s-polarized SFG output, s-polarized visible input, and p-polarized infrared input) and ppp. The spectral resolution is 5 cm-1. A near total internal reﬂection geometry (Figure S2) was used to enhance the SFG intensity. All of the SFG spectra were measured after the interaction between GALA and lipid bilayers reached equilibrium. The spectra were averaged over 100 runs at each point and normalized by the intensities of the input IR and visible beams. 3. Results and Discussion 3.1 The pH Response of GALA in Zwitterionic Phosphocholine Lipid Bilayer 6


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We first investigated the interaction between GALA and neutral fluid-phase POPC lipid bilayer. Initially, ∼5 µL of GALA solution (in methanol with a concentration of 2 mg/mL) was injected into the subphase of the lipid bilayer that was prepared in DI water at pH=6.9(denoted as pH1). The amide I and amide III signals were collected after the interaction between GALA and lipid bilayer reached equilibrium. After that, we added a certain amount of HCl (0.1 mol/L, ~25µL) to get a targeted subphase pH of 3.4 (denoted as pH2) and collected amide I and amide III signals in equilibrium again. Finally, we replaced the subphase solution by DI water (pH~ 6.9, denoted as pH3) to examine the capability to switch membrane-embedded GALA off at neutral pH. Figure 1A shows ssp amide I spectra at different pH. The corresponding ppp amide I spectra are put in Figure S3. The amide I spectra at all three pH values are dominated by a peak centered at ~1655 cm-1, indicating both the membrane surface-bound GALA and membrane-embedded GALA adopt a loop or α-helical structures.46 To distinguish the α-helical and loop structures, we measured the SFG spectra in the amide III region. Figure 1B presents the ssp amide III signals at different pH. The amide III spectra show one broad peak at 1255cm-1(denoted as Peak1) and one narrow peak at ~1300 cm-1(denoted as Peak2). These two peaks are assigned to loop and α-helical structures,25,26 respectively, indicating that GALA adopts both loop and α-helical structures in POPC lipid bilayer. Strong amide I and amide III signals have been observed at neutral pH1, implying that GALA already targets the fluid-phase POPC lipid bilayer and undergoes coil-helical transition in membrane at neutral pH, which is rather different from the previous report on GALA in bulk solution that GALA adopts random-coil structure at pH>6.6 In order to qualitatively analyze the changes in Figure 1 and Figure S3, we fitted the spectra using a 7



standard equation, Eq.(S2). Some fitting parameters are given in Table S1. It is evident that ) and 1255 cm-1(χ(2) ) increase more than the fitted strength of the peaks at 1655 cm-1(χ(2) 1655 1255 1.6 times when the pH is changed from pH1=6.9 to pH2=3.4(Table S1). Considering that the ppp and ssp intensity ratio of the ~1655 cm-1 peak keeps almost the same (1.5) at different pH, the intensity increase is mainly contributed by the increase in the molecular number of membrane-bound/embedded GALA in POPC lipid bilayer. GALA forms more loop structures )/(χ(2) ) at pH2=3.4 is larger than that at at acid environment because the ratio of (χ(2) Peak1 Peak2 pH1=6.9(see Table S1). It is worth noticing that the intensity of the ssp and ppp spectra at both amide I and amide III region remains nearly unchanged when the pH is changed from pH2=3.4 to pH3=6.9. This result reveals that GALA does not exit from the POPC lipid bilayer at neutral pH after membrane insertion, which is opposite to the results of GALA at water/DPPC lipid monolayer interface that exhibits reversible activation.1 B

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Figure 1. The ssp SFG spectra in amide I (A) and amide III (B) region after the interaction between GALA and POPC lipid bilayer reaches equilibrium at different pH.

