# Atomic Scale Interactions between RNA and DNA Aptamers with the TNF-α Protein

Authors: Homayoun Asadzadeh; Ali Moosavi; Georgios Alexandrakis; Mohammad R. K. Mofrad
Journal: BioMed Research International
 (2021)

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## Abstract

Interest in the design and manufacture of RNA and DNA aptamers as apta-biosensors for the early diagnosis of blood infections and other inflammatory conditions has increased considerably in recent years. The practical utility of these aptamers depends on the detailed knowledge about the putative interactions with their target proteins. Therefore, understanding the aptamer-protein interactions at the atomic scale can offer significant insights into the optimal apta-biosensor design. In this study, we consider one RNA and one DNA aptamer that were previously used as apta-biosensors for detecting the infection biomarker protein TNF-α, as an example of a novel computational workflow for selecting the aptamer candidate with the highest binding strength to a target. We combine information from the binding free energy calculations, molecular docking, and molecular dynamics simulations to investigate the interactions of both aptamers with TNF-α. The results reveal that the RNA aptamer has a more stable structure relative to the DNA aptamer. Interaction of aptamers with TNF-α does not have any negative effect on its structure. The results of molecular docking and molecular dynamics simulations suggest that the RNA aptamer has a stronger interaction with the protein. Also, these findings illustrate that basic residues of TNF-α establish more atomic contacts with the aptamers compared to acidic or pH-neutral ones. Furthermore, binding energy calculations show that the interaction of the RNA aptamer with TNF-α is thermodynamically more favorable. In total, the findings of this study indicate that the RNA aptamer is a more suitable candidate for using as an apta-biosensor of TNF-α and, therefore, of greater potential use for the diagnosis of blood infections. Also, this study provides more information about aptamer-protein interactions and increases our understanding of this phenomenon.


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## Body

## 1. Introduction

Severe blood infections leading to sepsis are one of the major causes of death, especially among hospitalized patients [1, 2]. Patients suffering from blood infections are characterized by complex pathophysiology and heterogeneous phenotypes with respect to response to treatment, symptoms, and outcomes. Blood infections are clinically difficult to diagnose due to the multiple factors contributing to their emergence [3], and definitive diagnosis techniques, risk determination tools, treatment selections, and evaluation methods, or outcome prediction procedures are to be found for these infections [4]. Biomarkers are recognized as natural molecules, genes, or characteristics that can be used as a basis for the detection of specific physiologic or pathologic processes. Clinically speaking, biomarkers are deemed valuable only when they can contribute to decision making. Ideal biomarkers are characterized by fast kinetics, high affinity and specificity, detectability by automated technologies, and inexpensive bedside testing [4]. Many protein biomarkers have been identified that can be used to detect blood infections, such as C-reactive protein (CRP) [5], Interleukin 6 (IL-6) [6], Procalcitonin (PCT) [7], Interleukin 10 (IL-10) [8], Interferon-gamma (IFN-γ) [8], and tumor necrosis factor-alpha (TNF-α) [9–11].The multiple proinflammatory cytokines are identified as possible biomarkers of blood infection [11], and TNF-α is one of the most promising candidates [10], as its serum levels increase significantly during an infection [9]. Tumor necrosis factor-α (TNF-α) is an important proinflammatory cytokine that contributes to acute phase reaction. Although TNF-α can be secreted by many cell types such as NK cells eosinophils, neurons, CD4+ lymphocytes, neutrophils, and mast cells, it is secreted primarily by macrophages [12]. TNF-α is a subordinate member of the TNF superfamily which embodies different types of transmembrane proteins with a homology domain [13]. Mature human TNF-α is secreted after cleavage of a 76-residue peptide from the amino terminus of the prohormone and the mature protein contains single intramolecular disulfide bridge [14]. Previous studies have shown TNF-α to be a trimer in solution [14, 15]. The several crystal forms in which the molecule has been obtained have either crystallographic or noncrystallographic 3-fold symmetry, suggesting that the cytokine forms trimers in the solid-state as well [16, 17]. The TNF-α subunit is a β-sheet sandwich constructed almost entirely of antiparallel β-strands.Due to the known involvement of TNF-α in sepsis, many bioassays have been targeting its molecular structure with various probes to reveal its presence in serum [18]. TNF-α, including fluorescence immunoassay, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, and time-resolved immune-fluorometric assay, is, thanks to a number of commonly used traditional immunoassays, easy to detect and quantify [19, 20]. Nevertheless, creation of sandwich immunoassays in the traditional methods is dependent on antibody pairs. Moreover, these methods are characterized by multiple washing steps and high-cost readout signal development procedures.Aptamers are single-stranded DNA or RNA oligonucleotides that have been developed as powerful and reasonably priced alternatives to traditional antibodies for TNF-α targeting. Aptamers are actually recognized as high-affinity and high-specificity biomolecule binding agents. These oligonucleotides are characterized by their modifiability and their potential to introduce affinity and signal transducing moieties into the same molecule. Aptasensors with analyte capturing and signal transducing potential have already been addressed in the literature [21, 22]. Detection of aptasensor-based cell biomarkers has received a lot of attention over the last few years [23, 24].Analytical and numerical methods can provide further insight into observed biologically oriented experiments or biological phenomenon, which are difficult to study experimentally [25–29]. Among these available techniques, molecular dynamics and docking technique which are strong computational tools that provide valuable complementary to experiment information about the details of the atomistic interactions in biological phenomena has attracted attention recently. Up to now, various researches have been performed a lot of research regarding the molecular dynamic and docking simulation to investigate the behavior of protein, aptamer, ligands, and peptides in the atomic scale [30, 31]. In 2021, He et al. [32] performed molecular docking simulations for an electrochemical impedimetric sensing platform based on a peptide aptamer, and they found the binding capacity of peptide aptamers by molecular docking and demonstrated that docking was an effective tool to screen peptide aptamers for amino acid-binding capacity.In this study, we investigate the interactions of TNF-α, with aptamer candidates (DNA and RNA) via molecular dynamics (MD) method. For this purpose, two-dimensional (2D) and three-dimensional (3D) structures of aptamers were calculated and were optimized through a 200 ns MD simulation. In the next step, the interactions of these aptamers with different sides of the TNF-α protein were investigated using molecular docking and MD simulations and binding free energy calculations. The results of this study provide useful information for identifying an aptasensor with high selectivity and affinity to TNF-α.


