Molecular Dynamics Study on Encapsulation of Double Stranded

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C: Physical Processes in Nanomaterials and Nanostructures

Molecular Dynamics Study on Encapsulation of Double Stranded Nucleic Acids into Carbon Nanotube Yanxiao Han, and Chuanlu Yang J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b05754 • Publication Date (Web): 26 Jul 2018 Downloaded from http://pubs.acs.org on July 26, 2018

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The Journal of Physical Chemistry

Molecular Dynamics Study on Encapsulation of Double Stranded Nucleic Acids into Carbon Nanotube Yanxiao Han†, Chuan-Lu Yang‡* †

Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607, USA

‡

School of Physics and Optoelectronics Engineering, Ludong University, Yantai 264025,

People's Republic of China

ABSTRACT: By using molecular dynamics simulations, we investigate the spontaneous encapsulation and offloading of the double-stranded DNA and RNA with carbon nanotubes (CNTs). The bare double-stranded DNA and RNA show deficiency in the process of encapsulation into CNT. Here, we propose an encapsulation method of nucleic acid cargo by the aid of the positively charged canoe of CNT and an offloading step driven by a thinner CNT. The spontaneous encapsulation and offloading processes are characterized by the change of nucleic acid length, opens of base pairs, the distance between the DNA/RNA and the CNT, interaction energy and free energy between nucleic acids and CNT. The present findings can be used for nucleic acids delivery and storage.

1. INTRODUCTION *

Corresponding author. E-mail address: [email protected](C. L. Yang). 1 ACS Paragon Plus Environment

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Carbon nanotubes (CNTs) with multiple properties have become a functional material in diverse areas since its first discovery in 19911. Because of the large surface area, unique surface properties, needle-like shape, and biocompatibility, CNTs were also considered to be promising candidates as molecular transporter systems2-3 and nanocarriers for therapeutic and diagnostic purposes in gene therapy4. The various possibilities for CNT surface modifications 5, leading to functional CNT (f-CNT), make CNTs ideal for delivering a whole host of nucleic acids in the gene therapy

6-7

. Successful transfection of DNA8, small interfering

RNA (siRNA)9-10, oligonucleotides (ODNs) and aptamers4 by f-CNTs are reported both in vitro and in vivo experiments. However, the transfection efficacy is still low, only 10 times higher than the naked gene transferring8, because of the nucleic acid degradation by nucleases, rapid excretion by kidney when less than 21 base pairs11, inefficient endocytosis by targeted cell, and the inefficient release from endosomes12. To increase the gene transfection, a more stable and higher payload method is needed to protect gene from degradation as the transferred nucleic acid usually electrostatically complex, covalently link or adsorb on the surface of CNT13-14, which increase the chance of nucleic acid hydrolysis, especially for RNA with an extra hydroxy group in the 2’ position of pentose ring11. Considering the novel structure and a nature cavity inside CNT, we propose that the nucleic acid cargo is encapsulated into CNT prior to gene transfection to well protect and 2 ACS Paragon Plus Environment

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delivery gene effectively. Meanwhile, a spontaneous cargo off-loading mechanism is designed to overcome the inefficient release of nucleic acids after entering the cell. The molecular dynamics (MD) simulations for the self-assembly of nucleic acids on CNT outside wall15-19 were performed, and the translocation of short dsDNA (8 base pairs) and short dsRNA (22 base pairs) through CNT20-21 were studied. Due to the high free energy barrier, the translocation of dsDNA inside CNT is challenging21, which can only be realized by gel electrophoresis in experiment22.

As the experiments verified that the safe length threshold for

transfecting dsRNA is between 21 and 30 base pairs (varying by the infected cell type)12, we use the long (30 base pairs) double-stranded DNA (dsDNA) and dsRNA to carry out the MD simulations of our proposed spontaneous encapsulation and off-loading processes. 2. COMPUTATIONAL DETAILS 2.1 Atomic models of simulation systems The dsDNA/RNA is first put on a novel canoe, then translocated into the CNT capsule facilitated by the carrying of the canoe. The canoe is made of a (15,15) CNT with the length of 14 nm and diameter of 2 nm by longitudinally slicing the pristine CNT 23, as shown in Figure 1. The CNT of (30,30) with the length of 14 nm and diameter of 4 nm is used as a capsule. To strengthen the interaction

