Choline Ion Interactions with DNA Atoms Explain Unique Stabilization

Article pubs.acs.org/JPCB

Choline Ion Interactions with DNA Atoms Explain Unique Stabilization of A−T Base Pairs in DNA Duplexes: A Microscopic View Miki Nakano,† Hisae Tateishi-Karimata,† Shigenori Tanaka,‡ and Naoki Sugimoto*,†,§ †

Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, 7-1-20, Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan ‡ Graduate School of System Informatics, Department of Computational Science, Kobe University, 1-1, Rokkodai, Nada-ku, Kobe, 657-8501, Japan § Faculty of Frontier of Innovative Research in Science and Technology (FIRST), Konan University, 7-1-20, Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan S Supporting Information *

ABSTRACT: Under physiological conditions, G−C base pairs are more stable than A−T base pairs. In a previous study, we showed that in the hydrated ionic liquid of choline dihydrogen phosphate, the stabilities of these base pairs are reversed. In the present study, we elucidated the unique binding interactions of choline ions with DNA atoms from a microscopic viewpoint using molecular dynamics simulations. Three times more choline ions bind to the DNA duplex than sodium ions. Sodium ions bind closely but not stably; in contrast, the choline ions bind through multiple hydrogen bonding networks with DNA atoms stably. The affinity of choline ion for the minor groove of A−T base pairs is more than 2 times that for other groove areas. In the narrow A−T minor groove, choline ion has high affinity for the ribose atoms of thymine. Choline ions also destabilize the formation of hydrogen bonds between G−C base pairs by binding to base atoms preferentially for both of duplex and single-strand DNA, which are associated with the bonds between G−C base pairs. Our new finding will not only lead to better control of DNA stability for use in DNA nanodevices, but also provide new insight into the stability of DNA duplexes under crowding conditions found in living cells.

1. INTRODUCTION The structures formed by DNA via Watson−Crick base pairing have enormous potential for use in biosensors, biodevices, and biocircuits.1,2 Researchers have taken advantage of structural changes induced by cationic molecules to develop DNA-based nanodevices.3−9 In physiological levels of ions and at near neutral pH, G−C base pairs are more stable than A−T base pairs due in part to the difference in the number of hydrogen bonds between complementary bases. We have recently shown that A−T base pairs are more stable than G−C base pairs in the hydrated ionic liquid of choline dihydrogen phosphate (choline dhp).10 We hypothesized that choline ions stabilized A−T-rich DNA duplex structures by binding in the grooves and destabilized G−C-rich DNA duplexes by preferentially binding to guanines in the singlestranded form.10 Some alkylammonium ions such as tetramethylammonium and tetraethylammonium stabilize A−T-rich duplexes by preferentially binding to the minor groove of A−T base pairs.11−14 Glycine betaine, which is a zwitterionic osmolyte with alkylammonium-derivative ions, destabilizes G−C-rich duplexes by binding to single-stranded DNA, especially unpaired guanines at high salt concentrations.15−18 However, these mechanisms have not been elucidated sufficiently at the atomic level. Molecular ions like choline bind to DNA in more varied modes than do metal ions. Because of their molecular size, configuration, and the characteristics of the atoms exposed on their surfaces, molecular ions can interact not only through electrostatic © 2013 American Chemical Society

interactions between point charges, but also through hydrogen bonding, van der Waals interactions, and hydrophilic or hydrophobic interactions. Therefore, unlike metal ions which interact via point charges, molecular ions can recognize specific atoms or configurations of target DNA. Choline ion is an important biological cation that helps maintain the structure of cell membranes and has roles in cell signaling and as a neurotransmitter.19 Ionic liquids based on choline ion ensure long-term stability of biomolecules like DNA and proteins.20−22 Both cations and anions included in ionic liquid may affect the chemical stability of these molecules. Fujita et al. showed that the nature of anion appears to be more important to the stability of proteins than the type of cation.21 It is likely, however, that cations are more important to the stability of DNA, because cationic molecules are required to reduce the repulsive forces between the phosphate groups of DNA strands. Identification of the interactions that occur between choline ion and DNA at the atomic level will provide insight into how these ions regulate the stability of DNA duplexes in living cells where choline ions play active roles and will further our ability to control DNA duplex stability in molecular machines. Molecular dynamics (MD) simulation is the useful tool to reveal the microscopic properties of biomolecules such as protein Received: July 5, 2013 Revised: October 30, 2013 Published: October 30, 2013 379