To determine whether the GALA is inserted into the lipid bilayer or not, we further conducted another set of experiments by replacing subphase H2O solution by D2O solution with pD=6.9 and pD=3.4, respectively. Earlier studies have confirmed that the amide proton of peptide residues absorbed at the lipid/water interfaces or in solution can rapidly undergo hydrogen-deuterium exchange when the peptide is exposed to D2O solution, resulting that the frequency of amide III signals shifts to below 1000 cm-1. However, the part inserted into the hydrophobic core of lipid bilayer does not exchange in the 3-4 days.47,48 Therefore, we can use this method to identify insertion of GALA into POPC bilayer by observing whether the amide III signals in the frequency of 1200-1300cm-1 disappear or not. Figure 2 and Figure S4 show the amide III and amide I signals at pD=6.9 and pD=3.4. It can be seen that the intensity of amide I signals keeps almost the same after replacing the subphase H2O solution by D2O solution. And the amide III signals at ~1300 cm-1 do not disappear. These findings suggest that GALA is inserted into POPC bilayer at both pH=6.9 and 3.4. Herein, GALA’s structure and action in POPC lipid bilayer at different pH can be described by Figure 2C.

A

0.4

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SFG Intensity

SFG Intensity

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0.2 0.0 1150 1200 1250 1300 1350 -1

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9



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Figure 2. The ssp amide III SFG spectra in POPC bilayer in D2O solution at (A) pD=6.9 and (B) pD=3.4. (C) A scheme of GALA’s structure and action in POPC lipid bilayer at different pH.

Using the same experimental procedures, we studied the interaction between GALA and neutral gel-phase DPPC lipid bilayer. Figure 3 shows the ssp amide I and amide III signals after the interaction between GALA and DPPC lipid bilayer reaches equilibrium at different subphase pH. The ppp amide I spectra are presented in Figure S5. With the exception of the total intensity, the spectral features in amide I and amide III region are similar to the results of GALA in POPC bilayer, i.e., the amide I spectra are dominated by the ~1655 cm-1 peak and the amide III spectra show two peaks (~1240 and ~1295 cm-1) originated from the loop and α-helical structures, respectively. Both the intensity of amide I and amide III increases as the pH is changed from pH1 to pH2, but does not change while the pH is changed from pH2 to pH3, indicating that pH response of GALA in DPPC bilayer is also irreversible. Furthermore, more (2)

(2)

loop structure of GALA is formed in DPPC bilayer in terms of the ratio of χPeak1 /χPeak2 of the ssp amide III spectra (Table S2). Figure S6 depicts the experimental results in D2O 10


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solution. The amide III signals in the frequency ranging from 1150 cm-1 to 1350 cm-1 completely disappear at pD=6.9, but the signals at pH=3.4 and pD= 3.4 maintain almost the same, which indicate that GALA is only absorbed at the surface of DPPC bilayer at pH1=6.9 while it is inserted into DPPC bilayer at pH2=3.4(described by Figure 3C). In general, some common peptides are either hydrophobic or hydrophilic. If the peptides are hydrophobic, it should be inserted into the bilayer chains, instead of laying on the hydrophilic bilayer surface. If they are hydrophilic, it should not be inserted in the membrane hydrophobic chains. However, different from the common peptides, GALA can both lay on the hydrophilic surface of lipid bilayer and insert into the lipid hydrophobic alkyl chain environments. This is because GALA is a 30 amino acid synthetic peptide with a repeating sequence of Glu-Ala-Leu-Ala. The hydrophobic periodicity of the Glu-Ala-Leu-Ala repeats allows the peptide to fold into a stable amphiphilic helix/loop structure with hydrophobic leucine and alanine sites on one, and hydrophilic glutamic acids on the other side of the helix/loop structure.16,17 Therefore, GALA can adjust its hydrophilic or hydrophobic sides by forming the dimer or oligomer conformations. When the GALA inserts into hydrophobic membrane leaflets, its hydrophobic sides may face to the lipid alkyl chain side. In contrast, when GALA lay on the hydrophilic membrane surface, GALA may turn its hydrophilic sides to the lipid head group to reduce the interaction energy. In addition, it needs to mention that the intensity of the ssp amide I spectra at pH1=6.9 in DPPC lipid bilayer is much weaker than the case in POPC lipid bilayer. The intensity of GALA in POPC bilayer is about 20~30 times larger than that in DPPC bilayer, which implies that GALA prefers to interact with fluid-phase POPC bilayer, rather than gel-phase DPPC bilayer. This phenomenon is further illustrated by the strong interaction 11



