DNA Compaction and Charge Inversion Induced

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DNA Compaction and Charge Inversion Induced by Organic Monovalent Ions Wenyan Xia, Yanwei Wang, Anthony Yang and Guangcan Yang * School of Physics and Electronic Information, Wenzhou University, Wenzhou 325035, China; [email protected] (W.X.); [email protected] (Y.W.); [email protected] (A.Y.) * Correspondence: [email protected]; Tel.: +86-577-8668-9033; Fax: +86-577-8668-9010 Academic Editor: Helmut Schlaad Received: 14 January 2017; Accepted: 23 March 2017; Published: 30 March 2017

Abstract: DNA condensation and charge inversion usually occur in solutions of multivalent counterions. In the present study, we show that the organic monovalent ions of tetraphenyl chloride arsenic (Ph4 As+ ) can induce DNA compaction and even invert its electrophoretic mobility by single molecular methods. The morphology of condensed DNA was directly observed by atomic force microscopy (AFM) in the presence of a low concentration of Ph4 As+ in DNA solution. The magnetic tweezers (MT) measurements showed that DNA compaction happens at very low Ph4 As+ concentration (≤1 µM), and the typical step-like structures could be found in the extension-time curves of tethering DNA. However, when the concentration of Ph4 As+ increased to 1 mM, the steps disappeared in the pulling curves and globular structures could be found in the corresponding AFM images. Electrophoretic mobility measurement showed that charge inversion of DNA induced by the monovalent ions happened at 1.6 mM Ph4 As+ , which is consistent with the prediction based on the strong hydrophobicity of Ph4 As+ . We infer that the hydrophobic effect is the main driving force of DNA charge inversion and compaction by the organic monovalent ion. Keywords: DNA compaction; charge inversion; monovalent ions; hydrophobic effect

1. Introduction DNA is one of the most important biological polyelectrolytes, and is highly negatively charged in solution. The highly-charged stiff polymer can be condensed into compact structures by multivalent ions and many other condensing agents [1–3]. The understanding of DNA compaction is not only important for the study of fundamental biological processes such as chromosome compacting, but also for the development of new gene carriers in therapeutic applications [4–6]. On the other hand, DNA compaction is closely related with its charge screening, because structural packaging requires an effective screening of the negative charges on DNA. The process is generally considered to be related to the neutralization—or more likely overcompensation—of the DNA electric charge [7–9]. Overcompensation or charge inversion occurs when the charge of counterions surrounding the DNA surface are greater than the bare charge of polyelectrolyte itself. For DNA systems, the effect seems to be found almost exclusively in the case of multivalent ions in aqueous solution [7,10–14]. Charge inversion was also observed in mixtures of charged polyelectrolytes with oppositely charged oligomers [7,15], polymers [16–24], colloids [18,25–28], or micelles [29]. The related DNA compaction from bulk solution critically depends on the valence of the counterions, and a valence of three or larger is required to overcome the inherently large electrostatic repulsive barrier between the like-charged polyelectrolytes [30–33]. However, the underlying microscopic mechanism of attraction between like-charged macroions such as DNA and their charge inversion is still controversial. There have been a number of theoretical studies aimed at elucidating the fundamental physical mechanisms responsible for DNA compaction Polymers 2017, 9, 128; doi:10.3390/polym9040128