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between canoe and dsDNA/RNA, the canoe is charged with the same amount of opposite charges of dsDNA/RNA. To achieve the offloading of dsDNA/RNA, we use the thinner (10,10) CNT with the length of 15 nm as a piston to eject the nucleic acids. The piston diameter depends on the diameter of the canoe and CNT capsule. Our previous work examined carefully how to choose piston diameter 24. To make the piston more competitive to bind with CNT during ejection, the proper piston diameter should be less than 4 nm (CNT diameter) and bigger than 2 nm (canoe diameter), here we use 2.8 nm. The nucleic acid sequence is 5’- CGG CCT GGC CTT ACA TCT CGG CCT GGC CTT-3’ for dsDNA, while replace all the T by U for dsRNA. We study several systems for dsDNA/RNA translocation into CNT: (1) dsDNA with CNT capsule only, (2) dsRNA with CNT capsule only, (3) dsDNA translocate into CNT capsule with charged canoe, (4) dsRNA translocate into CNT capsule with charged canoe, (5) dsDNA translocate into CNT capsule with noncharged canoe, (6) dsRNA translocate into CNT capsule with noncharged canoe, (7) ejection of dsDNA in system 3, (8) ejection of dsRNA in system 4. The system details are summarized in Table 1. All the systems are immersed in a water box with 150 mM NaCl ions. 2.2 MD simulations


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First, 2000 steps of minimization are performed to remove the steric clashes between solvent molecules. Then, temperature reassignment is performed to set the system to the desired temperature (310 K) by periodically reassigning the velocities in the system. Next, ions and water molecules are equilibrated for 2 ns around dsDNA/RNA which are restrained using harmonic forces with a spring constant of 1 kcal/(mol Å2). The last frames of restrained equilibration are used to start simulations of free dsDNA/RNA and CNTs. The equilibration of the system is performed with NAMD225 in the NPT ensemble (p = 1 bar and T = 310 K), using the Langevin dynamics (γLang = 1 ps-1) with a time step of 2.0 fs. The nucleic acids are described with CHARMM36 force field26 and CNT atoms described as benzene-type atoms (CA). Explicit TIP3P water model is used to solvate the system27. Non-bonded interaction is truncated at 12 Å, the X-PLOR switching function for electrostatic and vdW calculations is activated at 10 Å. The long-range Coulombic interactions are evaluated with the Particle Mesh Ewald (PME)28 method. The simulations for encapsulation of dsDNA/RNA with charged canoe last for 112 ns and 100 ns, respectively. Other simulations are run for 20 ns. 2.3 Potential of mean force (PMF) calculation To validate the obtained translocation, and to gain insight into the translocation dynamics, we calculate the Gibbs free energy of the binding of the canoe to CNT capsule, using umbrella sampling (US) method. To obtain the reaction coordinate, 5 ACS Paragon Plus Environment


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a collective variable (colvar)29 based simulation is performed. The center of mass distance between canoe and CNT capsule is selected as colvar components with initial distance 147 Å and final distance 120 Å. After 2 ns colvar based simulation, we get the trajectory of pulling canoe (with nucleic acid) into CNT capsule. The reaction coordinate is then partitioned into 13 windows with 2 Å of width. Biasing harmonic potentials are introduced to obtain a sampling of less probable states with a force constant of 5 kcal/(mol Å2). Each US window is run for 10 ns, which amounts to a total time of 130 ns. After sorting the resulting trajectories into the specified windows, we use the weighted histogram analysis method30 (WHAM) to reconstruct PMF. 2.4 Data analysis method Both the initial dsDNA and RNA structures with B-form are generated with 3DNA program31. The base pair open is measured by using CURVES+

32

program.