## 2. Method and Materials

### 2.1. Preparation and Calculation the Input Structural Files

In this study, we studied the interactions of two DNA and RNA aptamers with TNF-α. The structure of TNF-α was obtained from the RCSB database (PDB id: 1TNF) [14]. The missing residues of TNF-α were modeled by the SWISS-MODEL web tool and, then, simulated for 200 ns [33, 34]. On the basis of previous experimental studies, we selected two aptamers that specifically interact with TNF-α [35, 36]. The sequence of aptamers is illustrated in the following:25-mer DNA: 5′-TGGTGGATGGCGCAGTCGGCGACAA-3′.28-mer RNA: 5′-GGAGUAUCUGAUGACAAUUCGGAGCUCC-3′.The secondary structures of DNA and RNA aptamers were predicted by the use of the Mfold [37] web server and ViennaRNA [38] web services, respectively. The SimRNA [39] web server was used to predict the 3D structure of RNA aptamers from the 2D pattern. Furthermore, the 3D structure of DNA aptamer was modeled using the method of Iman Jeddi and colleagues [40]. Finally, each 3D structure model of DNA and RNA aptamers was modeled for 200 ns, and the final data were employed for docking with the equilibrated TNF-α conformation.


### 2.2. Molecular Docking

In order to obtain the basic information about the possible binding locations of aptamers to protein, we performed molecular shape complementarity docking using the PatchDock [41] web server. Prepared Protein Data Bank (PDB) files of aptamers and receptor were provided to a PatchDock server at a default value of 4.0 for clustering Root-Mean-Square Deviation (RMSD) and default complex types. PatchDock represents the Connolly’s surface of docking as flat, convex, and concave patches and conforms them to produce candidate transformations [41]. For each aptamer-protein complex, we extracted two select cluster with higher score for using as the initial set of coordinates in the MD simulations [42–44].


### 2.3. Molecular Dynamics (MD) Simulation

All the simulations in the present investigation were performed via the GROMACS 5.1.4 package [45]. Also, the Amber ff99SB-ILDN force fields were used in this study [46]. In order to neutralize the system, we add the appropriate number of the ions (sodium and chloride) that were added to the system. The periodic boundary conditions (PBCs) were applied to each system in all the spatial directions [47]. Additionally, to solvate the system, the transferable intermolecular potential 3-point (TIP3P) water model was utilized [48]. Using LINCS algorithms, all of the covalent bonds lengths were constrained [49]. Simulations were performed using a short-distance electrostatic interplay and a distance cutoff of 1.2 nm for the van der Waals interaction. Using the Particle Mesh Ewald algorithm (PME), the long-range electrostatic forces were calculated [50]. All systems’ energy was minimized via the steepest descent algorithm. The considered cases were then allowed to reach the equilibrium state by a subsequent NVT 500 ps run. After that, all the cases equilibrated through the NPT ensemble. Using Nose-Hoover algorithm temperature [51], the temperature was maintained at 310 K in this process [52, 53]. Over the course of the NPT equilibration, the Parrinello-Rahman barostat was used to maintain the pressures at 101.3 kPa via [54]. Complementary details about simulation systems are provided in Table 1.Table 1
List of abbreviations and key physical parameter values for the performed simulations. In all states, the length of simulation and the temperature are 200 ns and 310 K, respectively.
Simulation compoundsAcronymNo. of water moleculesTNF-αTNF30260RNA aptamerRNA10004TNF-RNA-1TR131425TNF-RNA-2TR230581DNA aptamerDNA7674TNF-DNA-1TD130681TNF-DNA-2TD229451


### 2.4. The g_mmpbsa Method

The interactions between the protein (nonpolar and polar) can be determine via the binding free-energy evaluation and other biological macromolecules. The binding free-energy was calculated by using the MM-PBSA method and the g_mmpbsa tool [55–58].By adding the nonpolar (∆Gnonpolar) and the polar (∆Gpolar) interaction free-energy differences, the overall binding free-energy (∆G) was obtained as the following:(1)∆Gtotal=∆Gpolar+∆Gnonpolar,where ∆Gpolar and ∆Gnonpolar are given by(2)∆Gpolar=∆Gps+∆Gelec,(3)∆Gnonpolar=∆Gnps+∆GvdW,where∆Gps and ∆Gnps are the difference of the polar and nonpolar solvation energies, respectively. Also, ∆GvdW is the difference of the energy related to the van der Waals interactions, and ∆Gelec stands for the difference of the energy associated with the electrostatic interactions. 500 snapshots taken from the last 100 ns of each aptamer-protein complex simulation were used in these binding free-energy calculations.


