Evaluating the Effect of Lidocaine on the Interactions of C-reactive

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Evaluating the Effect of Lidocaine on the Interactions of C-reactive Protein with Its Aptamer and Antibody by Dynamic Force Spectroscopy Zhiping Li, Qing Wang, Xiaohai Yang, Kemin Wang, Shasha Du, Hua Zhang, Lei Gao, Yan Zheng, and Wenyan Nie Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03960 • Publication Date (Web): 24 Feb 2017 Downloaded from http://pubs.acs.org on February 25, 2017

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Analytical Chemistry

Evaluating the Effect of Lidocaine on the Interactions of C-reactive Protein with Its Aptamer and Antibody by Dynamic Force Spectroscopy Zhiping Li, Qing Wang*, Xiaohai Yang, Kemin Wang*, Shasha Du, Hua Zhang, Lei Gao, Yan Zheng and Wenyan Nie 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 *(Q.W.) Fax: +86-731-88821566, E-mail: [email protected]; *(K.W.); Fax: +86-731-88821566, E-mail: [email protected]. ABSTRACT: Effects of medicine on the biomolecular interaction have been given extensive attention in biochemistry and biomedicine because of the complexity of the environment in vivo and the increasing opportunity of exposure to medicine. Herein, the effect of lidocaine on the interactions of C-reactive protein (CRP) with its aptamer and antibody under different temperature was investigated through dynamic force spectroscopy (DFS). The results revealed that lidocaine could reduce the binding probabilities and binding affinities of CRP-aptamer and CRP-antibody. An interesting discovery was that lidocaine had differential influences on the dynamic force spectra of CRP-aptamer and CRP-antibody. The energy landscape of CRP-aptamer turned from two activation barriers to one after the treatment of lidocaine, while the one activation barrier in energy landscape of CRP-antibody almost remained unchanged. In addition, similar results were obtained for 25oC and 37oC. According to the result of molecular docking, the reduction of binding probabilities might be due to the binding of lidocaine on CRP. Additionally, the alteration of dissociation pathway of CRP-aptamer and the change of binding affinities might be caused by the conformational change of CRP, which was verified through synchronous fluorescence spectroscopy. Furthermore, differential effects of lidocaine on the interactions of CRP-aptamer and CRP-antibody might be attributed to the different dissociation processes and binding sites of CRP-aptamer and CRP-antibody, and different structures of aptamer and antibody. This work indicated that DFS provided information for further research and comprehensive applications of biomolecular interaction, especially in the design of biosensors in complex systems.

As one of the most ubiquitous reactions, biomolecular interaction takes place in protein-protein, protein-nucleic acid and nucleic acid-nucleic acid. The characterization of these recognition processes is of paramount importance for basic biomedicine and affinity-based analytics.1-3 Generally, most of the research on the subject of biomolecular interaction is in near-physiological condition, leaving out of consideration about the influence of external factors.4-6 However, external interference that includes electric field,7 temperature8 and metal ion,9 especially medicine,10-12 could influence biomolecular interaction. Considering that medication has become a universal phenomenon and the research on the mechanism of medicine effect is still not systematic, it is indispensable to investigate medicine effects on the interaction of biomolecule. Recently, researchers have paid extensive attention to the exploration of the effect of medicine on biomolecular interaction.10-14 Stuhlmeier found that differential effects of quinacrine on the interactions of nuclear factor κB-DNA and the activation protein-1-DNA with an electrochemical method.13 De la Cruz et al. discovered that the interaction of platelet-leukocyte could be enhanced by the combination of the aspirin and dipyridamole.14 However, comparing with these traditional ensemble methods,13, 14 the usage of single-molecule techniques allows more detailed insight in the study of medicine effects on the biomolecular interactions. Since AFM has emerged as a unique tool for monitoring the forces between or within biomolecules with picoNewton (pN) sensitivity,15, 16 it becomes an excellent tool for investigating medicine effects on biomolecular interaction. Recently, Ferrari discovered that hesperetin interfered with transforming growth factor-β ligand-receptor interaction.17 Fang and co-works found that the