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and charge inversion [34,35]. All these systems are strongly correlated; the electrostatic interactions are strong and the effects of thermal motions, translational, or conformational entropy of chains is small. It has been proposed that electrostatic correlations are dominant and lead to charge inversion in the case of counterions with valences larger than two near strongly-charged interfaces [8]. In the case of monovalent ions, electrostatic correlations are weak and no charge inversion is expected. Recently, Martin-Molina et al. [36] proposed a new mechanism for the charge inversion of colloids in electrolyte solutions based on the hydrophobic effect. They observed charge inversion due to the organic monovalent ion Ph4 As+ in colloids, and the effect was attributed to the hydrophobic effect. In the present study, we introduce the organic monovalent ion Ph4 As+ into a DNA system and find that it not only provokes the charge inversion of DNA, but also leads to DNA compaction, which is the first experimental evidence for DNA compaction induced by monovalent cations. The phenomenon seems to be similar to the poor solvent effect of neutral condensing agents such as ethanol in DNA solution [37]. 2. Experimental Procedures 2.1. Materials Tetraphenylarsonium chloride (Ph4 AsCl) is a tetrahedron consisting of a central As atom covalently bonded to four hydrophobic groups (the phenyl –C6 H5 rings). This highly hydrophobic cation is widely employed as a reference cation in electrochemistry because of the relatively small importance of electrostatics in its hydration free energy in different media; this is due to its large size (diameter d = 0.94 nm), its symmetry, and its monovalent character. Tetraphenylarsonium chloride (C24 H20 AsCl·HCl·xH2 O), sodium bromide (NaBr), and MgCl2 were purchased from Sigma-Aldrich, (Sain Louis, MO, US). Bacterial λ-DNA was purchased from New England Biolabs (Ipswich, MA, US), and it had a concentration of 500 ng/µL as obtained from the manufacturer. We could use λ-DNA directly without further treatment in atomic force microscopy (AFM), but DNA modification was necessary before pulling in magnetic tweezers (MT). The solvent we used was 1 mM sodium bromide aqueous solution, and water was deionized and purified by a Millipore system and had a conductivity less than 1 × 10–6 Ω–1 cm–1 . Mica for AFM imaging was cut into approximately 1 cm2 square pieces, and their surfaces were always freshly cleaved before use. All chemical agents were used as received, and all measurements were repeated at least twice to obtain consistent results. 2.2. AFM Imaging The sample preparation procedure is similar to the one in our previous work [36]. It can be briefly described as follows: mica disks of diameter one centimeter attached to magnetic steel disks were used as substrates for DNA adsorption. For each sample, the final concentration of DNA was 1 ng/µL, corresponding to 3 µM of phosphate groups, and a drop of about 15 µL of Ph4 As+ mixture was deposited for 3 min on a freshly-cleaved mica surface. The surface was rinsed with distilled water and dried with a gentle flow of nitrogen gas. A multi-mode atomic force microscope (SPM-9600, Shimadzu, Kyoto, Japan) was used for DNA imaging in the presence of Ph4 As+ . All AFM images were captured in the conventional ambient tapping mode, with scan speeds of ≈2 Hz and data collection at 512 × 512 pixels. All the images were processed manually using off-line analysis software equipped with the microscope. 2.3. Magnetic Tweezers Experiment Magnetic tweezers are common tools for manipulating single molecules, such as tethering a condensed DNA molecule [38]. A transverse MT system was composed of an inverted microscope and charge coupled device (CCD) controlled by a personal computer, which is schematically shown in Figure 1. In our experiments, the ends of DNA were coated with biotin and digoxin by biochemical method, and the surfaces of paramagnetic beads were covered with streptavidin. A coverslip with one

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and functionalized with anti-digoxin in order to link with the dig-end of DNA. The other end of DNA was linked to a paramagnetic bead by the streptavidin–biotin bond. The force was applied to the paramagnetic bead by adjusting the permanent magnet which is installed on a Polymers 2017, 9, 128 3 ofthe 10 micromanipulator. A video camera was used to monitor the image of the tethered structure, and positions of paramagnetic beads were recorded in real-time. The analysis of the extension and force waspolished determined a tracking algorithm based onslides, correlation function [39]. side wasby sandwiched between two glass and was used as a flow chamber by sealing Various concentrations of Ph 4AsCl solution were mixed with NaBr solution (1 mM), then an the sealing the open sides of the structure. The polished sidewall was silylated and functionalized equalanti-digoxin volume of inDNA containing 10 mM MgClThe 2 were added for magnetic tweezers with ordersolutions to link with the dig-end of DNA. other end of DNA was linked to a measurement. The solution was incubated for 30 min at least at room temperature, and introduced paramagnetic bead by the streptavidin–biotin bond. The force was applied to the paramagnetic bead into the flow cell by using a syringe pump. In a typical measurement, we moved the by adjusting the permanent magnet which is installed on a micromanipulator. A videomagnet camera from was some distance to some position close to a paramagnetic bead, thus applying a magnetic force on the used to monitor the image of the tethered structure, and the positions of paramagnetic beads were suspended bead. When a fixed magnetic force was applied to the bead, we monitored the end-to-end recorded in real-time. The analysis of the extension and force was determined by a tracking algorithm lengthon ofcorrelation DNA in real-time measure its conformational change. based functionto[39].