## 2.1. Preparation and Calculation the Input Structural Files

In this study, we studied the interactions of two DNA and RNA aptamers with TNF-α. The structure of TNF-α was obtained from the RCSB database (PDB id: 1TNF) [14]. The missing residues of TNF-α were modeled by the SWISS-MODEL web tool and, then, simulated for 200 ns [33, 34]. On the basis of previous experimental studies, we selected two aptamers that specifically interact with TNF-α [35, 36]. The sequence of aptamers is illustrated in the following:25-mer DNA: 5′-TGGTGGATGGCGCAGTCGGCGACAA-3′.28-mer RNA: 5′-GGAGUAUCUGAUGACAAUUCGGAGCUCC-3′.The secondary structures of DNA and RNA aptamers were predicted by the use of the Mfold [37] web server and ViennaRNA [38] web services, respectively. The SimRNA [39] web server was used to predict the 3D structure of RNA aptamers from the 2D pattern. Furthermore, the 3D structure of DNA aptamer was modeled using the method of Iman Jeddi and colleagues [40]. Finally, each 3D structure model of DNA and RNA aptamers was modeled for 200 ns, and the final data were employed for docking with the equilibrated TNF-α conformation.


## 2.2. Molecular Docking

In order to obtain the basic information about the possible binding locations of aptamers to protein, we performed molecular shape complementarity docking using the PatchDock [41] web server. Prepared Protein Data Bank (PDB) files of aptamers and receptor were provided to a PatchDock server at a default value of 4.0 for clustering Root-Mean-Square Deviation (RMSD) and default complex types. PatchDock represents the Connolly’s surface of docking as flat, convex, and concave patches and conforms them to produce candidate transformations [41]. For each aptamer-protein complex, we extracted two select cluster with higher score for using as the initial set of coordinates in the MD simulations [42–44].


## 2.3. Molecular Dynamics (MD) Simulation

All the simulations in the present investigation were performed via the GROMACS 5.1.4 package [45]. Also, the Amber ff99SB-ILDN force fields were used in this study [46]. In order to neutralize the system, we add the appropriate number of the ions (sodium and chloride) that were added to the system. The periodic boundary conditions (PBCs) were applied to each system in all the spatial directions [47]. Additionally, to solvate the system, the transferable intermolecular potential 3-point (TIP3P) water model was utilized [48]. Using LINCS algorithms, all of the covalent bonds lengths were constrained [49]. Simulations were performed using a short-distance electrostatic interplay and a distance cutoff of 1.2 nm for the van der Waals interaction. Using the Particle Mesh Ewald algorithm (PME), the long-range electrostatic forces were calculated [50]. All systems’ energy was minimized via the steepest descent algorithm. The considered cases were then allowed to reach the equilibrium state by a subsequent NVT 500 ps run. After that, all the cases equilibrated through the NPT ensemble. Using Nose-Hoover algorithm temperature [51], the temperature was maintained at 310 K in this process [52, 53]. Over the course of the NPT equilibration, the Parrinello-Rahman barostat was used to maintain the pressures at 101.3 kPa via [54]. Complementary details about simulation systems are provided in Table 1.Table 1
List of abbreviations and key physical parameter values for the performed simulations. In all states, the length of simulation and the temperature are 200 ns and 310 K, respectively.
Simulation compoundsAcronymNo. of water moleculesTNF-αTNF30260RNA aptamerRNA10004TNF-RNA-1TR131425TNF-RNA-2TR230581DNA aptamerDNA7674TNF-DNA-1TD130681TNF-DNA-2TD229451


## 2.4. The g_mmpbsa Method

The interactions between the protein (nonpolar and polar) can be determine via the binding free-energy evaluation and other biological macromolecules. The binding free-energy was calculated by using the MM-PBSA method and the g_mmpbsa tool [55–58].By adding the nonpolar (∆Gnonpolar) and the polar (∆Gpolar) interaction free-energy differences, the overall binding free-energy (∆G) was obtained as the following:(1)∆Gtotal=∆Gpolar+∆Gnonpolar,where ∆Gpolar and ∆Gnonpolar are given by(2)∆Gpolar=∆Gps+∆Gelec,(3)∆Gnonpolar=∆Gnps+∆GvdW,where∆Gps and ∆Gnps are the difference of the polar and nonpolar solvation energies, respectively. Also, ∆GvdW is the difference of the energy related to the van der Waals interactions, and ∆Gelec stands for the difference of the energy associated with the electrostatic interactions. 500 snapshots taken from the last 100 ns of each aptamer-protein complex simulation were used in these binding free-energy calculations.