dx.doi.org/10.1021/jp406647b | J. Phys. Chem. B 2014, 118, 379−389

The Journal of Physical Chemistry B

Article

effects of choline ions on the stabilities for both consecutive and mixed sequences. All oligodeoxynucleotides were high-performance liquid chromatography grade (Japan Bio Service). Concentrations of DNA oligonucleotide single strands were determined from the absorbance at 260 nm measured at 80 °C and single-strand extinction coefficients calculated from the mononucleotide and dinucleotide data according to the nearest-neighbor approximation model.37 The absorbance was measured using a Shimadzu 1700 spectrophotometer connected to a thermoprogrammer. The hydrated ionic liquid, choline dhp, was purchased from Ionic Liquids Technologies Co. Ltd. and used without further purification. 2.2. Analysis of the Thermal Stability of DNA Duplexes. Ultraviolet (UV) absorbance was measured on a Shimadzu 1700 spectrophotometer equipped with a temperature controller. UV melting curves at 260 nm were measured in buffers containing 50 mM MES (pH 6.0), 1 mM Na2EDTA, and 4 M NaCl, 4 M choline chloride, or 4 M choline dihydrogen phosphate (choline dhp). Because it was difficult to adjust the pH value of Na dhp solution due to its acidity, we also carried out the UV experiments in the solution containing 4 M Na dhp only. The melting temperatures (Tms) were estimated from the UV melting curves. Samples were heated at a rate of 0.5 °C min−1 because the melting curve was unaffected by heating rates between 0.2 °C min−1 and 0.5 °C min−1 (data not shown). Before the measurements, the DNA samples were heated to 80 °C, cooled to 0 °C at a rate of 2 °C min−1, and incubated at 0 °C for 30 min. 2.3. Materials of Molecular Dynamics Simulations. To investigate the differences of the cation−DNA interaction between A−T base pairs and G−C base pairs, we carried out MD simulations with ODN1, ODN3, and ODN4. It was difficult to obtain a Tm value for the duplex form of ODN4, which consists only of G−C base pairs, because these sequences form G-quadruplexes, i-motifs, and other aggregates.28,38,39 Using MD simulations, we were able to observe the distribution of ions around G−C base pairs in the B-form DNA duplex rather than noncanonical structures. Therefore, we used ODN4, which consists of only G−C base pairs, for MD simulations to clarify the interaction of choline ion with these base pairs. To investigate the binding preferences of choline ion to unpaired DNA bases, we also performed MD simulations of single-stranded DNAs (A10, G10, T10, and G10) in solvent including choline ions. The effect of dihydrogen phosphate (dhp) on the stability of DNA duplexes is of interest; however, it is likely that cations are more important to the stability of DNA structures than anions as cations reduce the repulsive forces between the phosphate groups of DNA strands. In addition, it was technically impossible to perform the MD simulation on the system including dhp. Because of the large polarity within dhp, the deviations of atoms with one time step in MD simulation were very large. Therefore, in this study, the computational study focused on the role of choline ions on DNA duplex stability. The DNA duplexes were constructed using the Build and Edit Nucleic Acid module included in Discovery Studio 3.1.40 In our previous study, we confirmed that both A−T-rich and G−C-rich DNA oligonucleotides used in this study adopt the B-form conformation by CD analysis of these oligonucleotides in different salt concentrations (0.1 to 4 M) of NaCl or choline dhp solutions.10 Therefore, the initial structures used in our MD simulations were B-form DNA duplexes. These DNA duplexes were merged in TIP3P water molecules,41 and 200 choline ions or sodium ions were added to the solvent. The cation

and DNA. Using MD simulations, we can extract atomic-level information on interactions and dynamics that are difficult to obtain from experiments. The nanoscale views of biomolecules obtained from MD simulations has been used to guide rational drug design and development of nanodevices.23−26 To date, MD simulation studies of proteins have vastly outnumbered those of nucleic acids, because of the smaller number of available nucleic acid structures, slower development of their force fields, and limits to computing power.27,28 Several MD simulations of interactions between metal ions and nucleic acids have been reported.29−36 Kirmizialtin and Elber demonstrated that behaviors of Na+ and Mg2+ differ: the tight binding of Mg2+ occurs via a solvation shell, whereas Na+ can bind directly to RNA.36 Li et al. showed that Mg2+ ions interact with DNA mainly through hydrogen bond interactions, which are sensitive to the local environment.32 However, the microscopic study of the interactions between molecular ions such as choline ion and DNA is just beginning. To our knowledge, this study is the first analysis of the interactions between choline ions and DNA atoms at the atomic level. In this study, we examined experimentally that choline ions reverse the stability of A−T and G−C base pairs. Using MD simulations, we demonstrated the differences between interactions of DNA with choline ion and the sodium ion, which is a typical cation and abundant in living cells. In addition, we clarified that the unique binding mode of choline ion to DNA atoms results in the stabilization of A−T base pairs and the destabilization of G−C base pairs. We could provide a rational elucidation how choline ion affects the DNA base pair stability from a microscopic viewpoint. Our results will guide the development of novel functional materials using the stability switches controlled by molecular ion−DNA interactions. In addition, our new findings may also be relevant to the environment in which DNA is found in cells because osmolytes, including choline ions and glycine betaine, are abundant in cells.

2. MATERIALS AND METHODS 2.1. Experimental Materials. Three different 10-mer DNA duplexes were used experimentally in this study (Table 1). Table 1. Experimental Melting Temperatures (Tms) of DNA Duplexes at Strand Concentrations of 10 μM in the Presence of Indicated Ions Tm[°C]b

name ODN1 ODN2 ODN3 ODNm1 ODNm2

sequence 5′-A10-3′/5′-T10-3′ 5′-A7C3-3′/5′-G3T7-3′ 5′-A5C5-3′/5′-G5T5-3′ 5′-TTATAACCTA-3′/ 5′-TAGGTTATAA-3′ 5′-CGGCAAGCGC-3′/ 5′-GCGCTTGCCG-3′

A−T content [%]a

NaCl

choline dhp

choline Cl

100 70 50 80

31.1 37.3 46.0 30.1

53.1 40.2 34.0 37.5

44.8 45.2 41.5 40.9

20

53.7

31.0

49.1

a

The ratio of A−T base pairs in 10 mer DNA duplex. bAll the experiments were carried out in buffer containing 50 mM MES (pH 6.0) and 1 mM Na2EDTA with 4 M NaCl, 4 M choline chloride, or 4 M choline dhp.

ODN1 was 100% dA (adenine) and dT (thymine), ODN2 was 70% dA and dT, and ODN3 was 50% dA and dT. A−T base pairs in these sequences were consecutive. In addition, we prepared two mixed sequences, ODNm1 and ODNm2, to evaluate the 380



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Calculations were performed on the SGI UV 1000 system at the Research Center for Computational Science, Okazaki, and on DELL Precision T5400 system at Kobe University.

concentrations were 1.7 M. One hundred eighty-two chloride ions were added to neutralize the simulation system. In our previous study, we demonstrated that in choline dhp solution in excess of 1 M, A−T base pairs are stabilized, whereas G−C base pairs are destabilized compared to NaCl solution. Tm values of the A−T-rich sequence and the G−C-rich sequence were completely reversed at 4 M choline dhp concentration.10 In the simulation systems at 4 M salt, some technical problems occurred. The sodium and chloride ions formed a crystal-like structure, and choline ions and DNA atoms were entangled. Therefore, we performed our MD simulations with 1.7 M salt concentration, where these effects were minimized. The systems including choline ions are referred to as C-ODN1, C-ODN3, and C-ODN4, and those including sodium ions are referred to as N-ODN1, N-ODN3, and N-ODN4. In addition, we prepared C-ODNm1 and C-ODNm2 for the system including choline ions. The system names and their configurations used in our MD calculations are listed in Table 2. The