Figure Figure1.1.A Aschematic schematicdiagram diagramof ofmagnetic magnetictweezers. tweezers.

2.4. Electrophoretic Mobility Measurement by Dynamic Scattering (DLS) Various concentrations of Ph4 AsCl solution were Light mixed with NaBr solution (1 mM), then an equal volume of DNA solutions containingmeasurements 10 mM MgCl2 were for magnetic tweezers measurement. The electrophoresis-mobility wereadded carried out by using a dynamic light The solution was incubated for 30 min at least at room temperature, and introduced into the flow cell scattering device of Malvern Zetasizer Nano ZS90 (Malvern Instruments Ltd., Worcestershire, UK) by using a syringe pump. In a typical measurement, we moved the magnet from some distance to equipped with the patented M3-PALS technique. The laser source is a He-Ne gas laser (λ = 633 nm) some position close to a paramagnetic bead, thus applying a magnetic force on the suspended bead. and the light scattering is collected by an avalanche photodiode mounted on the goniometer arm to When a fixed magnetic force was applied to DNA the bead, we monitored the end-to-end length of in the direction of the incident radiation. The samples were diluted to a concentration ofDNA 1 ng/μL real-time to measure its conformational change. in a buffer solution containing 1 mM NaBr and 5 mM MgCl2; then, different concentrations of Ph4As+

were added. All measurements were carried out after 5 min incubation at room temperature. During 2.4. Electrophoretic Mobility Measurement by Dynamic Light Scattering (DLS) the measurement, 1 mL of DNA solution was used, and the sample cell was kept at 25 °C The electrophoresis-mobility measurements were carried out by using a dynamic light scattering temperature. device of Malvern Zetasizer Nano ZS90 (Malvern Instruments Ltd., Worcestershire, UK) equipped 3. Results and Discussion with the patented M3-PALS technique. The laser source is a He-Ne gas laser (λ = 633 nm) and the light scattering is collected by an avalanche photodiode mounted on the goniometer arm to the direction 3.1. Observation by AFM The DNA samples were diluted to a concentration of 1 ng/µL in a buffer of the incident radiation. + were solution 1 mM NaBr 5 mM MgCl2 ; then,ofdifferent concentrations ofinPh + complexes 4 As We containing attempted to observe theand change in morphology the DNA–As the presence added. All measurements carried out after 5 min incubation at room temperature. During the of different concentrationswere of Ph 4As+ in solution. In our experiment, MgCl2 solution (5 mM) was ◦ C temperature. measurement, 1 mL of DNA solution was used, andsurface. the sample cell was kept of at 25 + complexes added into the buffer to adsorb DNA on the mica The AFM images DNA–As on the mica surface are shown in Figure 2. For reference, we start by imaging DNA alone in the same 3. Results and Discussion buffer condition as used for the complexes (Figure 2A). We can see that the DNA molecules are well separated on the and have relaxed morphologies with no compaction loops. From Figure 3.1. Observation by surface, AFM 2B–F, the corresponding molar ratio between arsenic cations and the+ phosphate group of DNA We attempted to observe the change in morphology of the DNA–As complexes in the presence varies from 3.33 to 333.3. As shown in Figure 2B, there are few intermolecular contacts at lower As+ of different concentrations of Ph4 As+ in solution. In our experiment, MgCl2 solution (5 mM) was concentrations, but individual molecules have an increased number of intramolecular loops with added into the+buffer to adsorb DNA on the mica surface. The AFM images of DNA–As+ complexes increasing As concentration. However, when [As+] > 0.1 mM, typical condensed structures were on the mica surface are shown in Figure 2. For reference, we start by imaging DNA alone in the same buffer condition as used for the complexes (Figure 2A). We can see that the DNA molecules are well separated on the surface, and have relaxed morphologies with no compaction loops. From Figure 2B–F,