interaction of thrombomodulin with thrombin was hindered by cigarette smoke extract 18 and cigarette carcinogens. 19 In our recent work, atomic force microscopy (AFM) was applied to the investigation of medicine effects on the interactions of myoglobin with its recognition probes.20 According to the force histogram of myoglobin-aptamer and myoglobin-antibody, different influences of medicine were observed. Recently, AFM based dynamic force spectroscopy (DFS) has been successfully applied to provide new insights into the molecular dynamics of recognition process. In so-called DFS, the rate of the increasing force (loading rate) is varied during the single-molecule force measurement. DFS can provide information on the dissociation dynamics of ligand-receptor and the prominent barriers traversing in the energy landscape along its force-drive dissociation pathways,21-23 which is helpful for precisely understanding the biomolecular interaction.24 Presumably, DFS may offer a new insight in to the study of medicine effect on biomolecular interaction. C-reactive protein(CRP) is a major acute-phase reactant protein, which can be used as a general inflammatory biomarker in diagnosing inflammatory responses.25, 26 Lidocaine as a local anesthetic can reduce the production of both pro- and anti-inflammatory cytokines which influences acute-phase inflammation reactions.27, 28 It was reported that the level of CRP significantly lowed in the postoperative period under the effect of lidocaine.29, 30 However, it is unclear that whether lidocaine has impact on the interaction of CRP with its recognition probes. In the present work, the effect of lidocaine on the interactions of CRP with its aptamer and antibody was studied predominately by DFS. Lidocaine enabled the binding probabilities of CRP-aptamer -1-

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and CRP-antibody to reduce, but the differential effects of lidocaine on the interactions of CRP-aptamer and CRP-antibody were not obvious. These were different from medicine effects on the interactions of myoglobin with its recognition probes.20 Interestingly, distinguishing effects of lidocaine on the dynamic force spectra of CRP-aptamer and CRP-antibody were observed. Additionally, surface plasmon resonance (SPR) was applied to investigate lidocaine effect on the binding affinities of CRP-aptamer and CRP-antibody. The present work will not only give new evidence for better understanding of medicine effect on biomolecular interaction at the single-molecule level, but also provide valuable information for design of biosensors in complex systems. EXPERIMENTAL SECTION Materials and Reagents. DNA aptamer against CRP (5’-CGA AGG GGA TTC GAG GGG TGA TTG CGT GCT CCA TTT GGT G-(T)12-NH2-3’ and 5’-CGA AGG GGA TTC GAG GGG TGA TTG CGT GCT CCA TTT GGT G-3’) which was obtained by our group using a platform that integrated microbead-assisted SELEX with microfluidics technology,31 was synthesized by Sangon Biotech (Shanghai, China). C-reactive protein was purchased from Biovesion (USA). Anti-CRP was obtained from Beijing Biosynthesis Biotechnology Co., Ltd (China). Lidocaine (purity: 99%) was obtained from Shanghai Future Industry Limited by Share Co., Ltd (China). (3-Mercaptopropyl) trimethoxysilane (MPTMS) was obtained from Sigma (USA). N-Hydroxysuccinimidepolyethylenglycol maleimide (NHS-PEG-MAL, MW 3400) and 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were purchased from Nanocs (USA) and Amresco (USA), respectively. All of the other chemical reagents were of analytical grade or higher. Ultrapure water (18.2 MΩ·cm) was used in all experiments. Functionalization of AFM Tips. AFM probes (silicon nitride tips with normal spring constant ~ 0.06 N/m) were functionalized with aptamer or antibody according to the previously reported procedure.32 First, the cantilevers were cleaned by chloroform, HF acid and alkaline solution, sequentially. Then, the cleaned tips were hydroxylated by treating them with piranha solution (98% H2SO4/H2O2, 7:3 = v/v) and silanized by soaking them in 1.0% (v/v) MPTMS/toluene solution. Subsequently, they were rinsed with excess toluene and activated through incubation with 1 mg/ml NHS-PEG-MAL, which was used as a tether for the aptamer or antibody,33 in dimethyl sulfoxide for 3 h. Afterward, the activated tips were immersed in an amine group modified aptamer or antibody solution for 1 h. Finally, the tips were washed with binding buffer (20 mM HEPES, 120 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, pH 7.35) to remove any unbound aptamer or antibody, and stored in this buffer at 4 °C until use. Preparation of CRP-modified gold substrates. Gold substrates were modified with CRP as previously reported.34, 35 In brief, the gold substrates were successively treated with acetone, ethanol and piranha solution (98% H2SO4/H2O2, 7:3 v/v). Each step was followed by a washing with ultrapure water and a drying with nitrogen gas. Then, the gold substrates were incubated with 13.3 µg/ml CRP solution at 4 °C overnight. For investigation of lidocaine effect, the CRP-modified substrates were incubated with 5 µg/ml lidocaine for different time (1 h, 2.5 h and 4 h, respectively). After being rinsed with buffer, the interactions of CRP-aptamer and CRP-antibody were researched using AFM force spectroscopy.