3. RESULTS 3.1. Melting Temperature Measurement. We first investigated the sequence dependence of DNA stability by evaluation of ultraviolet (UV) melting curves in aqueous buffer and in hydrated ionic liquid. The melting temperatures (Tms) of 10-mer DNA duplexes with different A−T base pair contents were measured in solutions containing 4 M NaCl or 4 M choline dhp. At this concentration at room temperature, choline dhp has the properties of an ionic liquid: low vapor pressure, high viscosity, and low dielectric constant.47 Many studies with choline dhp solution have been performed at this concentration.21,48−50 In addition, in our previous study, we demonstrated that stability of A-T-rich and G-C-rich sequences are reversed in 4 M choline dhp compared to those in aqueous buffer.10 Therefore, we measured Tm values at 4 M salt concentration. The Tm values for 10-mer DNA duplexes with A−T base pair content ranging from 20 to 100% are given in Table 1. The Tm values for DNAs in NaCl solution decreased with increasing A− T base pair content except for ODNm1. By contrast, the Tm values in choline dhp solution increased with increasing A−T base pair content for both A−T base pairs consecutive and mixed sequences. These trends were observed at higher and lower DNA concentrations (data not shown). To gain insight into the mechanism of choline ion action, we estimated the Tm values of DNA duplexes in 4 M choline chloride, which is not a hydrated ionic liquid. Tm values measured in choline chloride solution are also shown in Table 1. The stabilities of ODN1 (100% A−T base pair composition) and ODN2 (75% A−T base pair composition) were enhanced in choline chloride relative to those in NaCl solution. The stability of ODN3 (50% A−T base pair composition) was lower in 4 M choline chloride than in NaCl solution. These results indicate that choline ions stabilize A−T base pairs in a DNA duplex and destabilize G−C base pairs. These effects are strengthened in the solution of choline dhp. In addition, we also estimated the Tm values of DNA duplexes for ODN1, ODN2 and ODN3 in 4 M Na dhp. In Na dhp solution, Tm values of ODN1 and ODN2 were 37.5 and 17.7 °C, respectively, indicating the dihydrogen phosphate (dhp) stabilized AT base pairs and destabilized the GC base pairs. Because ODN3 did not show the clear melting transition in 4 M Na dhp (data not shown), the dhp effect on the DNAs would be not simple, and further investigations would be required. Although dhp affects the stability of the DNA duplex, the effect to stabilize AT base pairs is more strongly influenced by the presence of choline ions than by the anion. Therefore, in this study, we performed a computational study focused on the role of choline ions on DNA duplex stability at the atomic level. 3.2. Properties of Ions around DNA Duplexes Determined Using MD Simulations. To characterize the properties of cations around DNA duplexes, we defined following DNA(atom) quantities, ncation where D ̅ NA and nc̅ ation

Table 2. List of the System Names, Sequences, and Numbers of Solvent Molecules and Ions Employed in the Molecular Dynamics Simulations name C-ODN1 C-ODN3 C-ODN4 C-ODNm1

sequence

5′-A10-3′/5′-T10-3′ 5′-A5C5-3′/5′-G5T5-3′ 5′-C10-3′/5′-G10-3′ 5′-TTATAACCTA-3′/ 5′-TAGGTTATAA-3′ C-ODNm2 5′-CGGCAAGCGC-3′/ 5′-GCGCTTGCCG-3′ N-ODN1 5′-A10-3′/5′-T10-3′ N-ODN3 5′-A5C5-3′/5′-G5T5-3′ N-ODN4 5′-C10-3′/5′-G10-3′ A10 A10 G10 G10 T10 T10 C10 C10

number of waters

number of cations

number of anions

6569 6463 6540 6780

choline 200 choline 200 choline 200 choline 200

Cl− 182 Cl− 182 Cl− 182 Cl− 182

6785

choline 200

Cl− 182

6518 6182 6480 6614 6608 6408 6343

Na+ 200 Na+ 200 Na+ 200 choline 200 choline 200 choline 200 choline 200

Cl− 182 Cl− 182 Cl− 182 Cl− 191 Cl− 191 Cl− 191 Cl− 191

initial structure of ODN1 in the TIP3P box including choline ions for our MD simulation and a chemical structure of choline ion are shown in Figure S1. 2.4. Protocols for MD Simulations. MD simulations were carried out with the AMBER12 software package.42 AMBERff03.r1 force field was applied for DNA,43 and the force field of choline ion was generated using the antechamber and gaff module included in AMBER Tools.44,45 The protocols of structural optimization and MD simulations were as follows: First, the optimization of water molecules and ions was carried out in 1000 steps with the conformation of DNA fixed. Second, the whole system was energetically minimized in 2500 steps without any constraints. Third, the system was heated to 300 K for 20 ps. Finally, constant-pressure and constanttemperature MD simulations were carried out at 1 atm and 300 K for 20 ns without any constraints. Throughout these MD simulations, periodic boundary conditions and SHAKE algorithm were applied.46 The simulation time step was 2 fs, and the nonbonded cutoff length was set at 10 Å. It is wellknown that a G-rich sequence such as ODN4 adopts G-quadruplex structures under solution conditions used here. During MD simulations of ODN4, the initial B-form duplex structure was maintained during the 20 ns MD simulations (data not shown).

DNA cation nDNA (t1 ̅

< t < t 2 , r < r0) =

∑ cation

Θ(r < r0) t1< t < t 2

(1)

and 381



Article cation

DNA(atom) ncation (t1 ̅

< t < t 2 , r < r0) =

of the MD simulations where system was in equilibrium (Figure S2). In addition, we calculated the average existing time, τcation ̅ (t1 < t < t2, r < r0), during which each cation was within the distance r0 from the DNA atoms during the time interval from t1 to t2.