appeared (Figure 2C), and DNA highly looped around this point, as shown in Figure 2D. The compaction grew gradually when the concentration of As+ increased further (Figure 2E), and at the highest concentration (Figure 2F), we can see even more compacting patterns (e.g., globules) on the mica surface. AFM may modify the actual morphology of DNA in As+ solution. We have previously shown Polymers 2017, 9, 128 4 of 10 that the treatment of condensed DNA adsorbed to a surface only reduces the molecules’ heights but maintains their lateral dimensions [37]. Thus, AFM images flatten the morphology of DNA condensates with their original compacting structure. the condensed is adsorbed on the corresponding molar ratio between arsenic cations However, and the phosphate groupDNA of DNA varies from a mica surface onlyinbinds thethere surface loosely. Despite thecontacts interactions between the substrate 3.33 to 333.3. Asand shown Figureto2B, are few intermolecular at lower As+ concentrations, and individual the polyelectrolytes canincreased modify the structure of complexes),loops there with is a nice correlation but molecules(which have an number of intramolecular increasing As+ + between the macroscopic phase [As diagram and thetypical AFM observations. We thinkwere that observed, the reasonand for concentration. However, when ] > 0.1 mM, condensed structures phenomenon similar towere the still mechanism of alcohol [37] leading to DNA compaction. Since athis part of the DNAisstructures in the coiled conformation. A condensed center appeared DNA is2C), a semiflexible molecule andthis the point, attractive forces in between segments are rather (Figure and DNA polymer highly looped around as shown Figure DNA 2D. The compaction grew weak, the when DNA the molecules in poor tend tofurther form compact toroidal inconcentration solution due gradually concentration ofsolvent As+ increased (Figure 2E), and atstructures the highest to the equilibrium exclusion and bending (Figure 2F), we canbetween see eventhe more compacting patterns energy. (e.g., globules) on the mica surface.

2+ solution; Panels (B–F) Figure 2. AFM observation of DNA complexes. complexes. Panel (A) λ-DNA λ-DNA in in 55 mM mMMg Mg2+ + DNA conformations at different concentrations concentrations (0.01, (0.01, 0.05, 0.05, 0.1, 0.1, 0.5 0.5 and and 11 mM, mM, respectively) respectively) of ofAs As+ in 2+ 5 mM Mg 2+ buffer. buffer.

+ solution. To enhance DNA adsorption on the micaof surface forAs imaging, we We used a buffer containing AFM may modify the actual morphology DNA in have previously showna 2+ high concentration of Mg (5 mM). To rule out its effect on DNA compaction, we performed the that the treatment of condensed DNA adsorbed to a surface only reduces the molecules’ heights 2+—the lowest concentration to deposit DNA on a mica surface. The same experiments in 1 mM of Mg but maintains their lateral dimensions [37]. Thus, AFM images flatten the morphology of DNA results are similar, andoriginal are shown in Figure 3. We can see that DNA condensed from loose condensates with their compacting structure. However, thethe condensed DNA is adsorbed on + concentration in the low concentration of structures to highly-compact globules with increasing As a mica surface and only binds to the surface loosely. Despite the interactions between the substrate Mg2+the . Thus, we can deduce that the compaction is caused by arsenicthere ion rather than the effect and polyelectrolytes (which canDNA modify the structure of complexes), is a nice correlation + is much weaker than spermine, of magnesium ion. Actually, the DNA condensing ability of Ph 4 As between the macroscopic phase diagram and the AFM observations. We think that the reason for this as shown in Figure 4, where effect of 0.5 As+[37] is comparable with 0.01 mM spermine. phenomenon is similar to thethe mechanism of mM alcohol leading to DNA compaction. Since DNA is

a semiflexible polymer molecule and the attractive forces between DNA segments are rather weak, the DNA molecules in poor solvent tend to form compact toroidal structures in solution due to the equilibrium between the exclusion and bending energy. To enhance DNA adsorption on the mica surface for imaging, we used a buffer containing a high concentration of Mg2+ (5 mM). To rule out its effect on DNA compaction, we performed the same experiments in 1 mM of Mg2+ —the lowest concentration to deposit DNA on a mica surface. The results are similar, and are shown in Figure 3. We can see that the DNA condensed from loose structures to highly-compact globules with increasing As+ concentration in the low concentration of Mg2+ . Thus, Figure 3. The structure of DNA in mixture ((A)the 0.1 effect mM; (B) mM; we can deduce that the DNA compaction is solution caused of byvarious arsenicconcentrations ion rather than of 0.5 magnesium + and 1 mM Mg2+. + (C) 1 mM) of As ion. Actually, the DNA condensing ability of Ph4 As is much weaker than spermine, as shown in Figure 4, where the effect of 0.5 mM As+ is comparable with 0.01 mM spermine.