## 3. Results and Discussion

### 3.1. Elucidation of the Structure and Dynamics of Individual Aptamers and TNF-α Proteins

Based on the previous studies, we selected two aptamers that have specific interactions with TNF-α [35, 36]. These RNA and DNA aptamers were 28 and 25 nucleotides in length, respectively. In the next step, the secondary structure and folding of each aptamer were predicted using the Mfold [38] web server and ViennaRNA [38] web services. The Gibbs free energy of folding for DNA and RNA, respectively, and suggest the folding pattern with the most favorable energy. Then, the 3D structure of aptamers predicted based on the folding pattern that calculated in the previous step. Finally, in order to validate the obtained 3D structure, 200 ns molecular dynamics simulation performed on modeled structures. In Figure 1, the 2D, 3D, and equilibrated 3D structures are shown. The ΔG energy of folding for DNA and RNA aptamers is -4.81 kcal/mol and -10.6 kcal/mol, respectively. Many previous computational studies used the RMSD as a parameter to illustrate the equilibration of the system. Hence, in order to investigate the stability of conformations arrived at by our MD simulations, the RMSD value was computed for C-α in TNF-α and the backbone atoms of aptamers. The resulting RMSD time trajectories are illustrated in Figure 2. As shown in Figure 2(a), after about 20 ns, the structural fluctuation of the protein became stable and RMSD values plateaued. Furthermore, aptamers had higher fluctuations in their structure than TNF-α, due to their structural features that make them more flexible than proteins [59].Figure 1
The 2D, 3D (presimulation), and equilibrated 3D (postsimulation) structures of (a) RNA and (b) DNA aptamers.
(a)(b)Figure 2
Time course of RMSD during the MD simulations for (a) TNF-α and (b) aptamers. The RMSD value of each chain of TNF-α was computed separately, and only their average is reported.
(a)(b)In Figure2(b), the DNA aptamer has higher fluctuation in own structure than the RNA aptamer. These results are in line with the value of ΔG energy of folding. In total, the RNA aptamer has a more stable structure than the DNA aptamer. Also, previous experimental works have shown that the RNA folded structure has more stability than the single-strand DNA folded structure [60]. Furthermore, some researchers have employed the MD and docking simulation in their studies. In 2016, Torabi et al. [61] conducted a MD and docking simulation to achieve a better understanding of specific binding interactions of the target protein (RBP4) and RBA. They computed RMSD as a function of time and compare them for the two states, one lone RBA and RBA in the complex with RBP4, and second lone RBP4 backbone and RBP4 backbone in the complex with RBA. The resulting RMSD time trajectories are shown that after about 20 ns, the structural fluctuation of the protein became stable, and it can be concluded that the RBA has more fluctuation than the RBA in the complex with the RBP4. Also, in 2018, Autiero et al. [62] conducted a research via the MD and docking simulation technique and evaluated the dynamics of the complex between the S8 protein and the aptamer. The RMSD values of trajectory structures vs. complex, aptamer, and protein was computed and compared with each other. The RMSD time trajectories shown that the overall system undergoes a little rearrangement after 50 ns, whereas both the protein and the aptamer remain virtually unchanged. Thus, according to these results and our study, it can be concluded that RMSD in MD simulation is an important parameter which can demonstrate the equilibration of the system [30–58].


### 3.2. Studying Protein-Aptamer Interactions by Molecular Docking Simulations

To investigate further the interactions of aptamers with TNF-α in terms of identifying the possible binding locations of aptamers to the protein, we performed molecular docking computations using the PatchDock server. Many previous studies have used the PatchDock server to investigate the interaction of nucleic acids with proteins [63–65]. The coordinates of TNF-α, RNA aptamer, and DNA aptamer were extracted from the last snapshot of the prior MD simulations and used for performing docking computations. For each RNA-protein and DNA-protein complex, we selected two clusters with a higher score relative to other clusters. More detailed information from the docking computation results is shown in Table 2, with both clusters of RNA aptamer having a higher binding affinity to TNF-α than the DNA clusters. The RNA aptamer had more interface area with TNF-α, which shows that more nucleotides of the aptamer are in the direct contacts with protein. Also, the RNA aptamer had higher binding energy with TNF-α than the DNA aptamer. The results of docking calculations are in agreement with previous experimental studies that have illustrated the RNA molecules to have stronger interactions with proteins due to the Ribose carbohydrate in their backbone [66, 67].Table 2
Results of the molecular docking calculations between aptamers and TNF-α.
Geometric shape complementarity score [68]Approximate interface area of the complex (A2) (area)Atomic contact energy [69] (kcal/mol) (ACE)TR1131601870.90-235.58TR2128761736.30-166.63TD1108651532.40-153.24TD298641498.80-142.57As shown in Figure3, the binding locations of aptamers onto the protein surface differed in each cluster. Furthermore, the orientation of aptamers was different in clusters. Both aptamers in cluster 1interacted with two side chains of TNF-α, but aptamers in cluster 2 interacted with one chain only. This is probably why the aptamers in cluster 2 had a smaller interface area with TNF-α relative to the aptamers in cluster 1.Figure 3
Graphical representation of the aptamer-TNF-α complexes computed by PatchDock.