∑ Θ(r < r0) atom

t1< t < t 2

(2)

for ⎧1 for r < r0 ⎫ ⎬ Θ(r < r0) = ⎨ ⎩ 0 for r ≥ r0 ⎭ ⎪

⎪

⎪

⎪

cation

τ̅

cation

(t1 < t < t 2 , r < r0) =

∑ Θ(r < r0)Δt atom

(3)

t1< t < t 2

(4)

where r is the distance between the target cation and the DNA atom, and r0 is the threshold distance of r. We set r0 to 3.5 Å in this study. n̅cation DNA (t1 < t < t2, r < r0) is the average number of cations within the distance r0 from DNA atoms during time interval from t1 to t2 for each DNA duplex. nDNA(atom) (t1 < t < t2, c̅ ation r < r0) is the average number of DNA atoms within the distance r0 from the cations during time interval from t1 to t2 for each cation. Because the hydroxyl group of choline ion has high ability to form hydrogen bonds due to its high polarity, the hydroxyl group directs the orientation with respect to the DNA. Therefore, the position of oxygen atom was used as the indicator of the position of choline ion. Figure 1 shows the schematic views of the target

Δt in eq 4 is the time step of MD simulation, which was 2 fs in this DNA(atom) study. Table 3 shows the values of ncation , and τcation D ̅ ̅ NA , nc̅ ation DNA(atom) Table 3. ncation , and τcation Values Obtained from D ̅ ̅ NA , nc̅ ation MD Trajectories Representing the Properties of Ions around the DNA Duplexesa

name

n̅cation DNA

C-ODN1 C-ODN3 C-ODN4 C-ODNm1 C-ODNm2 N-ODN1 N-ODN3 N-ODN4

17.63 ± 0.02 17.65 ± 0.02 18.17 ± 0.02 18.90 ± 0.02 16.98 ± 0.02 5.01 ± 0.01 5.08 ± 0.01 5.47 ± 0.01

n̅DNA(atom) cation 3.037 ± 0.003 2.954 ± 0.003 3.037 ± 0.003 3.408 ± 0.003 3.089 ± 0.003 2.337 ± 0.004 2.378 ± 0.005 2.138 ± 0.003

τcation [ps] ̅ 2957.5 ± 372.5 3151.6 ± 363.0 2641.1 ± 307.8 3499.8 ± 461.71 3121.4 ± 382.77 792.1 ± 88.2 913.7 ± 113.8 844.7 ± 97.0

n̅DNA(atom) , and τcation are defined by eqs 1−4 in the text, and cation ̅ schematic views of the alignments of the target cations and the DNA DNA(atom) are shown in Figure 1. All the values atoms for n̅cation DNA and n̅cation were calculated with t1 = 15 ns, t2t2 = 20 ns, and r0 = 3.5 Å. a cation n̅DNA ,

calculated for each duplex and cation combination. The values of DNA(atom) n̅cation for choline ions were larger than those of DNA and n̅cation sodium ions. Likewise, τcation values were longer for the choline ̅ ions than for sodium ions. Because choline ion interacts with DNA atoms by forming hydrogen bonds, this ion can interact with many atoms of DNA. These multiple bonding possibilities retain the choline ion near the DNA duplex for a longer time than the sodium ion is retained. In general, strengths of hydrogen bonds are weaker than those of ionic bonds; however, our simulations showed that the interaction between choline ions and DNA atoms is likely more stable than the interaction between sodium ions and DNA. 3.3. Free Energy Profile of Cations around DNA. To understand the quantitative interaction between cations and DNA duplexes, free energy profiles of cations around DNA at 300 K (ΔG300K) were estimated. The values of ΔG300K(r) were converted from the radial distribution functions gcation D ̅ NA (r) (eq 5) using potential of mean force defined by eq 6.52 cation gDNA (t1 ̅

Figure 1. Schematic views of the alignments of the target cations and the DNA(atom) ́ (r < 3.5 Ǻ ). All DNA atoms for (a) n̅cation DNA (r < 3.5 Å) and (b) n̅cation DNA atoms are shown as gray sticks. Oxygen atoms of choline ions are shown in red, nitrogen atoms in blue, and carbon atoms in yellow. Hydrogen atoms of choline ions were omitted for clarity. Dashed circles represent the area within 3.5 Å of the target atoms. These figures were created with PyMOL.51

< t < t2 , r) =

ρ(r )cation DNA(atom) ρ0

⎛ g cation (r ) ⎞ ̅ DNA ⎟ ΔG300K (r ) = −RT ln⎜⎜ cation (∞) ⎟⎠ ⎝ gDNA ̅

DNA

t1< t < t 2

(5)

(6)

ρ(r)cation DNA(atom)

Here, is the local density of the cation at a distance r from the DNA atom, and ρ0 is the average density of targeted cation. R is the molar gas constant, and T (= 300 K) is the absolute temperature. We took the value of ΔG300K(∞) to zero as the reference. t1 and t2 were set to be 15 and 20 ns in this study,

cations and the DNA atoms. Figure S2 shows the accumulated averages of the number of cations within 3.5 Å from DNA atoms ́ (n̅cation DNA (0 < t < t2, r < 3.5 Å). For each system, we analyzed 25000 snapshots taken from the trajectories during 15−20 ns intervals 382