same experiments in 1 mM of Mg2+—the lowest concentration to deposit DNA on a mica surface. The results are similar, and are shown in Figure 3. We can see that the DNA condensed from loose structures to highly-compact globules with increasing As+ concentration in the low concentration of Mg2+. Thus, we can deduce that the DNA compaction is caused by arsenic ion rather than the effect Polymers 2017, 9, 128 5 of 10 of magnesium ion. Actually, the DNA condensing ability of Ph4As+ is much weaker than spermine, as shown in Figure 4, where the effect of 0.5 mM As+ is comparable with 0.01 mM spermine.

Figure 3. The The structure structure of of DNA DNA in in mixture mixture solution solution of of various various concentrations concentrations ((A) ((A) 0.1 0.1 mM; mM; (B) (B) 0.5 0.5 mM; mM; + and 1 mM Mg2+ + 2+. . (C) 1 mM) of As and 1 mM Mg Polymers 2017, 9, 128 5 of 9

Figure 4. DNA structures in solution of (A) 0.01 mM spermine and (B) 0.5 mM As+. Figure 4. DNA structures in solution of (A) 0.01 mM spermine and (B) 0.5 mM As+ .

3.2. Tethering of Single DNA Molecules 3.2. Tethering of Single DNA Molecules DNA tethering was achieved in a magnetic tweezers (MT) setup, as described above. We first DNA tethering was achieved in a magnetic tweezers (MT) setup, as described above. We first put the microsphere-bound DNA molecules in NaBr buffer (1 mM) and magnesium ion (5 mM) into put the microsphere-bound DNA molecules in NaBr buffer (1 mM) and magnesium ion (5 mM) into the flow cell and incubated them for 30 min at room temperature. Then, we could find that some the flow cell and incubated them for 30 min at room temperature. Then, we could find that some paramagnetic beads had tethered to the surface of the sidewall through a single DNA molecule. In paramagnetic beads had tethered to the surface of the sidewall through a single DNA molecule. In the the absence of any condensing agents, we found that the extension of DNA was close to 16 μm under absence of any condensing agents, we found that the extension of DNA was close to 16 µm under high extension (>10 pN). Then, about 400 μL of different concentrations of Ph4AsCl solution were high extension (>10 pN). Then, about 400 µL of different concentrations of Ph4 AsCl solution were loaded into the flow cell and stretched the DNA by approaching the magnet to the bead, or releasing loaded into the flow cell and stretched the DNA by approaching the magnet to the bead, or releasing the bead by moving back the magnet while the extension of the DNA molecule was monitored in the bead by moving back the magnet while the extension of the DNA molecule was monitored in real-time. The results of the DNA tethering are shown in Figures 5 and 6. From Figure 5A, we can see real-time. The results of the DNA tethering are shown in Figures 5 and 6. From Figure 5A, we can see the stepwise shrinking of the DNA extension when a constant 0.5 pN pulling force was exerted upon the stepwise shrinking of the DNA extension when a constant 0.5 pN pulling force was exerted upon the bead at low concentration of Ph4AsCl (1 μM). After the DNA molecule condensed to a compact the bead at low concentration of Ph4 AsCl (1 µM). After the DNA molecule condensed to a compact state, we had to apply much larger force (5.4 pN in this case) to unravel the condensed structure, as state, we had to apply much larger force (5.4 pN in this case) to unravel the condensed structure, as shown in Figure 5B. We could repeat the shrinking and unraveling many times, and the processes shown in Figure 5B. We could repeat the shrinking and unraveling many times, and the processes was was reversible in our experiment. When the concentration of Ph4AsCl in the buffer increased, the reversible in our experiment. When the concentration of Ph4 AsCl in the buffer increased, the tethering tethering force increased accordingly to unravel the condensed DNA structures. We could still see force increased accordingly to unravel the condensed DNA structures. We could still see the stepwise the stepwise structure in the shrinking or pulling curves of DNA molecules when the concentration structure in the shrinking or pulling curves of DNA molecules when the concentration of Ph4 AsCl is of Ph4AsCl is not greater than 0.5 mM, as shown in Figures 6 and 7. However, the stepwise structure not greater than 0.5 mM, as shown in Figures 6 and 7. However, the stepwise structure disappeared in disappeared in the shrinking curves when the concentration of As+ reached 1 mM, as shown in the shrinking curves when the concentration of As+ reached 1 mM, as shown in Figure 7C. We infer Figure 7C. We infer than a more compact structure (e.g., globule) was formed in the high than a more compact structure (e.g., globule) was formed in the high concentration condition. concentration condition.