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AFM Force Measurements. All force measurements were carried out on JPK biological AFM (JPK Instruments AG, Germany) in liquid cells in contact mode. And the temperature of the system was controlled using a commercial temperature controller for AFM (BioCell II, JPK Instruments, Berlin, Germany). For the Force Mapping mode, each data set was comprised of 1024 (32×32) individual force-distance (FD) curves taken over a (5×5) µm2 area. Force measurements at different loading rates were achieved by changing the cantilever velocity ranging from 150 nm/s to 800 nm/s. For each experiment, at least 3000 force-distance curves were collected and then the most probable force was calculated, along with a 95% certainly confidence limit. Force curves were analyzed with the JPKSPM Data processing (Version 5.1.8) and the spring constants of the cantilevers were calibrated using the thermal fluctuation method.36 Molecular docking details. The crystal structure of human C-reactive protein was downloaded from the RCSB protein data bank (PDB code: 1B09) and the protein was prepared in a standard manner using AutoDock4.2. Molecular docking study was carried out for the CRP and lidocaine using AutoDock4.2. In general, the docking parameters for AutoDock4.2 were kept as default values. A 60 Å × 60 Å × 60 Å grid map was used in all docking calculations centered on the active site [145.704, 66.348, 34.581]. For verifying the importance of Asn61 and Phe66 in the binding event of CRP and lidocaine, computational site-directed mutagenesis was conducted using PyMOL (Mutagenesis). When mutagenesis processes were finished, the minimum energy structure was selected as the initial structure for analyzing mutagenesis results using molecular operating environment (MOE). Finally, the results were analyzed and visualized using PyMOL (http://pymol.sourceforge.net/). Fluorescence quenching of CRP by lidocaine. The change in the micro-environment of the chromophores of CRP during binding with lidocaine can be investigated using synchronous fluorescence spectroscopy.37 Different concentrations (0, 0.1, 1, 2.5 and 5 µg/ml) of lidocaine and CRP were co-incubated for 1 h at room temperature. Then, synchronous fluorescence spectra of CRP were recorded using a Hitachi F-7000 fluorescence spectrophotometer by setting ∆λ = 15 nm and 60 nm for tryptophan and tyrosine residues, respectively. The widths of both the excitation and the emission slit were set to 5.0 nm.

Figure 1. Schematic diagram of single-molecule force measurement with a aptamer- or antibody-modified AFM tip on CRP-coated gold substrate (A) The AFM tip functionalization procedure. (B) CRP was immobilized on the gold substrate by physical absorption, and then, the modified substrate was treated. (C) Representative force curves acquired with aptamer or antibody modified AFM tips on CRP-coated gold substrate and after the system was blocked with free aptamer or antibody solution (inset). 2


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Surface plasmon resonance analysis. The dissociation constants (Kd) of CRP-aptamer and CRP-antibody for the CRP with and without lidocaine treatment were determined by a homemade surface plasmon resonance instrument (Changchun Dingcheng Technology, China). In brief, 200 µg/ml of CRP was first immobilized on uncoated Au film. After being blocked by BSA, different concentrations of aptamer or antibody (from 0 nM to 200 nM) were added and incubated for 30 min at 25 °C, and the Au film surface was repeatedly washed with binding buffer. Then, the resonance angle of SPR for each sample was recorded and analyzed. For investigation of lidocaine effect, the CRP-modified Au film was co-incubated with 5 µg/ml lidocaine for 1 h before the Au substrate was blocked by BSA. RESULTS Specific interactions of CRP-aptamer and CRP-antibody. A single-molecule force measurement was first carried out to study the interactions of CRP with its aptamer and antibody according to the schematics illustrated in Figure 1A. Aptamer or antibody was first covalently attached to the AFM tip through a PEG linker (~ 30 nm). Then, the rupture forces of CRP-aptamer and CRP-antibody were detected when the AFM tip and the CRP-coated gold substrate were brought into and out of contact. To achieve single-molecule force measurements, the experiment conditions, such as the immobilization density of aptamer or antibody on the tip, tip-substrate contact time and the concentration of CRP were optimized and the binding probability was under 30%. 38 Typical force-distance curves are shown in Figure 1C, of which the first randomly appearing peaks were caused by the nonspecific force between the tip and the substrate,39 whereas the second peaks represented the specific 0.18 0.16