The evaluation of solvent accessible surface area (SASA) of the system of canoe and dsDNA/RNA is performed with the built-in SASA VMD plugin33, where a vdW radius of 1.4 Å is assigned to atoms to identify the points on a sphere that are accessible. 3. RESULTS AND DISCUSSION


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The present study examines how to encapsulate dsDNA/RNA with CNT. The early study

17

shows that the 8 base pairs of dsDNA need to be placed initially a

part inside the CNT to achieve the further translocation. Here, we place the 30 base pairs of dsDNA/RNA initially 13 Å away from one end of CNT with different distance to the side wall as shown in Figure 2a-d. The simulation lasts for 20 ns during which both nucleic acids and CNT are free. It is possible that dsDNA attaches to the side wall of CNT with the open base pair, but the further translocation leads to the unzipping of dsDNA chains as shown in Figure 2e. The inset figure shows that the unzipped base pairs of dsDNA stack to the CNT side wall with a single nucleic acid chain attaching to the inside wall. Figure 2f shows that although dsRNA has an open base pair, it leaves the CNT due to the further distance to CNT sidewall (Figure 2d). Given that the spontaneous translocation of the bare dsDNA/RNA into the CNT is unfavorable, a more efficient method is needed to encapsulate nucleic acids. 3.1 Encapsulation of dsDNA/RNA into carbon nanotube by using a positively charged canoe. The dsDNA/RNA is pre-placed in the middle of canoe charged by 60 positive charges. Considering that the maximum of vdW attraction appears at about 4 Å for the CNT atom, we put the canoe carrying dsDNA/RNA 4 Å away from the CNT. Figure 3 shows the snapshots of the translocation of the canoe into CNT capsule, 7 ACS Paragon Plus Environment


during which relative motion of dsDNA/RNA occurs on canoe surface. The translocation starts at 1.2 ns/5.5 ns for dsDNA/RNA with a canoe. The complete encapsulation with canoe takes about 2.4 ns and 6.7 ns for dsDNA and dsRNA, respectively. The simulations are up to 100 ns after the completion of encapsulation, during which dsDNA/RNA are well confined inside of canoe with slightly conformational change (Figure S1 and S2). To well understand the encapsulation process which happens rapidly, the first 20 ns of the trajectory is analyzed. Figure 4a shows that dsDNA/RNA relax to 107 Å/117 Å before translocation starts, then get compressed to 94 Å/100 Å when the canoes completely fit into the CNT (at 2.4 ns and 6.7 ns), finally relax back to the original length and stretch during the analyzed 20 ns. Figure 4b shows the distance between the front end of dsDNA/RNA and the same end of the canoe (head to head distance), which has an opposite trend as Figure 4a. The head to head distance of dsDNA/RNA with canoe reaches the maximum of about 42 Å/29 Å, when the dsDNA/RNA length reaches the minimum. With the process of translocation, the solvent accessible area of dsDNA/RNA with canoe (Figure 4c) shows slight decrease, then drops to 19,000 Å2 and 19,500 Å2, respectively, after the completion of translocation. As CNT capsule is neutral, the electrostatic energy between CNT and others is zero, the driven force of the translocation arises only from the vdW interaction. Figure 4d shows that the vdW interaction energy between canoe and 8 ACS Paragon Plus Environment

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CNT reaches the same maximum value when the translocation completes. To understand better the interaction between CNT and canoe, we calculate the Gibbs free energy of binding of the canoe with dsDNA to the CNT capsule. The obtained binding free energy is -180 kcal/mol, when the distance between the canoe and CNT centers reduces to 121 Å. The huge decrease of free energy indicates that the encapsulation of CNT canoe is spontaneous (Figure 5). To examine the effect of charge on the canoe to the encapsulation of dsDNA/RNA, we simulated the dsDNA/RNA with a neutral canoe as the control group. The results are shown in Figure 6. For the neutral canoe, the translocations start at 2.2 and 3.3 ns for dsDNA and dsRNA, respectively. During the translocation, the front end of dsDNA/RNA slides back to the back end of the canoe, leading to the bending of nucleic acids. The head to head distance between dsDNA/RNA and canoe increases and separates to 99/61 Å when the canoe translocate into the CNT at 2.8/4.2 ns, and keeps a relative constant value during the next 15 ns (Figure 7a). Although the SASA of the dsDNA/RNA with neutral canoe reduces to a similar number as the case of the charged canoe (Figure 7b and 4c), the structure of dsDNA/RNA with neutral canoe bends severely. To check the deformation of dsDNA/RNA during translocation, we calculate the open degree of base pairs in the charged and uncharged canoe cases (Figure 8a and c). There are more open base pairs in the dsRNA than dsDNA on the charged 9 ACS Paragon Plus Environment