between GALA and DLPC bilayer (with a phase transition temperature of -1 °C) (Figure S7, Table S3). In general, the interaction between peptides and lipid bilayers obeys a common rule of hydrophobic matching.49,50 However, the length(4.5 nm) of an helix composed of 30 amino acid residues is closer to the hydrophobic length of DPPC bilayer (3.6 nm) than POPC (2.7 nm) and DLPC(1.95nm) bilayers. Therefore, hydrophobic matching is not the key factors for the interaction between GALA and lipid bilayers.

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Figure 3. The ssp SFG spectra in amide I (A) and amide III (B) region after the interaction between GALA and DPPC lipid bilayer reaches equilibrium at different pH. (C) A scheme of GALA’s structure and action in DPPC lipid bilayer at different pH.

3.2 The pH Response of GALA in Positive Ethylphosphocholine Lipid Bilayer We turned to investigate the interaction between GALA and positive gel-phase DPEPC lipid bilayer. Figure 4 shows ssp amide I and amide III signals after the interaction between GALA and DPEPC lipid bilayer reaches equilibrium at different subphase pH. The ppp amide I spectra are presented in Figure S8. Similar to the results in POPC and DPPC bilayer, GALA also adopts a mixed loop and helical structures in DPEPC bilayer. Compared to the weak signals at pH=6.9 for the gel-phase DPPC bilayer, very strong signals are observed at pH=6.9 for the case in gel-phase DPEPC bilayer. Because GALA’s Glu sidechains are negatively charged at neutral pH environment, therefore, it is expected that the interaction between GALA and positive DPEPC is strong because of the electrostatic attractive interactions.51 Indeed, cationic lipids have been employed to facilitate the accumulation of the GALA peptide on the cell surface and thus promote cellular uptake.19,52,53 As the pH decreases from 6.9 to 3.4, the fitted strengths of the peaks at 1655 cm-1 (χ(2) ) increase 1.3 and 1.9 times for 1655 the ssp and ppp polarization combinations, respectively(Table S4). Contrary to the results of GALA in DPPC and POPC bilayers, the amide I and III spectra return to the original state at pH1 =6.9 when the pH is changed from 3.4 to 6.9, which means that the pH response of GALA in DPEPC lipid bilayer is reversible. Figure S9 shows the experimental results in D2O solution. The amide III signals in the frequency ranging from 1150 cm-1 to 1350 cm-1 completely disappear at pD=6.9, while do not at pD= 3.4(Figure S9). These results prove that 13



GALA is absorbed at the surface of DPEPC bilayer at pH=6.9 while it is partially inserted into DPEPC bilayer at pH=3.4(described by Figure4C). B

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Figure 4. The ssp SFG spectra in amide I (A) and amide III (B) region after the interaction between GALA and DPEPC lipid bilayer reaches equilibrium at different pH. (C) A scheme of GALA’s structure and action in DPEPC lipid bilayer at different pH.