0.18

A

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Probability

Probability

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Binding probability

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0.20 0.15 0.10 0.05 0.00

D

0.25

binding force of CRP-aptamer or CRP- antibody. After the blocking experiment (adding free aptamer or antibody solution into the AFM liquid cell), no specific adhesion peak appeared in the retract curve (the inset of Figure 1C). This was because the added aptamer and antibody molecules had occupied the binding site on CRP in advance. Referring to the previous works,40, 41 the histogram of binding force has been analyzed, and the values of the most probable force were determined from a fit of the main peak in the histogram by a Gaussian distribution. For the CRP-aptamer, the force distribution histogram is shown in Figure 2A and the probability in the histogram was calculated from the counts of the binding force in the different ranges divided by all the specific binding events. The most probable force of CRP-aptamer was about 103.2 ± 5.0 pN (Figure 2A) and the binding probability was 24.5 ± 3.5% at the loading rate of 3.52×104 pN/s (Figure 2C). When the CRP-modified gold substrate was blocked by aptamer, only force curves with nonspecific adhesion peak was observed and thebinding probability decreased to 3.6 ± 0.8%. Besides, the binding probability markedly decreased to 2.1 ± 0.2% when the aptamer-modified tip was first incubated with CRP. A control experiment was performed under the same condition without aptamer attached to the end of the PEG, the binding probability decreased to 3.8 ± 0.8%, which was similar to the value (2.6 ± 0.4%) when the aptamer-modified tip and the unmodified substrate were used. Blocking experiments and control experiments confirmed the specificity of the interaction between CRP and aptamer. For the CRP-antibody, the most probable force was about 99.7 ± 2.4 pN (Figure 2B) and the binding probability (Figure 2D). After blocking the CRP-modified gold substrate with the antibody or blocking the antibody-modified tip with CRP, the specific rupture peak in force curves disappeared and the binding probability decreased obviously to 5.5 ± 0.2% and 3.1 ± 1.0%, respectively. To further determine the specificity of these force events, control experiments were performed under the same condition without antibody attached to the end of the PEG or without CRP on the gold substrate. The binding probability decreased to 6.4 ± 0.2% and 3.8 ± 0.8%, respectively. These results demonstrated that the interaction force recorded for the CRP-antibody was also specific. Effect of lidocaine on the binding of CRP-aptamer and CRP-antibody. The effect of lidocaine on the interactions of CRP with its aptamer and antibody was then investigated. After the CRP was treated with lidocaine for different time (1, 2.5 and 4 h, respectively), the interactions of CRP-aptamer and CRP-antibody

0.20 0.15 0.10 0.05 0.00

a

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d

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Figure 2. Force measurements of CRP with its aptamer and antibody at a loading rate of 3.52×104 pN/s. Histogram of binding forces of CRP-aptamer (A) and CRP-antibody (B). Binding probabilities of CRP-aptamer (C) and CRP-antibody (D) under different conditions. (a) Aptamer or antibody modified on the AFM tip, CRP immobilized on the gold substrate; (b) PEG-modified tip without attaching aptamer or antibody, CRP immobilized on the gold substrate; (c) aptamer or antibody modified on the AFM tip, gold substrate without CRP; (d) aptamer or antibody modified on the AFM tip, CRP on substrate blocked with aptamer or antibody; (e) aptamer or antibody modified on the AFM tip and blocked with CRP, CRP immobilized on the gold substrate. Error bars showed the standard deviation of measurement taken from three independent experiments.

Table 1. Binding probabilities and the most probable forces of CRP-aptamer and CRP-antibody under the effect of lidocaine. Error bars showed the standard deviation of measurement taken from three independent experiments. Incubating time Aptamer-CRP Aptamer-CRP/Lidocaine

The most probable force (pN)

22.5 ± 1.3

103.4 ± 2.8

1h

24.5 ± 2.6

96.0 ± 3.6

2.5 h 4h

19.5 ± 1.4 14.6 ± 0.9 27.1± 1.6 18.5 ± 1.1 18.6 ± 2.9 18.3 ± 2.2

100.7 ± 2.7 98.1 ± 6.9 99.7 ± 2.4 96.2 ± 5.3 93.5 ± 6.4 95.3 ± 1.2

Antibody-CRP Antibody-CRP/Lidocaine

Binding probability (%)

1h 2.5 h 4h

3



Force (pN)

Force (pN)

100 80 60 40

60 40 20 0

2.5 h

Time

180

A 160

80

0 1h

P>0.05

100

20 0h

180

140 B 120

140

25 ¡æ 37 ¡æ

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0h

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160

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11.0

180

C

25 ¡æ 37 ¡æ

160

140 120 100

D

25 ¡æ 37 ¡æ

9.5

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140 120 100 80

80

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0h

10.5

lnr (pN/s)

60 0.00

100

11.0

Force (pN)