canoe surface, while the number of the open base pairs of dsDNA/RNA is similar to each other on the uncharged canoe. The snapshots of base pair interacting with canoe are shown in Figure 8b and d. The bases within 3 Å of CNT are shown in atomic details. Most base pairs of the dsDNA on the charged canoe (Figure 8b up) are well paired, only a few bases at the end of dsDNA chain are open and interact with canoe, while the base pairs in dsRNA are not well ordered with random bases interacting with CNT (Figure 8b down). Figure 8d shows that bases at the bend of dsDNA/RNA opens randomly and those at the two ends of nucleic acids interact more with CNT. Comparing the charged canoe with the neutral one, the former can provide a binding force to dsDNA/RNA by electrostatic interaction which minimizes the nucleic acids deformation. 3.2 Spontaneous offloading of the dsDNA/RNA The offloading of the encapsulated dsDNA/RNA from the CNT is needed to make the encapsulation applicable. Here, the encapsulated dsDNA/RNA with the charged canoe in CNT is used to investigate the offloading process. The mechanism is based on the competitive binding of CNT piston to the CNT capsule, which is similar to the previous report

24

. The snapshots of the ejection of

dsDNA/RNA with canoe are shown in Figure 9. The thinner CNT (green) working as the piston is placed within 4 Å of CNT capsule (gray). The pushing of dsDNA with canoe starts at 0 ns and lasts for 4.2 ns, while the pushing of dsRNA starts at 10 ACS Paragon Plus Environment

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3.8 ns and completes at 10 ns. The interaction energy of the whole system, the energy between canoe and CNT capsule, between piston and CNT capsule are shown in Figure 10. During the offloading of dsDNA with canoe, the interaction energy between the piston and CNT capsule is increasing, while the energy between canoe and CNT capsule is decreasing, as a result, the net interaction energy of the system increases and reaches a stable value at 4.2 ns (Figure 10a). Similarly, after 3.8 ns of preparation, the ejection of dsRNA with canoe starts with the decreasing interaction energy between canoe and CNT capsule and the increasing interaction energy between the piston and CNT canoe (Figure 10b). The increasing net interaction energy of both systems during the ejection implies that the binding of the piston to the CNT capsule is more competitive than the binding of the canoe to CNT capsule. This can be explained by the essentially more carbon atoms in the CNT piston than the canoe inducing more vdW interaction. To use this mechanism in the real system, we suggest that the CNT piston links to the CNT capsule by disulfide bonds that can be cleaved by the acid environment of endosome in the cell. Once the complex assembly in Figure 9 reaches endosome, the rapid offloading starts after disulfide bonds cleavage, which makes dsDNA/RNA easy to release. As our proposed encapsulation-offloading dynamics is controlled mainly by vdW interaction between canoe/piston and CNT capsule, it is expected to eliminate the influence of salt concentration gradient during nucleic 11 ACS Paragon Plus Environment


acid delivery. As shown in Figure S5 and S6, the encapsulation and offloading of dsDNA with charged canoe still work in pure water without NaCl. Chakrabarti et. al.16, 19 showed that the salt concentration influences the nucleic acid loading ability by changing the conformation of nucleic acid on the CNT outside surface. In our cases, nucleic acids are always confined inside of canoe and CNT, which maintains the loading ability of CNT. Even if conformation of nucleic acids changed, offloading still works by a selected piston with proper diameter24. 4. CONCLUSION In the present study, we have examined in atomic detail for the processive dsDNA/RNA translocation in the CNT capsule by the aid of CNT canoe and the offloading of dsDNA/RNA by the CNT piston. Positively charged canoe can well maintain the conformation of dsDNA/RNA, while the neutral canoe causes the deformation of the nucleic acid structure during the translocation. The dsDNA/RNA can be physically adsorbed on the canoe by coulombic interaction. The vdW interaction between the canoe and CNT capsule results in the spontaneous encapsulation of dsDNA/RNA with the canoe. The free energy profile indicates that translocation of the canoe into the CNT capsule is energy favorable. The encapsulation of dsDNA/RNA is promising to reduce the nucleic acids hydrolysis caused by its surrounding and prevent the offloading of cargo during the nucleic acids delivery. The simulation of nucleic acids ejection by the CNT piston 12 ACS Paragon Plus Environment

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can be used to trigger the dissociation of dsDNA/RNA from its protective CNT capsule.