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energy barrier (ΔΔG300K) to escape the bound state to DNA was 0.86 kcal mol−1. This value could be easily overcome by thermal motions at physiological temperature, because the RT value is about 0.6 kcal mol−1 at 300 K. There was no barrier for r > 3.0 Å in the sodium ion free energy profile, and the ΔG300K values became monotonically lower as distance from the DNA increased. These results indicate that the DNA-bound sodium ions can exchange rapidly with sodium ions in the bulk solution. For choline ion, there were no significant barriers in the free energy profile over the distance of 3.5 Å from the DNA. Because the diffusion coefficient of choline ion is smaller than that of sodium ion, choline ions close to the DNA remain there even though the barrier height at 3.0 Å is relatively low (0.28 kcal mol−1). The small steps in choline ion free energy profiles, where the distance to the DNA is very close (r = 1.95 Å, indicated by the red arrow), as shown in Figure 2b, correspond to the state in which choline ion approaches the imino hydrogen atoms of the bases: dG(H22), dC(H42), and dA(H62). Figure S3 shows the bases, the sugar, backbone, and atom numberings used in this work. 3.4. Binding Sites of Cations around DNA. Figure 3 shows binding sites of cations around ODN1 and ODN4 drawn by MD trajectories. Sodium ions mainly approached the phosphate groups of DNA strands to neutralize their negative charge both for N-ODN1 and N-ODN4. The distributions of choline ions around ODN1 and ODN4 were remarkably different from the sodium ion distributions. Around ODN1, which consisted of only A−T base pairs, choline ions bound preferentially in the minor groove (C-ODN1 minor side). By contrast, around ODN4, which consisted of only G−C base pairs, the choline ions were preferentially bound in the center of the major groove (C-ODN4 major side). We also show the binding map for ODN3, ODNm1, and ODNm2, which consist of both A−T and G−C base pairs in Figure S4. The preference binding of choline ions and sodium ions to the base pairs was the same of those for ODN1 and ODN4 as shown in Figure 3.

Figure 2. Free energy profiles as functions of the distances from DNA atoms of (a) sodium ion and (b) oxygen atom of choline ion around ODN1 (red line), ODN3 (blue line), and ODN4 (green line). Blue arrows indicate the basin positions for each cation. Black arrows indicate the free energy barriers for each cation to escape the bound state to DNA. Red arrow indicates the small steps in choline ion free energy profiles, where the distance to the DNA is 1.95 Å.

respectively. Figure 2 shows the ΔG300K(r) values for sodium ion and choline ion around all DNA atoms. The basin positions of sodium ions (r = 2.35 Å) were closer to DNA atoms than those of oxygen atoms of choline ions (r = 2.65 Å) for all sequences. These results indicate that sodium ions bind closely to DNA atoms, whereas choline ions wander near the DNA atoms at a longer distance. For sodium ion, the free

Figure 3. Binding sites of cations around the DNA duplexes. Upper figures: major groove sides of ODN1 and ODN4. Lower figures: minor groove sides of ODN1 and ODN4. DNA atoms are shown as white surfaces. Blue dots represent the binding sites of cations. Yellow dashed lines represent the regions of enhancing the concentration of cholines that gather to the major groove of ODN4 or minor groove of ODN1. These figures were created with VMD.53 383



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+

(O) Figure 4. Average number of oxygen atoms of choline ions (n̅choline DNA(atom)) around each atom of DNA shown for each nucleotide calculated from MD trajectories for C-ODN1 and C-ODN4; (a) adenine, (b) thymine, (c) guanine, and (d) cytosine. Phosphate atoms are colored in red, ribose in green, base atoms within the major groove in pink, and base atoms within the minor groove in blue.

3.5. Base Component Dependence of Choline Ion for Binding Site to DNA Duplex. To quantify the binding preferences of choline ions to DNA atoms, we counted the average number of choline ions (shown as oxygen atoms of choline ions) around each DNA atom for ODN1 and ODN4,

Figure 5 shows the representative alignments of choline ions around A−T and G−C base pairs. Movies of these choline ions bound to DNAs are available as Supporting Information (Movies S1 and S2). Choline ions located around G−C base pairs disrupt the hydrogen bonds between G and C from both minor and major groove sides. On the other hand, the choline ions in the minor groove of A−T base pairs form multiple hydrogen bonds with DNA atoms that do not disrupt base pairing. These minor groove choline ions appear to stabilize the groove conformation and do not interact with the hydrogen bond forming atoms of the A−T base pair. 3.6. Groove Width Measurement. We measured the distances between phosphorus atoms across the minor grooves in snapshots obtained from our simulations (Figure 6a). The average widths of the minor grooves of N-ODN1, N-ODN4, C-ODN1, and C-ODN4 were 11.31 ± 1.74 Å, 13.85 ± 1.25 Å, 11.99 ± 1.36 Å, and 13.70 ± 1.13 Å, respectively. Both in the solvents including sodium ions and choline ions, the widths of minor groove of A−T base pairs were narrower than those of G−C base pairs. Figure 6b shows the choline ions buried in the minor groove of ODN1. These choline ions fit well into the minor groove of A−T base pairs. Movie S3 (Supporting Information) shows that choline ions bound in the A−T base pair minor groove tract remained over the time course of the simulation. The narrow groove of DNA duplex consists of A−T base pairs allows multiple hydrogen bonds between choline ion and DNA atoms. This mode of choline ion binding would sustain the conformation of A−T-rich DNA duplex by interacting with both strands.

+

(O) n̅choline DNA(atom) (Figure 4). The definitions of atom names and their +

(O) sites are shown in Figure S3. Total values of n̅choline DNA(atom) around phosphate groups were not significantly different between A−T +

+

(O) choline (O) base pairs (n̅choline DNA(PO2) = 9.44) and G−C base pairs (nD ̅ NA(PO2) = +

(O) 9.17). However, n̅choline DNA(atom) values around the atoms of each base were very different. Around G−C base pairs (ODN4), choline ions binded to the atoms associated with the hydrogen bond formation between the bases in the pair, dG(O6), dG(N2), dG(H21), dG(H22), dC(N4), dC(H41), dC(H42), dC(O2). +

(O) Around A−T base pairs (ODN1), the values of n̅choline DNA(atom) for the atoms involved in the hydrogen bonding between A−T base pair were not significantly higher than those for other atoms, dA(N6), dA(H61), dA(H62), dT(O4). In addition, the values of +

(O) n̅choline DNA(atom) for dT(H4′) and dT(H1′) were significantly higher than those for other atoms of thymine. These atoms are exposed in the minor groove of DNA duplex. To clarify the preference of choline ion for the grooves, we calculated the radial distribution functions of choline ion near the grooves of ODN1 or ODN4 (Figure S5). The results indicate that choline ion strongly preferred to bind to the major groove of G−C base pairs, whereas it has a higher affinity for dT(H4′) and dT(H1′) in the A−T minor groove.