0.25

without blocking blocking with free antibody

D

Force (pN)

without blocking blocking with free aptamer

Binding probability

0.30

C

10.0

120

lnr (pN/s) 180

0.30

9.5

140

60

9.5

4h

25 ¡æ 37 ¡æ

80

60

4h

B 160

Force (pN)

P>0.05

A

120

Force (pN)

140

Binding probability

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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60

9.5

0h

1h

2.5 h

4h

Time

Figure 3. Analysis the effect of lidocaine on the interactions of CRP with its aptamer and antibody. Binding force of CRPaptamer (A) and CRP-antibody (B) when the CRP was treated by lidocaine for different incubating time (0, 1 h, 2.5 h and 4 h, respectively). No significant difference in the binding force of CRP-aptamer (CRP-antibody) with and without lidocaine treatment (P > 0.05 in the Student t test). Binding probability of CRP-aptamer (C) and CRP-antibody (D) with different incubating time of lidocaine (0, 1 h, 2.5 h and 4 h, respectively). Binding probabilities of CRP-aptamer and CRP-antibody were higher than those in the blocking experiments. Error bars showed the standard deviation of measurement taken from three independent experiments. were determined at the loading rate of 3.52×104 pN/s. The results are shown in Figure 3 and Table 1. More detailed experimental data is shown in the Table S1 and Table S2 of Supporting information. For the CRP-aptamer, the most probable force almost remained unchanged after CRP was co-incubated with lidocaine (Figure 3A and Table 1). When the incubating time was 1 h, it had little impact on the binding probability of the CRP-aptamer (24.5 ± 2.6%). However, as the incubating time was over 1h, the binding probability decreased with the increase of time (Figure 3C). Then, a two-tailed student’s t-test was used in the statistical analysis to determine the significance of the difference in the most probable force before and after the treatment of lidocaine, using a confidence interval of 95%. There was no significant difference in the binding force of CRP-aptamer with or without the treatment of lidocaine. Therefore, it could be concluded that lidocaine did not affect the CRP-aptamer binding force, instead it decreased the binding probability. For the CRP-antibody, the most probable force also remained unchanged after CRP was co-incubated with lidocaine (Figure 3B and Table 1). When the incubating time was 1 h, the binding probability decreased to 18.5 ± 1.1%. As the incubating time of lidocaine prolonged to 2.5 h and 4 h, the binding probability of the CRP-antibody was unchanged compared with the binding probability at 1 h (Figure 3D). These data indicated that lidocaine had little influence on the binding force of CRP-antibody, but it reduced the binding probability. Effect of lidocaine on the dynamic force spectra of CRP-aptamer and CRP-antibody. The effect of lidocaine on the dynamic interactions of CRP with its aptamer and antibody at

10.0

10.5

11.0

10.5

11.0

lnr (pN/s)

lnr (pN/s)

Figure 4. Plot of the binding force for the interactions of CRP-aptamer against the logarithm of the loading rate for the CRP without (A) and with (B) lidocaine treatment at 25 °C (solid line) and 37 °C (dashed line). Plot of the binding force for the interactions of CRP-antibody against the logarithm of the loading rate for the CRP without (C) and with (D) lidocaine treatment at 25 °C (solid line) and 37 °C (dashed line). Error in force value showed the standard deviation of measurement taken from three independent experiments. different temperature was further explored using dynamic force spectroscopy. By measuring the binding forces of CRP-aptamer and CRP-antibody at different loading rates ranging from 1.32×104 pN/s to 7.04×104 pN/s, the dynamic spectra and the information of dissociation dynamic could be obtained. The dynamic force spectrum of CRP-aptamer is displayed in Figure 4A. With the increasing loading rate, the binding force of CRP-aptamer displayed an initial gradual increase followed by a more-rapid increase at higher loading rates. By contrast, with the administration of lidocaine, the binding force increased in only one linear region over the whole loading rate (Figure 4B). For the CRP-antibody, the most probable force before and after the treatment of lidocaine, increased in only one linear region over the whole loading-rate range, and the spectra were almost overlapped (Figure 4C and 4D). In addition, the results showed that Table 2. Kinetic parameters characterizing energy landscape of CRP-aptamer and CRP-antibody under the effect of lidocaine at 25 °C.