3.3 The pH Response of GALA in Negative Phosphoglycerol Lipid Bilayer

14


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Finally, we investigated the interaction between GALA and negative phosphoglycerol lipid bilayer. Figure 5 shows ssp amide I and amide III signals after the interaction between GALA and DPPG lipid bilayer reaches equilibrium at different subphase pH. The ppp amide I spectra are put in Figure S10. Because of electrostatic repulsive interactions, GALA does not interact with DPPG bilayer at pH=6.9. Therefore, a rather weak peak at ~1640 cm-1 is detected at pH =6.9. The ~1640 cm-1 peak is assigned to random-coil structure without connecting with α-helix and arises from bulk solution in the vicinity of DPPG lipid bilayer. However, when the pH is tuned from pH1=6.9 to pH2=3.4, the SFG amide I and amide III signals largely increase. At pH=3.4, the amide I spectra is dominated by a peak centered at ~1655cm-1 and the amide III spectra exhibit two peaks at ~1250 cm-1 and ~1300 cm-1, implying GALA transforms from a random-coil structure in neutral solution to a mixed loop and α-helical structure in DPPG bilayer at acidic conditions. When the subphase pH is changed from pH2 =3.4 to pH3=6.9, both the amide I and amide III spectral signals disappear, indicating GALA exits from the DPPG bilayer and shows a reversible activation. The experimental results at pD=3.4(Figure S11) confirm that GALA is inserted into DPPG lipid bilayer at pH=3.4. By comparing the intensity change of the ~1250 cm-1 peak between pH=3.4 and pD=3.4, it can be concluded that the α-helical structure of GALA is fully inserted into hydrophobic core of DPPG lipid bilayer while the loop structure may locate near the hydrophilic head groups of DPPG lipid bilayer (Figure 5C), which is similar to the case in POPC lipid bilayer at acid conditions. It is worth mentioning that although the insertion of negative peptides into negative lipid bilayers is energetically unfavorable, the GALA can insert into negative fluid-phase DLPG bilayer at neutral pH and form α-helical structure, as 15



indicated by the amide I and amide III spectra of GALA in DLPG lipid bilayer at pH= 6.9 and 3.4(Figure S12 and Table S6). However, at the acidic conditions, GALA interacts much more strongly with gel-phase DPPG (see fitting parameters in Table S5) than fluid-phase DLPG bilayer, which is completely different with the case for the zwitterionic phosphocholine lipid bilayers. It is worth mentioning that the lipid bilayer is slightly disturbed but not disrupted or damaged during the pH-response processes. Figure S13 shows the ssp and ppp spectra from the lipid alkyl chain after the interaction between GALA and DPPG/d-DPPG lipid bilayer at different pH values reaches equilibrium. It is evident that the ssp and ppp intensity from the CH3 group at pH3=6.9 decreases less than 25% compared to the one at pH1=6.9(Figure S13A and S13B). For the ssp and ppp spectra in the 1950-2300 cm-1 region(Figure S13C and S13D), the intensity of the broad band between 2100-2220 cm-1 (contributed to CD2, and the Fermi resonance of CD2 and CD3) decreases a little bit when the intensity from the CD3 group keeps almost the same at pH1 and pH3.

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Figure 5. The ssp SFG spectra in amide I (A) and amide III (B) region after the interaction between GALA and DPPG lipid bilayer reaches equilibrium at different pH. (C) A scheme of GALA’s structure and action in DPPG lipid bilayer at different pH.

4. Conclusion We have performed a detailed study to investigate the interactions between GALA and the lipid bilayers with different chain length and head groups by measuring the second-order amide I and amide III spectra. It is found that GALA can insert into fluid-phase lipid bilayers at neutral pH, but lies down on the gel-phase lipid bilayer surface. In acidic conditions, GALA inserts into both fluid-phase and gel-phase lipid bilayers. GALA adopts a mixed loop and α-helical structures in all kinds of lipid bilayers. Besides, the reversible activation of GALA in lipid bilayers depends on the nature of lipid. After membrane insertion, GALA shows reversible activation in the negative PG and positive EPC lipid bilayers. But GALA does not exit from the neutral PC lipid bilayers. ASSOCIATED CONTENT

Supporting Information: Fitting of SFG-VS Signals, Figure S1-Figure S13, Table S1-Table 17



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S6. This information is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Email: [email protected]; Tel: 086-551-63603462; Fax: 086-551-63603462

Notes The authors declare no competing financial interest.

Acknowledgements This work was supported by the National Natural Science Foundation of China (21473177, 21633007), National Key Research and Development Program of China (2017YFA0303502), Fundamental Research Funds for the Central Universities (WK2340000064), and the Key Research Program of the Chinese Academy of Sciences.

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