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3.7. MM-GBSA (Molecular Mechanics - Generalized Born Surface Area) Analysis. The MM-GBSA method is used for energy analysis using continuum solvent models.54 The molecular mechanical (MM) energies are determined with the sander program from AMBER12 and represent the internal energies (bond, angle, and dihedral) and van der Waals and electrostatic interactions. The electrostatic contributions to the solvation free energy are calculated by generalized Born (GB) methods. The nonpolar contributions to the solvation free energy are determined with solvent-accessible-surface-areadependent (SA) terms. Using the MM-GBSA module included in Amber12, we calculated the binding energy between DNA strands (ΔE0), DNA duplex and cations (ΔE1), and DNA strands with cations (ΔE2), respectively. ΔE0 = E(duplex) − E(DNA1) − E(DNA 2)

(7)

ΔE1 = E(duplex + cation) − E(duplex) − E(cation) (8)

ΔE2 = E(duplex + cation) − E(DNA1 + cation/2) − E(DNA 2 + cation/2)

(9)

For calculation of ΔE1, we selected 18 cations closest to DNA atoms for each system; this was the required number of cations to neutralize the system. For ΔE2, we divided the cations selected for ΔE1 between each DNA strand equally. To estimate the effect of cations on stabilization of the DNA duplex, we calculated the values of ΔE2 − ΔE0 for each system. In this study, we assumed the dielectric constant of solvent, including ions, was 78. We used 2500 snapshots from each MD trajectory for MM-GBSA analyses. Table 4 shows ΔE0, ΔE1, and ΔE2 − ΔE0 values for ODN1,

Figure 5. Representative alignments of choline ions bound to (a) an A−T base pair and (b) a G−C base pair. Phosphorus, oxygen, nitrogen, and hydrogen atoms are shown in gold, red, blue, and white, respectively. The carbons of choline ions are shown in yellow, and the carbons of DNA are in green. Hydrogen bonds are shown as yellow dotted lines. These figures were created with VMD.53

Table 4. ΔE0, ΔE1, and ΔE2 − ΔE0 Values for Each System Consisting of DNA Duplex and 18 Cations Closest to DNAa

C-ODN1 C-ODN3 C-ODN4 N-ODN1 N-ODN3 N-ODN4

ΔE0 [kcal mol−1]

ΔE1 [kcal mol−1]

ΔE2 − ΔE0 [kcal mol−1]

−38.90 ± 3.59 −64.40 ± 4.52 −97.50 ± 4.57 −39.63 ± 3.92 −64.66 ± 4.35 −92.03 ± 5.48

−146.48 ± 14.14 −139.76 ± 11.04 −129.28 ± 10.10 −47.00 ± 6.13 −47.45 ± 6.53 −45.34 ± 7.08

−61.65 ± 9.76 −58.98 ± 9.97 −60.87 ± 10.95 −5.12 ± 4.53 −0.82 ± 5.56 −2.80 ± 5.11

a ΔE0: binding energy between DNA strands, ΔE1: binding energy between DNA duplex and cations, ΔE2: binding energy between DNA strands with eighteen cations closest to DNA, ΔE2 − ΔE0: effect of cations on stabilization of the DNA duplex. These quantities are defined by eqs 7−9 in the text.

ODN3, and ODN4. As we expected, the binding energy between DNA strands, ΔE0, became stable with increasing of G−C content. However, the binding energy, ΔE1, showed a different tendency. Affinity of sodium ion to DNA duplex did not have a base component dependency; by contrast, choline ion had high affinity to A−T base pairs. The effects of cations on the stability of the DNA duplex, ΔE2 − ΔE0, are independent of the sequence. The effects of choline ion are larger than those of sodium ion. To investigate more local effect of choline ion, we selected single choline ions closest to the major groove or minor groove for ODN1 or ODN4, respectively. One choline ion was selected, which is the closest ion to dG(O6), dC(O2), dT(O2), or

Figure 6. (a) Schematic view of the measurement of minor groove widths. The numbers 1 through 10 indicate the bases from 5′ terminus to 3′ terminus of each DNA strand. The distances between phosphorus atom pairs connected with dashed lines were taken as the width of the minor groove. (b) Choline ions buried in the minor groove of A−T base pairs duplex. DNA atoms are shown as gray spheres. Carbon atoms of choline ions are shown in yellow, oxygen atoms of choline ions are shown in red, and hydrogen atoms are shown in white. 385



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Table 5. ΔE1, ΔE2_1 − ΔE0, and ΔE2_2 − ΔE0 Values for the Systems Consisting of ODN1 or ODN4 and One Choline Ion Closest to the Groove of DNA Duplexa name

ΔE1 [kcal mol−1]

ΔE2_1 − ΔE0 [kcal mol−1]

ΔE2_2 − ΔE0 [kcal mol−1]

AT-major AT-minor GC-major GC-minor

−7.51 ± 3.35 −17.83 ± 2.68 −9.81 ± 2.01 −9.58 ± 3.13

−3.70 ± 2.62 −11.62 ± 1.88 −9.83 ± 1.85 −5.60 ± 2.58

−5.51 ± 2.00 −9.01 ± 1.90 −3.89 ± 1.15 −6.61 ± 2.78

dC(O2) in GC-minor). These results will be described in the next section. 3.8. Preference Binding of Choline Ion to SingleStranded DNA. In our previous study, we supposed that choline ions destabilize G−C-rich duplexes by binding to guanines in a single strand.10 To investigate the binding preferences of choline ion to unpaired DNA bases, we performed MD simulations of single-stranded DNAs in solvent including choline ions. Figure 7 shows the radial distribution functions of choline ion around the atoms of single-stranded DNAs focusing on the atoms that are associated with hydrogen bond formation between Watson− Crick base pairs, calculated from 25 000 snapshots taken from the trajectories during 15−20 ns intervals of the MD simulations. Strong peaks were exhibited around oxygen atoms, dT(O2), dG(O6), and dC(O2), although dT(O4) did not show any significant peaks of choline ion. Among these oxygen atoms, only dT(O2) is not associated with the formation of a hydrogen bond between base pairs in the duplex. There were no significant peaks around hydrogen atoms of amino groups for any of the bases. There was the broad peak around dC(N3) around r = 3.5 Å. If this atom was blocked by choline ion, cytosine cannot form a base pair with guanine. Therefore, choline ions preferentially bind to atoms of guanine and cytosine favoring the singlestranded relative to the duplex conformation.