Aptamer-CRP AptamerCRP/Lidociane Antibody-CRP AntibodyCRP/Lidocaine

Loading rate range (pN/s) 1.32×1043.52×104 3.52×1047.04×104 1.32×1047.04×104 1.32×1047.04×104 1.32×1047.04×104

xᵦ (nm)

koff (s-1)

τ (s)

0.40 ± 0.03 0.048 ± 0.01 0.12 ± 0.01 0.17 ± 0.01 0.21 ± 0.02

0.15 ± 0.03 127.95 ± 47.98 45.18 ± 9.26 22.92 ± 3.28 11.51 ± 2.59

6.9 ± 2.0 0.01 ± 0.005 0.02 ± 0.007 0.05 ± 0.01 0.09 ± 0.03

∆∆E (kBT) 5.71 ± 0.24 -1.04 ± 0.16

-0.69 ±0.02

Here, the lifetime (τ) was calculated by1/ koff. Error bars showed the standard deviation of measurement taken from three independent experiments. 4


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temperature had little influence on the effect of lidocaine on the interactions of CRP with its aptamer and antibody. The dissociation kinetic parameters were obtained through the analysis of the dynamic force spectra. According to the Bell model,42 the measured force (F) of the ligand-receptor bond linearly depends on the logarithm of the loading rate r as follows:

∙

∙

(1)

Where koff(0) is the dissociation rate constant, xᵦ is the energy barrier width, and kB is the Boltzmann constant, T is the absolute temperature, F and r are the most probable force and the loading rate, respectively. Take the effect of lidocaine on the dynamic interactions of CRP with its aptamer and antibody at 25 oC for an example, Table 2 showed dynamic parameters derived by fitting Eq. 1 to the experimental data shown in Figure 4. For the CRP-aptamer, the dissociation rate constant koff (0) and the energy barrier width xᵦ were 0.15 ± 0.03 s-1 and 0.40 ± 0.03 nm in the lower force region and were 127.95 ± 47.98 s-1 and 0.048 ± 0.01 nm in the higher force region. However, the dissociation rate constant for the CRP treated with lidocaine was about 2-fold faster than that of the CRP without the treatment of lidocaine. For the CRP-antibody, the dissociation rate constant koff (0) and the energy barrier width xᵦ were 22.92 ± 3.28 s-1and 0.17 ± 0.01 nm for the CRP without lidocaine treatment, and were 11.51 ± 2.59 s-1 and 0.21 ± 0.02 nm for the CRP with lidocaine treatment. The difference in the dissociation rate constants before and after the treatment of lidocaine might be due to the different bind activation energy. According to transition state theory,43 the dissociation rate constant, koff (0), is a function of the activation energy ∆E, ∆

0 ∝

(2)

According to the previous works,44, 45 it was usually assumed that the pre-exponential factor in Arrehnius relationship was the same in two analogous systems. Here, we also assumed the pre-exponential factor in the unbinding processes between CRP and its recognition probe (aptamer or antibody) without lidocaine treatment was the same as that with lidocaine treatment. Then, the differences in the activation energy of CRP with its recognition probe (aptamer or antibody) before and after CRP was treated using lidocaine ∆∆E = ∆E(a) –∆E(b) can be determined by Eqs. 3 and 4: !/ #

For CRP-aptamer, ∆∆E = ∆E (Aptamer-CRP) – ∆E (Aptamer-CRP/lidocaine). For CRP-antibody, ∆∆E = ∆E (Antibody-CRP) - ∆E (Antibody-CRP/lidocaine). Thus, it was calculated that the difference in the energy barriers of CRP-aptamer and CRP-antibody before and after the treatment of lidocaine were 5.71 ± 0.24 kBT and -0.69 ± 0.02 kBT , respectively. According to the parameters in Table 2, the conceptual free-energy landscapes for the binding of CRP-aptamer and CRPantibody are drawn in Figure 5. For the CRP-aptamer, the dissociation passed through at least two energy barriers, and there was one intermediate state in this process. However, with the treatment of lidocaine, there was only one energy barrier to overcome from the binding state to the detachment, indicating no intermediate state existing in the unbinding process. The disappeared energy barrier in the dynamic force spectrum of CRP-aptamer indicated that different CRP structures existed in the CRP-aptamer when lidocaine was administrated. For the CRP-antibody, the dissociation passed through one activation barrier from the bound to the dissociated state for both the CRP with and without lidocaine treatment, and the minute effect of lidocaine on the dissociation energy barrier was observed. It could be concluded that the effect of lidocaine on the interaction of CRP- aptamer was different from that of CRP-antibody. Moreover, the lidocaine effect on the interaction of CRP with its aptamer and antibody at 37°C is shown in Table S-3 and Figure S-1of the Supporting Information. The results showed that lidocaine had similar effects on the interaction of CRP with its aptamer and antibody at 37°C. Effect of lidocaine on binding affinities of CRP-aptamer and CRP-antibody. To gain bulk solution measurement insight into the medicine effect, SPR was conducted to monitor lidocaine effect on the binding affinities of CRP with its aptamer and antibody. The CRP was first immobilized on the Au surface, and then, the affinity of CRP was evaluated by the binding capacity with different concentrations of aptamer or antibody. To calculate Kd, the change of resonance angle against aptamer (or antibody) concentrations, were plotted, and the data points were fitted with one-site model.31, 46 As showed in Figure 6, the change of resonance angle increased with the aptamer or antibody concentration and ultimately reached a plateau. For the 0.05 0.04