### 3.3. Molecular Dynamics Simulations

The atomic coordinates of aptamer-protein complexes obtained by molecular docking calculations were used as initial conditions for subsequent molecular dynamics simulations of 200 ns duration to help refine further our understanding of aptamer-TNF-α interactions.


### 3.4. Structural Analyses of Aptamer-Protein Complexes through MD Simulation

To investigating the effects of the conformational fluctuations of aptamers and TNF-α on the structural stability of the complexes they formed, the RMSD values of each entity were computed during their interactions over 200 ns MD simulation trajectories. First, we studied the fluctuations of the TNF-α structure during interactions with aptamers. As shown in Figure 4, the interactions of aptamers with TNF-α had no major effect on the structure of the protein. For each interaction pair, the RMSD value of three TNF-α chains was calculated separately, and the average value was reported. All interaction pairs reached stable states after about 15 ns, and the fluctuations of TNF-α structure in all simulation systems were almost equal and in the range of 0.2 nm to 0.4 nm. Hence, we can conclude that bound aptamers to the protein did not have any major effect on the structure of TNF-α. These findings are in line with other experimental and computational studies [70, 71].Figure 4
The RMSD value of TNF-α during interactions with (a) an RNA aptamer and (b) a DNA aptamer. To better represent the effect of aptamers on the protein structure, the RMSD plot of TNF-α alone was shown in both graphs.
(a)(b)Furthermore, the RMSD value of aptamers during interactions with TNF-α was also calculated. The RMSD was computed for only the backbone atoms of the aptamers. The results show that interactions with TNF-α reduced the structural fluctuations of the RNA aptamer (Figure 5(a)). In RNA-contained simulations, we can see that the fluctuations of free RNA aptamer are about 0.7 nm at the end of the simulation, but in the TR1 and TR2 systems, the structural fluctuations are 0.2 nm and 0.4 nm, respectively. By comparing these results with the docking results, we can conclude that the interactions between aptamer and TNF-α are stronger, and as a result, the structural fluctuations of the aptamer are reduced. For instance, the fluctuations of RNA aptamer in the TR1 system are lower than the fluctuation of the RNA aptamer in the TR2 system (Figure 5(a)), because the aptamer-protein complex in the TR1 system has a stronger binding affinity (Table 2). When the aptamer binds to the protein surface, the strong electrostatic interactions between basic residues of protein and negatively charged nucleotides make the structural fluctuations of the protein restrained. For the DNA-contained systems, similar behavior was observed (Figure 5(b)).Figure 5
RMSD plots in (a) RNA-contained simulation systems and (b) DNA-contained simulation systems.
(a)(b)


### 3.5. Aptamer-Protein Interactions during MD Simulations

For explaining the affinity binding of aptamers to TNF-α, the contact numbers between aptamers and protein were calculated during the simulation trajectories (Figure 6). Furthermore, to determine the role of different types of protein residues in interactions with aptamers, the average number of contacts between different types of residue (polar, nonpolar, aromatic, basic, and acidic) and each aptamer was computed (Figure 7). As shown in Figure 6, the RNA aptamer in both clusters has a higher number of contacts with TNF-α than the DNA aptamer. Also, the RNA aptamer in the TR1 system had the highest number of contacts between all simulation systems (Figure 6(a)). These findings are in good agreement with the results of our molecular docking computations and illustrate that the RNA aptamer had stronger interactions with protein relative to the DNA aptamer. The latter had almost equal number of contacts with protein in both clusters (Figure 6(b)).Figure 6
The number of contacts in (a) RNA-contained systems and (b) DNA-contained systems during 200 ns MD simulation trajectories (>0.6 nm).
(a)(b)Figure 7
Average number of contacts between different types of residues and aptamers in (a) RNA-contained systems and (b) DNA-contained systems (>0.6 nm).
(a)(b)Moreover, the results of the contact number analyses have shown the significant role of the basic residues of TNF-α that have in the interactions with aptamers. Previous investigations indicate that in the interactions with anionic molecules such as antimicrobial peptides and nucleic acids the basic residues in the proteins play a major role [72, 73]. In Figure 7, it has shown that in all systems, the basic residues had the highest number of contacts with aptamers which is due to the strong electrostatic interactions with negatively charged nucleotides. The basic residues of the TNF-α had higher numbers of contacts with the RNA and DNA aptamers. These findings also provide the insight that RNA aptamer interacted more strongly with TNF-α compared to DNA ones due to the more polar and less basic nature of the residues in the latter, while the number of aptamer contacts with TNF-α was comparable in both cases.To provide a better understanding of aptamer-protein complexes, the graphical representation of complexes at the end of the simulation was illustrated Figure8. As shown in Figure 8, aptamers in cluster 2 could not maintain their positions on the protein’s surface and settled at some distance from protein, especially their terminal nucleotides On the other hand, in cluster 1 systems, aptamers could conserve their interactions with TNF-α and have a higher number of contacts which are in line with results of our molecular docking computations (Table 2). In total, we can interpret from the results of MD simulation section that RNA aptamer can makes stronger interaction with TNF-α. Taken together, the number of contacts, free energy, and docking, all support the conclusion of stronger interactions with RNA. Our simulation findings are therefore congruent with the results of the previous experimental studies demonstrating the feasibility of using RNA aptamers as apta-biosensors tailored to bind target proteins with high specificity and at very low concentrations [36, 74].Figure 8
Graphical representation of aptamer-protein complexes at the end of the 200 ns MD simulations for (a) RNA-contained systems and (b) DNA-contained systems.
(a)(b)