a The definitions of ΔEs values and procedure to select the choline ion are described in the text (see section 3.7).

dT(O4) within the four bases in the center of the DNA strand for each snapshot. Table 5 shows the binding energies of choline ions, ΔE1, bound to the major and the minor groove of G−C base pairs and A−T base pairs, respectively. The choline ion had high affinity to the minor groove of A−T base pairs. Because we could not divide one ion to two DNA strands, we calculated the energies for the two types of binding modes of choline ion and the DNA strand. In the first mode, ΔE2_1, the choline ion was supposed to bind to thymine or cytosine. In the second mode, ΔE2_2, the choline ion was supposed to bind to adenine or guanine. Table 5 also shows ΔE2_1 − ΔE0 and ΔE2_2 − ΔE0 values, respectively. Choline ion binding to the minor groove of A−T bases has a larger stabilizing effect than binding in any other grooves. Notably, when choline ion was supposed to bind to the amino group within the base (dA(N6), dA(N61) and dA(N62) in AT-major, dG(N2), dG(H21) and dG(H22) in GC-minor, dC(N4), dC(H41) and dC(H42) in GC-major), the DNA duplex was less stabilized than when choline ion was supposed to bind to the carbonyl group (dT(O2) in AT-minor, dG(O2) in GC-major), and

4. DISCUSSION We demonstrated experimentally that choline ions stabilize A-T base pairs and destabilize G-C base pairs by measuring melting temperatures in 4 M NaCl, 4 M choline Cl, and in 4 M choline dhp. Although dihydrogen phosphate was partially responsible for this effect, in the present research, we focused on how choline ions affect the stability of Watson−Crick DNA base pairs by

Figure 7. Radial distribution function of oxygen atom of choline ion around the atoms of single-strand DNAs: (a) around A10, (b) around T10, (c) around G10, and (d) around C10. 386



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number of choline ions within 3.5 Å from dT(H4′) for each model using the following equation:

computational studies. Especially, we clarified the unique binding mode of choline ion to DNA atoms by emphasizing the difference with that of sodium ion and by highlighting their base component dependence from a microscopic viewpoint using MD simulation. Choline ions interact with the atoms belonging to all areas of DNA through multiple hydrogen bond networks, whereas sodium ion interacts with DNA phosphate atoms through electrostatic interactions between their point charges. The tightness of the interactions is reflected by the basin positions and depths shown in free energy profiles of cations around DNA (Figure 2). Although the interaction between sodium ion and DNA is tight, once this bond breaks due to thermal fluctuations, the force that retains the sodium ion near the DNA is lost. In contrast, a “single” choline ion forms multiple hydrogen bonds with DNA. Because this loose network is actually stable, choline ion lingers around DNA even though the strength of an individual hydrogen bond is weaker than that of a single electrostatic interaction. Therefore, the choline ion wanders near a DNA for significantly longer time than does a sodium ion. MM-GBSA analysis showed that choline ions stabilize a DNA duplex more effectively than do sodium ions. Li et al. showed that sodium ions have a less stable hydration shell compared to magnesium ions and interact with DNA nonspecifically.32 Kirmizialtin and Elber showed that sodium ions bind diffusely to RNA,36 and Manning demonstrated that there is no significant barrier to their translational motion around DNA within the diffusely bound state.55 Our data from simulations with sodium ions are in agreement with these previous studies. MM-GBSA method is insufficient to estimate the entropic effect; however, we previously reported that the stabilizing or destabilizing effect of choline ion on a DNA duplex enthalpically driven, our MMGBSA results are reliable in a qualitative manner.10 We have demonstrated here that choline ions bind preferentially in the minor groove of A−T base pairs, whereas they bind in the major groove of G−C base pairs. MM-GBSA analysis showed that the choline ion has a high affinity to the minor groove of A−T base pairs. An ion located there significantly stabilizes the duplex structure. In this analysis, the choline ion binding with the amino group in DNA bases was stabilized significantly relative to that binding with the oxygen atoms (Table 5). Because the strongly polarized hydroxyl group of choline ion is attracted to the negative charged oxygen atoms of the bases, this ion destabilizes the DNA base pairing. The adjacent dG(N7) and dG(O6) atoms create a negative potential environment in the major groove of G−C base pairs, whereas hydrogen atoms on the dA(N6) amino group create a positive potential environment in the major groove side of A−T base pairs.32 Previous studies showed that the minor groove of an A−T base pairs tract is narrow and deep compared to that of the minor groove of tract of G−C base pairs.14,22,56−59 An alkylammonium ion, which is in choline ion, fits well into the groove of A−T-rich duplex.11−14 We demonstrated that choline ions well fit into the minor groove of A−T base pairs by MD trajectory as shown in Movie S3. We also found that choline ion has strong affinity to the atoms of A−T minor groove, dT(H1′) and dT(H4′). Therefore, choline ion recognizes these attractive atoms, and locates stably the minor groove of A−T base pairs due to the electrostatically polar environment and the correct fitting. This choline ion stabilizes the minor groove conformation in A−T-rich regions through a hydrogen bonding network. To evaluate the correlation between MD results and DNA stabilities measured experimentally, we investigated that the

DNA +

choline (O) ndT(H4 (t1 < t < t 2 , r < r0) = ̅ ′)