∆∆

(3)

∆∆$ %& ' ∙ ( !/ #)

(4)

0.03

∆θ

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Figure 5. Conceptual binding free-energy landscapes of CRP-aptamer (A) and CRP-antibody (B) complexes for the CRP without (green line) and with (red line) lidocaine at 25 °C.

Figure 6. The change of resonance angle of CRP at various concentrations of aptamer (circle) or antibody (aquare) for the CRP without (solid line) and with (dashed line) lidocaine treatment. Error bars showed the standard deviation of measurement taken from three independent experiments. 5


CRP-aptamer, the dissociation constant was 11.09 nM, which was smaller than that of the CRP treated with lidocaine for 1 h (38.37 nM). In other words, the binding affinity of CRP toward aptamer reduced after the treatment of lidocaine. For the CRP-antibody,the dissociation constant was 40.03 nM for the CRP without lidocaine treatment, and was 113.37 nM for the CRP with lidocaine treatment. That was, lidocaine weakened the binding affinity of CRP-antibody. These phenomena demonstrated that the presence of lidocaine reduced the binding affinities of CRP-aptamer and CRP-antibody.

DISCUSSION According to the results, the interactions of CRP-aptamer and CRP-antibody were affected by lidocaine. Moreover, the effect of lidocaine on the interaction of CRP-aptamer was different from that of CRP-antibody. In detail, under the effect of lidocaine, the binding probabilities of CRP with its aptamer and antibody showed an analogous reduction. The binding probability of CRP-aptamer tended to remain steady before it reduced with the extension of lidocaine incubating time, while the binding probability of CRP-antibody reduced immediately once lidocaine was administrated, and then maintained unchanged. A noteworthy discovery was that lidocaine altered the dissociation pathway of CRP-aptamer. The dynamic force spectrum of CRP-aptamer turned from two linear regions to one after the treatment of lidocaine. For the CRP-antibody, the one linear region in the dynamic force spectrum was almost unchanged compared to the situation of CRP without lidocaine treatment. Additionally, the presence of lidocaine decreased the binding affinities of CRP toward aptamer and antibody. There were two possible reasons to explain the interference of lidocaine on the interactions of CRP with its aptamer and antibody. One was that the binding of lidocaine on CRP hampered

Figure 7. (A) Chemical structure of Lidocaine; (B) The predicted binding model of CRP with lidocaine and the predicted binding sites for CRP are ASN-61, PHE-66 and GLN139, (C) Computational site-directed mutagenesis of CRP in position 61 and the predicted binding sites for E61R are PHE66 and ARG61. (D) Computational site-directed mutagenesis of CRP in position 66 and the predicted binding sites for F66A are ALA66 and GLU138.

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Fluorescence intensity (a.u)