### 3.6. Binding Free Energy Calculations

Previous investigations have revealed that electrostatic interactions play a key role in biological macromolecule interactions, especially when these have opposite charges [56, 71, 75–77]. To get more information about the interaction of aptamers with the TNF-α via the g_mmpbsa tool (Table 3), the nonpolar, polar, and total binding free energy between the aptamers and the protein were computed. The table shows that the van der Waals (vdW) energy had a major contribution in nonpolar energy, while SASA energy had a small contribution. The vdW energy has a significant effect on the interactions between the aptamer and the protein, especially in TR1 and TR2 simulation systems. This is probably due to the favorable hydrophobic interactions of aptamers with protein. This is possibly due to the electrostatic interaction between the basic residues of TNF-α and the negatively charged nucleotides. The strongest electrostatic energy was found in TR1 and the weakest one in TD2. The polar solvation energy was positive in all the simulations. In total, by taking into account the total binding energy as a parameter to represent the strength of aptamer-protein complexes, the TR1 was the strongest complex and TD2 was the weakest. The results of these binding free energy calculations are in close agreement with the previous results and confirm that the RNA aptamer had stronger interactions with TNF-α than the DNA aptamer.Table 3
Binding free energy calculations of the aptamer-protein interactions. (kJ/mol).
Simulation systemsNonpolar binding free energyPolar binding free energyTotal binding free energyVan der Waals energySASA energyElectrostatic energyPolar solvation energyTR1-475.247-48.14-7291.52374.127-7740.783TR2-287.586-25.481-5761.59463.842-6010.819TD1-362.815-29.648-5634.12841.479-5985.112TD2-259.483-18.87-4861.24348.275-5091.321


## 3.1. Elucidation of the Structure and Dynamics of Individual Aptamers and TNF-α Proteins

Based on the previous studies, we selected two aptamers that have specific interactions with TNF-α [35, 36]. These RNA and DNA aptamers were 28 and 25 nucleotides in length, respectively. In the next step, the secondary structure and folding of each aptamer were predicted using the Mfold [38] web server and ViennaRNA [38] web services. The Gibbs free energy of folding for DNA and RNA, respectively, and suggest the folding pattern with the most favorable energy. Then, the 3D structure of aptamers predicted based on the folding pattern that calculated in the previous step. Finally, in order to validate the obtained 3D structure, 200 ns molecular dynamics simulation performed on modeled structures. In Figure 1, the 2D, 3D, and equilibrated 3D structures are shown. The ΔG energy of folding for DNA and RNA aptamers is -4.81 kcal/mol and -10.6 kcal/mol, respectively. Many previous computational studies used the RMSD as a parameter to illustrate the equilibration of the system. Hence, in order to investigate the stability of conformations arrived at by our MD simulations, the RMSD value was computed for C-α in TNF-α and the backbone atoms of aptamers. The resulting RMSD time trajectories are illustrated in Figure 2. As shown in Figure 2(a), after about 20 ns, the structural fluctuation of the protein became stable and RMSD values plateaued. Furthermore, aptamers had higher fluctuations in their structure than TNF-α, due to their structural features that make them more flexible than proteins [59].Figure 1
The 2D, 3D (presimulation), and equilibrated 3D (postsimulation) structures of (a) RNA and (b) DNA aptamers.
(a)(b)Figure 2
Time course of RMSD during the MD simulations for (a) TNF-α and (b) aptamers. The RMSD value of each chain of TNF-α was computed separately, and only their average is reported.
(a)(b)In Figure2(b), the DNA aptamer has higher fluctuation in own structure than the RNA aptamer. These results are in line with the value of ΔG energy of folding. In total, the RNA aptamer has a more stable structure than the DNA aptamer. Also, previous experimental works have shown that the RNA folded structure has more stability than the single-strand DNA folded structure [60]. Furthermore, some researchers have employed the MD and docking simulation in their studies. In 2016, Torabi et al. [61] conducted a MD and docking simulation to achieve a better understanding of specific binding interactions of the target protein (RBP4) and RBA. They computed RMSD as a function of time and compare them for the two states, one lone RBA and RBA in the complex with RBP4, and second lone RBP4 backbone and RBP4 backbone in the complex with RBA. The resulting RMSD time trajectories are shown that after about 20 ns, the structural fluctuation of the protein became stable, and it can be concluded that the RBA has more fluctuation than the RBA in the complex with the RBP4. Also, in 2018, Autiero et al. [62] conducted a research via the MD and docking simulation technique and evaluated the dynamics of the complex between the S8 protein and the aptamer. The RMSD values of trajectory structures vs. complex, aptamer, and protein was computed and compared with each other. The RMSD time trajectories shown that the overall system undergoes a little rearrangement after 50 ns, whereas both the protein and the aptamer remain virtually unchanged. Thus, according to these results and our study, it can be concluded that RMSD in MD simulation is an important parameter which can demonstrate the equilibration of the system [30–58].