∑ cation

Θ(r < r0) t1< t < t 2

(9)

where r is the distance from the atoms of dT(H4′). The definition of Θ(r < r0) is the same as eq 3. r0, t1, and r2 were set to be 3.5 Å, 15 ns, and 20 ns, respectively. The counts were averaged for 25 000 snapshots obtained from 15 to 20 ns MD trajectories + (O) values for ODN1(A−T 100%), for each model. n̅choline dT(H4′) ODNm1(A−T 80%), ODN3(A−T 50%), and ODNm2(A−T 20%) were 2.12, 1.56, 0.85, and 0.43 per snapshot. In section 3.7, we showed the stabilizing effect on DNA duplex formations due to a choline ion which locates in the minor or major groove side of A−T or G−C base pairs. Using ΔE2_1 − ΔE0 value for AT-minor (−11.62 kcal mol−1), which was supposed that the choline ion bound to the minor groove of + (O) (the thymine, we estimated the stabilizing effects of ncholine d̅ T(H4′) number of choline ions around dT(H4′)) on the duplex stability. The estimated values for ODN1, ODNm1, ODN3, and ODNm2 were −24.63, −18.13, −9.88, −5.00 kcal mol−1, respectively. Previously, we have estimated experimentally the stability of DNA duplexes (ΔGo25) for ODNm1 and ODNm2 in NaCl or choline dhp solutions. The stability differences of for ODNm1 and ODNm2 (ΔGo25 in choline dhp solution − ΔGo25 in NaCl solution) were −1.7 kcal mol−1, 4.3 kcal mol−1, respectively. Although it is difficult to directly compare the values obtained from experimental analyses and MD simulations, the choline ion locating in the minor groove of A−T base pair estimated by MD simulations contributes to effectively stabilize the DNA duplex formation. The results of MM-GBSA analysis and binding site preference indicate that choline competes to form hydrogen bonds with those atoms that interact to form a G−C base pair. However, these results do not show the positive evidence that this choline destabilizes the G−C base pair. Previous studies reported that alkylammonium-derivative ions bind to single-stranded DNA, especially unpaired guanines, at high salt concentrations.16,17 Holland et al. showed that Mg2+ binds more stably to guanine and cytosine than to adenine and thymine bases.59 Our MD simulations of single-stranded DNA and choline ions indicate that choline ions prefer to bind to the atoms of G−C bases pairs, dC(O2), dG(O6), and dC(N3). The destabilizing effect of choline ion on G-C base pairs is thus due to the specific binding to the atoms of the singlestranded DNA, which are involved with the formation of hydrogen bonds between guanine and cytosine in a duplex. In this study, we provided a microscopic picture of how choline ion affects DNA base pair stability. We characterized the unique interaction of choline ion with DNA atoms and identified the key atoms of the DNA that interact with choline ions. This microscopic information provides us with a better understanding of how to control DNA sensors and switches. This study is the first step in investigations of the interactions between choline ions and DNA atoms. It will be interesting to investigate the effect of choline ions on structures such as A-form duplexes, triplexes, and G-quadruplexes of DNA and RNA. Choline ions stabilize duplex structures in a sequence-dependent manner, and it is likely that these ions will have interesting effects on noncanonical DNA structures and RNA structures that may be harnessed to create nucleic acid-based machines. 387



5. CONCLUSION Here, we sought to understand how choline ions differentially affect the stabilities of A−T-rich and G−C-rich DNA duplexes. We demonstrated experimentally that choline ions stabilized A−T base pairs and destabilized G−C base pairs in DNA duplex by melting temperatures measurements. We then performed MD simulations to investigate the mechanism of this effect from a microscopic point of view. More choline ions than sodium ions were observed around DNA duplexes in simulations for both A−T base pairs and G−C base pairs. Choline ions resided near a DNA duplex longer than did sodium ions. Sodium ions bound to the atoms of phosphate group due to the strong electrostatic potential, whereas the choline ions formed multiple hydrogen bonds with DNA atoms. This loose network was actually more stable than the interaction between a sodium ion and the DNA atoms. Choline ions have a high affinity for the A−T base pair minor groove due to the narrower width and more electrostatically polar environment of this groove relative to the major groove. Furthermore, we specified dT(H1′) and dT(H4′) as the key atoms interacting with the choline ion. In contrast, choline ions preferentially bound with the atoms of guanine and cytosine that are involved in hydrogen bonding between the two bases for both duplex and singlet strands. As a result of these binding preferences, choline ions stabilize A−T base pairs and destabilize G−C base pairs. Our new findings in this study provide the atomic-level information about the binding mode of molecular ions to the atoms of DNA for the first time. An understanding of the variety of interactions of molecular ions with the bases and the backbone of DNA will lead to new technology to regulate DNA structural stability and will guide the development of novel functional materials using DNA. In addition, choline ion and other osmolytes are present in high concentrations in vivo. Thus our new findings will help us understand the stabilities of canonical DNA structures under the crowded conditions inside cells.

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ACKNOWLEDGMENTS

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REFERENCES

This work was partly supported by tGrants-in-Aid for Scientific Research, and the MEXT (Ministry of Education, Culture, Sports, Science and Technology)-supported Program for the Strategic Research Foundation at Private Universities (2009− 2014), Japan, and the Hirao Taro Foundation of the Konan University Association for Academic Research. The computations were partially performed at the Research Center for Computational Science, Okazaki, Japan. We thank Dr. Kyouhei Hayashi for collecting the UV melting data.

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1 shows the initial structure of C-ODN1 and chemical structure of a choline ion. Figure S2 shows the accumulated averages of the number of cations within 3.5 Å from the atoms of DNA. Figure S3 shows the structures and atom names of DNA bases and sugar phosphate backbone. Figure S4 shows the binding sites of cations around the ODN3, ODNm1, and ODNm2. Figure S5 shows the radial distribution function of oxygen atom of choline ion around the groove atoms. Movies S1 and S2 show the representative alignments of choline ions to an A−T base pair and a G−C base pair, respectively. Movie S3 shows the alignment of choline ions buried in the minor groove of ODN1. Complete author list for references 26, 42, 43, and 54 are also provided. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Mailing address: Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, 7-1-20, Minatojimaminamimachi, Chuo-ku, Kobe, 650-0047, Japan. E-mail: [email protected]; TEL: +81-78-303-1457; FAX: +81-78-303-1495. Notes

The authors declare no competing financial interest. 388



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