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Figure 8. Synchronous fluorescence spectra of CRP in the presence of different concentrations of lidocaine. (A) ∆λ = 15 nm and (B) ∆λ = 60 nm. C(CRP): 5 µg/ml. The arrow indicated the signal changes with increase of lidocaine concentration (0, 0.1, 1.0, 2.5, 5.0 µg/mL). its interaction with aptamer and antibody. As shown in Figure 7,three putative binding sites on CRP were obtained from molecular docking. The binding stability of lidocaine was attributed to the π-π interaction between the aromatic moiety of lidocaine and the Phe66 which existed in CRP, the hydrogen bond between the nitrogen in the tail group of lidocaine and Gln139 on CRP, as well as the hydrogen bond between the carbonyl of lidocaine and Asn61 on CRP. Meanwhile, computational site-directed mutagenesis was also carried out by replacing Phe66 and Asn61 to Ala and Arg, respectively. The results showed that the binding energy between CRP and lidocaine increased from -5.62 kcal/mol to -5.09 kcal/mol and -4.56 kcal/mol respectively (shown in Table S-4 of the Supporting Information). In addition, it was reported that the binding probability of receptor-ligand might be disturbed by small molecules through occupying the key binding sites or forming the steric-hindrance.18 Thus, it was presumably that the binding probabilities of CRP-aptamer (or CRP-antibody) might be decreased by lidocaine through occupying the binding sites of aptamer and antibody on CRP or forming the steric-hindrance. The other was that the conformational change of CRP caused the alteration in the dissociation pathway of CRP-aptamer. The disappeared free-energy barrier in the dynamic force spectrum of CRP-aptamer suggested that the structure of CRP was changed after the treatment of lidocaine. In order to verify the change of CRP conformation under the influence of lidocaine, the synchronous fluorescence spectra of CRP in the presence of lidocaine was also investigated. As shown in Figure 8, its fluorescence intensity decreased with the increasing concentration of lidocaine and the emission maximum of tyrosine and tryptophan residues showed a slight blue shift which indicated that the conformation of CRP was changed, and that the tyrosine and tryptophan residues were brought to a more hydrophilic environment. 47, 48 It could be concluded that the conformation of CRP changed under the effect of lidocaine, which is consistent with the influence of lidocaine on the dynamic force spectrum of CRP-aptamer. Since the binding affinity of protein may have a connection with the protein conformation,49 it was presumably that the CRP conformation changes may be one of the reasons which influenced the change of binding affinity. However, due to the complex relation between the protein structure and binding affinity, the mechanism for the decrease of binding affinity require further exploration. For differential effects of lidocaine on the interactions of CRP-aptamer and CRP-antibody, possible causes are as follows. First, the dissociation pathway of CRP-aptamer was different from that of CRP-antibody. As shown in Figure 4, the plot of the single-molecular force of CRP-aptamer against lnr consists of two linear regions, and the two distinct linear regions indicated that 6


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the dissociation of CRP-aptamer passed through at least two energy barriers and one intermediate state. However, the dynamic force spectrum of CRP-antibody has only one linear region, suggesting that there was only one energy barrier to overcome. The results suggested that the CRP-aptamer undergo a different dissociation processes from the CRP-antibody. Second, the binding sites of aptamer and antibody on CRP were different. According to our prior study,31 CRP which first specifically bonded with the aptamer, could continuously capture antibody, leading to a significant change in the resonance angle. Therefore, it could be concluded that the binding site of the aptamer to CRP was different from that of antibody to CRP. But for, the resonance angle would be unchanged. The third cause was the different structures of aptamer and antibody. 50 Aptamers are short singlestranded DNA or RNR that can bind specifically to proteins and other targets. 51 The flexible structure of aptamer made it better to perceive the effect of lidocaine on CRP than that of antibody.

CONCLUSIONS In summary, the effect of lidocaine on the interactions of CRP-aptamer and CRP-antibody was evaluated by DFS. After the treatment of lidocaine, the binding forces of CRP-aptamer and CRP-antibody were almost unchanged, and the binding probabilities showed an analogous reduction. Interestingly, the influence of lidocaine on the dynamic force spectrum of CRP-aptamer was different from that of CRP-antibody. The dynamic force spectrum of CRP-aptamer turned from two linear regions to one after the treatment of lidocaine, while the only one linear region in the dynamic force spectrum of CRP-antibody almost remained unchanged. Additionally, lidocaine exerted an inhibition effect on the binding affinities of CRP toward aptamer and antibody. The above results might be ascribed to the binding of lidocaine on CRP and the conformational change of CRP. Furthermore, different binding sites and dissociation pathways of aptamer and antibody toward CRP and different structures of aptamer and antibody contributed to the differential influences of lidocaine on the interactions of CRP-aptamer and CRP-antibody. This work provided new evidence for the study of medicine effects on the interactions of aptamer and antibody with their target proteins. Furthermore, the established method can be used to investigate the medicine effect on binding processes of biomolecules at the single-molecule level.

ASSOCIATED CONTENT

Supporting Information Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Details of lidocaine effect on the interaction of CRP-aptamer (Table S-1) and CRP-antibody (Table S-2); Lidocaine effect on the free-energy landscape of CRP-aptamer and CRP-antibody at 37 °C (Table S-3); The energy values for CRP and lidocaine (Table S-4) (PDF) Conceptual binding free-energy landscapes of CRP-aptamer and CRP-antibody at 37 °C (Figure S-1) (PDF)

AUTHOR INFORMATION Corresponding Author

*(Q.W.) Phone: +86-731-88821566. Fax: +86-731-88821566. E-mail: [email protected]

*(K.W.) Phone: +86-731-88821566. Fax: +86-731-88821566. E-mail: [email protected].

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21375034 and 21190040), and Natural Science Foundation for Distinguished Young Scholars of Hunan Province (2016JJ1008).

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Evaluating the Effect of Lidocaine on the Interactions of C-reactive

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