## 3.2. Studying Protein-Aptamer Interactions by Molecular Docking Simulations

To investigate further the interactions of aptamers with TNF-α in terms of identifying the possible binding locations of aptamers to the protein, we performed molecular docking computations using the PatchDock server. Many previous studies have used the PatchDock server to investigate the interaction of nucleic acids with proteins [63–65]. The coordinates of TNF-α, RNA aptamer, and DNA aptamer were extracted from the last snapshot of the prior MD simulations and used for performing docking computations. For each RNA-protein and DNA-protein complex, we selected two clusters with a higher score relative to other clusters. More detailed information from the docking computation results is shown in Table 2, with both clusters of RNA aptamer having a higher binding affinity to TNF-α than the DNA clusters. The RNA aptamer had more interface area with TNF-α, which shows that more nucleotides of the aptamer are in the direct contacts with protein. Also, the RNA aptamer had higher binding energy with TNF-α than the DNA aptamer. The results of docking calculations are in agreement with previous experimental studies that have illustrated the RNA molecules to have stronger interactions with proteins due to the Ribose carbohydrate in their backbone [66, 67].Table 2
Results of the molecular docking calculations between aptamers and TNF-α.
Geometric shape complementarity score [68]Approximate interface area of the complex (A2) (area)Atomic contact energy [69] (kcal/mol) (ACE)TR1131601870.90-235.58TR2128761736.30-166.63TD1108651532.40-153.24TD298641498.80-142.57As shown in Figure3, the binding locations of aptamers onto the protein surface differed in each cluster. Furthermore, the orientation of aptamers was different in clusters. Both aptamers in cluster 1interacted with two side chains of TNF-α, but aptamers in cluster 2 interacted with one chain only. This is probably why the aptamers in cluster 2 had a smaller interface area with TNF-α relative to the aptamers in cluster 1.Figure 3
Graphical representation of the aptamer-TNF-α complexes computed by PatchDock.


## 3.3. Molecular Dynamics Simulations

The atomic coordinates of aptamer-protein complexes obtained by molecular docking calculations were used as initial conditions for subsequent molecular dynamics simulations of 200 ns duration to help refine further our understanding of aptamer-TNF-α interactions.


## 3.4. Structural Analyses of Aptamer-Protein Complexes through MD Simulation

To investigating the effects of the conformational fluctuations of aptamers and TNF-α on the structural stability of the complexes they formed, the RMSD values of each entity were computed during their interactions over 200 ns MD simulation trajectories. First, we studied the fluctuations of the TNF-α structure during interactions with aptamers. As shown in Figure 4, the interactions of aptamers with TNF-α had no major effect on the structure of the protein. For each interaction pair, the RMSD value of three TNF-α chains was calculated separately, and the average value was reported. All interaction pairs reached stable states after about 15 ns, and the fluctuations of TNF-α structure in all simulation systems were almost equal and in the range of 0.2 nm to 0.4 nm. Hence, we can conclude that bound aptamers to the protein did not have any major effect on the structure of TNF-α. These findings are in line with other experimental and computational studies [70, 71].Figure 4
The RMSD value of TNF-α during interactions with (a) an RNA aptamer and (b) a DNA aptamer. To better represent the effect of aptamers on the protein structure, the RMSD plot of TNF-α alone was shown in both graphs.
(a)(b)Furthermore, the RMSD value of aptamers during interactions with TNF-α was also calculated. The RMSD was computed for only the backbone atoms of the aptamers. The results show that interactions with TNF-α reduced the structural fluctuations of the RNA aptamer (Figure 5(a)). In RNA-contained simulations, we can see that the fluctuations of free RNA aptamer are about 0.7 nm at the end of the simulation, but in the TR1 and TR2 systems, the structural fluctuations are 0.2 nm and 0.4 nm, respectively. By comparing these results with the docking results, we can conclude that the interactions between aptamer and TNF-α are stronger, and as a result, the structural fluctuations of the aptamer are reduced. For instance, the fluctuations of RNA aptamer in the TR1 system are lower than the fluctuation of the RNA aptamer in the TR2 system (Figure 5(a)), because the aptamer-protein complex in the TR1 system has a stronger binding affinity (Table 2). When the aptamer binds to the protein surface, the strong electrostatic interactions between basic residues of protein and negatively charged nucleotides make the structural fluctuations of the protein restrained. For the DNA-contained systems, similar behavior was observed (Figure 5(b)).Figure 5
RMSD plots in (a) RNA-contained simulation systems and (b) DNA-contained simulation systems.
(a)(b)