Figure 5. DNA extension–time curves under the influence of Ph4As+. (A) The shrinking of DNA at pulling force 0.5 pN; (B) unraveling of DNA condensed structures.

tethering force increased accordingly to unravel the condensed DNA structures. We could still see the stepwise structure in the shrinking or pulling curves of DNA molecules when the concentration of Ph4AsCl is not greater than 0.5 mM, as shown in Figures 6 and 7. However, the stepwise structure disappeared in the shrinking curves when the concentration of As+ reached 1 mM, as shown in Figure 7C. 9,We Polymers 2017, 128 infer than a more compact structure (e.g., globule) was formed in the 6high of 10 concentration condition.

+ Figure Ph44As Figure 5. 5. DNA DNA extension–time extension–time curves curves under under the the influence influence of of Ph As+. .(A) (A)The Theshrinking shrinkingof of DNA DNA at at pulling force 0.5 pN; (B) unraveling of DNA condensed structures. pulling force 0.5 pN; (B) unraveling of DNA condensed structures.

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Polymers 9, 128 the tethering curves, we concluded that the hydrophobic driving force mechanism 6 of 9 By2017, analyzing

is similar to the effect of poor solvent. However, the former is much stronger, since the tethering force is on the order of a few pN, while the shrinking force is difficult to measure in the case of ethanol solution [35]. For consistency and reproducibility, we repeated our tethering cycle under each condition at least 10 times. The curve of condensing force is presented in Figure 8. The details of pulling forces and DNA particle sizes are presented in Tables S1–S3.

Figure 6. DNA curves when (A) contracting (B) stretching various Ph4As+ concentrations. Figure 6. extension–time DNA extension–time curves when (A)and contracting andat(B) stretching at various + concentrations. Figure 6. DNA extension–time curves when (A) contracting and (B) stretching at various Ph4As+ concentrations. Ph4 As

Figure 7. DNA extension–time curves at high concentrations ((A) 0.1 mM; (B) 0.5 mM; (C) 1 mM) of Ph4As+. Figure7.7. DNA extension–time curves at high concentrations (B) 0.5 Figure DNA extension–time curves at high concentrations ((A) 0.1((A) mM;0.1 (B)mM; 0.5 mM; (C) mM; 1 mM)(C) of 1PhmM) 4As+. + of Ph4 As .

By analyzing the tethering curves, we concluded that the hydrophobic driving force mechanism is similar to the effect of poor solvent. However, the former is much stronger, since the tethering force is on the order of a few pN, while the shrinking force is difficult to measure in the case of ethanol solution [35]. For consistency and reproducibility, we repeated our tethering cycle under each condition at least 10 times. The curve of condensing force is presented in Figure 8. The details of pulling forces and DNA particle sizes are presented in Tables S1–S3. Figure 8. The curve of condensing force at different concentrations of Ph4As+. Figure 8. The curve of condensing force at different concentrations of Ph4As+.

3.3. Electrophoretic Mobility of DNA

3.3. Electrophoretic of DNA We measuredMobility the electrophoretic mobility of DNA in the mixture solution with different + concentrations of Ph 4Aselectrophoretic and 5 mM MgCl 2 by dynamic scattering The different result is We measured the mobility of DNA light in the mixture technique. solution with shown in Figure 9. We can see that the mobility of DNA changed from negative to positive values + concentrations of Ph4As and 5 mM MgCl2 by dynamic light scattering technique. The result is + with the As can concentration. mobility approached about 1.75 mM of Ph 4As+. shown in increasing Figure 9. We see that the The mobility of DNA changedzero fromatnegative to positive values For cation concentrations larger than this, mobility reversed from positive, with the increasing As+ concentration. Thethe mobility approached zeronegative at aboutto1.75 mM ofimplying Ph4As+. that charge inversion occurred. Meanwhile, the size of DNA complex decreased with As+ For cation concentrations larger than this, the mobility reversed from negative to positive, implying

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Figure 7. DNA extension–time curves at high concentrations ((A) 0.1 mM; (B) 0.5 mM; (C) 1 mM) of Ph4As+.