SUPPORTING INFORMATION The snapshots and data for encapsulation of dsDNA/RNA with charged canoe after 20 ns in 150 mM NaCl solution are provided (Figure S1-S4). Figure S5 and S6 show the snapshots of encapsulation and offloading of dsDNA with charged canoe in pure water. The videos for encapsulation and offloading of dsDNA/RNA with charged canoe in 150 mM NaCl solution (system 3, 4, 7 and 8) are provided. ACKNOWLEDGMENTS Y.H. acknowledges the resources of the Department of Chemistry, University of Illinois at Chicago. C. L. Yang acknowledges that this work was supported by the National Natural Science Foundation of China (NSFC) under Grant Nos. NSFC11574125, as well as the Taishan Scholars project of Shandong Province (ts201511055).

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(30) Grossfield, A. WHAM: the weighted histogram analysis method. University of Rochester Medical Center: Rochester, NY, 2012, accessed April 2017. (31) Lu, X.-J.; Olson, W. K. 3DNA: A software package for the analysis, rebuilding and visualization of three-dimensional nucleic acid structures. Nucleic Acids Res. 2003, 31, 5108−5121. (32) Lavery, R.; Moakher, M.; Maddocks, J. H.; Petkeviciute, D.; Zakrzewska, K. Conformational analysis of nucleic acids revisited: Curves+. Nucleic Acids Res. 2009, 37, 5917-5929. (33) Humphrey, W.; Dalke, A.; Schulten, K. VMD: visual molecular dynamics. J. Molec. Graphics 1996, 14, 33-38.

TABLE 1. Summary of the simulation setups. CNT System

dsDNA

1

2 3

4 5

6 7 8

dsRNA

Box

dimensions

(Å3)

Total atoms

76×76×327

168719

76×76×327

168752

capsule

Canoe

piston

charged

76×76×327

171763

charged

76×76×327

171796

neutral

76×76×327

171705

neutral

76×76×327

171738

charged

67×68×329

121951

charged

67×68×329

133026


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Figure 1. CNT canoe by sliding the CNT wall (The edges are marked in black circle).



Figure 2. dsDNA/RNA interact with CNT capsule. (a) dsDNA with initial distance of 13 Å from one side of CNT capsule; (b) side view of (a); (c) dsRNA with initial distance of 13 Å; (d) side view of (c); (e) dsDNA with CNT capsule after 20 ns simulation; (f) dsRNA with CNT capsule after 20 ns simulation. Inset figure: details of dsDNA interacting with CNT capsule.


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Figure 3. dsDNA/RNA encapsulate into CNT capsule facilitated by charged canoe. CNT capsule is in gray, canoe in cyan, and dsDNA/RNA in blue and red chains.



Figure 4. (a) length of dsDNA/RNA on charged canoe by measuring the distance between the first base pair and last base pair; (b) head to head distance between CNT capsule and charged canoe; (c) solvent accessible area of dsDNA/RNA with charged canoe; (d) vdW energy between CNT capsule and charged canoe.


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0

PME (kcal/mol)

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


-50

-100

-150

-200 145

140

135

130

125

120

Distance (Å)

Figure 5. Free energy profile. The reaction coordinate is defined as the center to center distance between the CNT capsule and canoe with dsDNA.



Figure 6. dsDNA/RNA encapsulates into CNT capsule facilitated by neutral canoe. CNT capsule is in gray, canoe in cyan, and dsDNA/RNA in blue and red chains.

Figure 7. (a) head to head distance between CNT capsule and neutral canoe; (b) solvent accessible area of dsDNA/RNA with neutral canoe.


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Figure 8. (a) base pair open of dsDNA/RNA on charged canoe; (b) details of interaction between dsDNA and charged canoe (up), dsRNA and charged canoe (down); (c) base pair open of dsDNA/RNA on neutral canoe; (d) details of interaction between dsDNA and neutral canoe (up), dsRNA and neutral canoe (down).



Figure 9. Offloading of dsDNA/RNA on charged canoe (CNT piston is in cyan cylinder).

Figure 10. Interaction energy between piston/canoe and CNT capsule. (a) dsDNA on charged canoe system; (b) dsRNA on charged canoe system. 24 ACS Paragon Plus Environment

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Molecular Dynamics Study on Encapsulation of Double Stranded

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