## 3.5. Aptamer-Protein Interactions during MD Simulations

For explaining the affinity binding of aptamers to TNF-α, the contact numbers between aptamers and protein were calculated during the simulation trajectories (Figure 6). Furthermore, to determine the role of different types of protein residues in interactions with aptamers, the average number of contacts between different types of residue (polar, nonpolar, aromatic, basic, and acidic) and each aptamer was computed (Figure 7). As shown in Figure 6, the RNA aptamer in both clusters has a higher number of contacts with TNF-α than the DNA aptamer. Also, the RNA aptamer in the TR1 system had the highest number of contacts between all simulation systems (Figure 6(a)). These findings are in good agreement with the results of our molecular docking computations and illustrate that the RNA aptamer had stronger interactions with protein relative to the DNA aptamer. The latter had almost equal number of contacts with protein in both clusters (Figure 6(b)).Figure 6
The number of contacts in (a) RNA-contained systems and (b) DNA-contained systems during 200 ns MD simulation trajectories (>0.6 nm).
(a)(b)Figure 7
Average number of contacts between different types of residues and aptamers in (a) RNA-contained systems and (b) DNA-contained systems (>0.6 nm).
(a)(b)Moreover, the results of the contact number analyses have shown the significant role of the basic residues of TNF-α that have in the interactions with aptamers. Previous investigations indicate that in the interactions with anionic molecules such as antimicrobial peptides and nucleic acids the basic residues in the proteins play a major role [72, 73]. In Figure 7, it has shown that in all systems, the basic residues had the highest number of contacts with aptamers which is due to the strong electrostatic interactions with negatively charged nucleotides. The basic residues of the TNF-α had higher numbers of contacts with the RNA and DNA aptamers. These findings also provide the insight that RNA aptamer interacted more strongly with TNF-α compared to DNA ones due to the more polar and less basic nature of the residues in the latter, while the number of aptamer contacts with TNF-α was comparable in both cases.To provide a better understanding of aptamer-protein complexes, the graphical representation of complexes at the end of the simulation was illustrated Figure8. As shown in Figure 8, aptamers in cluster 2 could not maintain their positions on the protein’s surface and settled at some distance from protein, especially their terminal nucleotides On the other hand, in cluster 1 systems, aptamers could conserve their interactions with TNF-α and have a higher number of contacts which are in line with results of our molecular docking computations (Table 2). In total, we can interpret from the results of MD simulation section that RNA aptamer can makes stronger interaction with TNF-α. Taken together, the number of contacts, free energy, and docking, all support the conclusion of stronger interactions with RNA. Our simulation findings are therefore congruent with the results of the previous experimental studies demonstrating the feasibility of using RNA aptamers as apta-biosensors tailored to bind target proteins with high specificity and at very low concentrations [36, 74].Figure 8
Graphical representation of aptamer-protein complexes at the end of the 200 ns MD simulations for (a) RNA-contained systems and (b) DNA-contained systems.
(a)(b)


## 3.6. Binding Free Energy Calculations

Previous investigations have revealed that electrostatic interactions play a key role in biological macromolecule interactions, especially when these have opposite charges [56, 71, 75–77]. To get more information about the interaction of aptamers with the TNF-α via the g_mmpbsa tool (Table 3), the nonpolar, polar, and total binding free energy between the aptamers and the protein were computed. The table shows that the van der Waals (vdW) energy had a major contribution in nonpolar energy, while SASA energy had a small contribution. The vdW energy has a significant effect on the interactions between the aptamer and the protein, especially in TR1 and TR2 simulation systems. This is probably due to the favorable hydrophobic interactions of aptamers with protein. This is possibly due to the electrostatic interaction between the basic residues of TNF-α and the negatively charged nucleotides. The strongest electrostatic energy was found in TR1 and the weakest one in TD2. The polar solvation energy was positive in all the simulations. In total, by taking into account the total binding energy as a parameter to represent the strength of aptamer-protein complexes, the TR1 was the strongest complex and TD2 was the weakest. The results of these binding free energy calculations are in close agreement with the previous results and confirm that the RNA aptamer had stronger interactions with TNF-α than the DNA aptamer.Table 3
Binding free energy calculations of the aptamer-protein interactions. (kJ/mol).
Simulation systemsNonpolar binding free energyPolar binding free energyTotal binding free energyVan der Waals energySASA energyElectrostatic energyPolar solvation energyTR1-475.247-48.14-7291.52374.127-7740.783TR2-287.586-25.481-5761.59463.842-6010.819TD1-362.815-29.648-5634.12841.479-5985.112TD2-259.483-18.87-4861.24348.275-5091.321


## 4. Conclusion

In this study, we investigated the interactions of RNA and DNA aptamers with TNF-α at the atomic scale by binding free energy calculations, molecular docking, and MD simulations. It was observed that the folding process of the RNA aptamer had more negative ΔG energy than the folding process of the DNA aptamer. Furthermore, the results of molecular docking calculations showed that the RNA aptamer had stronger interactions with TNF-α and got a higher score relative to the DNA aptamer. Computational structural analyses illustrated that during interactions, the aptamers did not have any negative effect on the TNF-α structure. Furthermore, the structural fluctuations of aptamers were reduced during interactions with the protein. The results of binding free energy calculations and MD simulations showed that the RNA aptamer had stronger interactions with protein than the DNA aptamer. The results revealed that basic residues of TNF-α had more contacts at the atomic scale with aptamer relative to other residue types. RNA aptamers, in turn, created more contacts with protein and thermodynamically had more favorable binding energy with TNF-α. Collectively, these findings illustrated that RNA aptamers are a more suitable candidate, compared to DNA aptamers, to use for constructing an apta-biosensor for sensing the presence of TNF-α in a biological sample, which is in agreement with previous experimental studies [40]. It is also important to point out that the computational methodologies workflow developed in this work could be generalizable to the design of other apta-biosensors to their target proteins.



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*Source: 9926128-2021-07-17.xml*