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σ Figure 8. 8. The The curve curve of of condensing force0at differentconcentrations concentrationsof ofPh Ph4As As++.. I Figure force 4 ccondensing = 0 exp( Δμ atkdifferent T) B

B

ed

(1)

mobility(um*cm/vs)

3.3. Electrophoretic Mobility of of DNA DNA 3.3. Electrophoretic Mobility is the difference in free energy between the dehydrated ion at close where ∆ = − We measured the electrophoretic mobility ofelectrolyte, DNA in in the the mixture solution with different proximity to the colloid and the hydrate mobility ion in bulk d is mixture the diameter of thewith counter ions, We measured the electrophoretic of DNA solution different + concentrations of charge Ph4As and 5 of mM MgCl 2 by light scattering The result is and σ0 is the bare DNA. For thedynamic current system, where d ≈technique. 1 nm, hydration energy +density concentrations of Ph 4 As and 5 mM MgCl2 by dynamic light scattering technique. The result is 2changed shown in Figure 9. We can see that the mobility of DNA from negative to positive values is about −5k BT, and bare surface charge is about 1.0 e/nm , one obtains = 1.6 mM. The measured shown in Figure 9. We can see that the mobility of DNA changed from negative to positive values +. with the the increasing As++ concentration. concentration. The mobility approached zero atwhere about charge 1.75 mM mM of Ph Ph4As Asis value of increasing charge inversion is about 1.75 mM, as shown in Figure inversion + with As The mobility approached zero9,at about 1.75 of . 4 For cation concentrations larger than this, the mobility reversed from negative to positive, implying observed for DNA in water at 298 K in the presence of low concentrations of a monovalent large For cation concentrations larger than this, the mobility reversed from negative to positive, implying that that charge occurred. Meanwhile, the size ofagreement DNA complex decreased with organic cationinversion [36]. The observed critical concentration iscomplex in withwith the predicted valueAs by+ charge inversion occurred. Meanwhile, the size of DNA decreased As+ concentration. + were concentration. When theofconcentrations As(1) 0.05, 0.1, 0.5, 1, 3 mM, the hydrophobicity. It is noticeable be 3applicable for2,a and weakly charged When the concentrations As+ that wereEquation 0.01,of 0.05, 0.1,might 0.5, 0.01, 1,only 2, and mM, the corresponding particle corresponding sizes of 310, DNA condensates were 310, 210, 180, 182,and and 180 nm, system. DNA condensates isparticle a highly charged system where the correlation is significant and a errors more sizes of DNA were 300, 210, 200, 180, 182, and300, 180effect nm, 200, respectively, the respectively, and the errors were ±20 nm. sophisticated theory is needed. In spite of the restriction, we can still use it as a preliminary estimation. were ±20 nm. The mechanism of DNA charge reversal is similar to colloid charge inversion, the hydrophobic effect as a driving force for charge inversion. This mechanism is able to induce charge inversion at 3 As++5mMMg2+ low concentrations of monovalent ions +containing hydrophobic functional groups, and saturation As 2 effects appearing at higher concentrations. Following the similar analysis in Reference [36], the critical concentration of counterions in 1 solution when charge inversion occurs can be expressed as 0 -1 -2

0.0

0.5

1.0

1.5

2.0

2.5

3.0

concentration(mM) Figure 9. 9. Electrophoretic Electrophoretic mobility mobility of of DNA DNA vs. vs. the the concentration concentration of of Ph Ph44As As++.. Figure

4. Conclusions The mechanism of DNA charge reversal is similar to colloid charge inversion, the hydrophobic we found for inversion. the first time that the organic ionsinversion of tetraphenyl effectInas summary, a driving force for charge This mechanism is able monovalent to induce charge at low chloride arsenic (Ph4As+) can induce DNAhydrophobic compactionfunctional and its groups, charge and inversion, which is concentrations of monovalent ions containing saturation effects contradictory to theconcentrations. common understanding that charge inversion occurs only if the valence of appearing at higher counterions is Zthe ≥ 3.similar These analysis findings in were confirmed magnetic tweezers measurements, atomic Following Reference [36],bythe critical concentration of counterions in force microscopy (AFM) imaging, and dynamic light scattering (DLS). solution when charge inversion occurs can be expressed as DNA compaction is usually involved the first-order phase transition between elongated and |σ0 | cause shrinkage compact states, although some chemical species c BI = exp(∆µ0 /k B T ) of DNA to be much different from (1) ed the transition [40]. DNA compaction occurs in one mode or mixed modes among all-or-none compaction, compaction, adsorption and wrapping, depending condensing where ∆µ0 =progressive µ0S − µ0B is the differenceand in free energy between the dehydrated ion at on close proximity agents and solvents. The DNA compaction in Ph 4As+ solution is in a progressive compaction mode, to the colloid and the hydrate ion in bulk electrolyte, d is the diameter of the counter ions, and σ0 is which directly shownofinDNA. their For AFM and 3). electrostatic interaction the bareischarge density theimages current(Figures system, 2where d ≈Since 1 nm,the hydration energy is about between monovalent ions and DNA is not able to induce its compaction or charge inversion, we 2 I –5kB T, and bare surface charge is about 1.0 e/nm , one obtains cB = 1.6 mM. The measured value infer that the main driving force of DNA charge inversion and compaction by the organic of charge inversion is about 1.75 mM, as shown in Figure 9, where charge inversion is observed for monovalent ion Ph4As+ is the hydrophobic effect. Supplementary Materials: The following are available online at www.mdpi.com/2073-4360/9/4/128/s1, Table S1: The condensing force of DNA at different concentrations of Ph4As+, Table S2: The unravelling force of DNA at different concentrations of Ph4As+, Table S3: The particle size of DNA at different concentrations of Ph4As+.

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DNA in water at 298 K in the presence of low concentrations of a monovalent large organic cation [36]. The observed critical concentration is in agreement with the predicted value by hydrophobicity. It is noticeable that Equation (1) might only be applicable for a weakly charged system. DNA is a highly charged system where the correlation effect is significant and a more sophisticated theory is needed. In spite of the restriction, we can still use it as a preliminary estimation. 4. Conclusions In summary, we found for the first time that the organic monovalent ions of tetraphenyl chloride arsenic (Ph4 As+ ) can induce DNA compaction and its charge inversion, which is contradictory to the common understanding that charge inversion occurs only if the valence of counterions is Z ≥ 3. These findings were confirmed by magnetic tweezers measurements, atomic force microscopy (AFM) imaging, and dynamic light scattering (DLS). DNA compaction is usually involved the first-order phase transition between elongated and compact states, although some chemical species cause shrinkage of DNA to be much different from the transition [40]. DNA compaction occurs in one mode or mixed modes among all-or-none compaction, progressive compaction, and adsorption and wrapping, depending on condensing agents and solvents. The DNA compaction in Ph4 As+ solution is in a progressive compaction mode, which is directly shown in their AFM images (Figures 2 and 3). Since the electrostatic interaction between monovalent ions and DNA is not able to induce its compaction or charge inversion, we infer that the main driving force of DNA charge inversion and compaction by the organic monovalent ion Ph4 As+ is the hydrophobic effect. Supplementary Materials: The following are available online at www.mdpi.com/2073-4360/9/4/128/s1, Table S1: The condensing force of DNA at different concentrations of Ph4 As+ , Table S2: The unravelling force of DNA at different concentrations of Ph4 As+ , Table S3: The particle size of DNA at different concentrations of Ph4 As+ . Acknowledgments: We thank Ruxia Wang and Yang Chen for their help in experiments and helpful discussions. This work is supported by the National Natural Science Foundation of China (11274245, 10974146, 11304232). Author Contributions: Guangcan Yang and Yanwei Wang conceived and designed the experiments; Wenyan Xia and Anthony Yang performed the experiments; Yanwei Wang and Wenyan Xia analyzed the data; Guangcan Yang, Wenyan Xia and Anthony Yang wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

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