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Colloids and Surfaces B: Biointerfaces 83 (2011) 61–68

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

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NA condensations on mica surfaces induced collaboratively bylcohol and hexammine cobalt

anwei Wanga,b, Shiyong Ranb, Baoyuan Mana, Guangcan Yangb,∗

School of Physics and Electronic Sciences, Shandong Normal University, Jinan 250014, ChinaSchool of Physics and Electronic Information, Wenzhou University, Wenzhou 325035, China

r t i c l e i n f o

rticle history:eceived 19 July 2010eceived in revised form 25 October 2010ccepted 25 October 2010vailable online 3 November 2010

eywords:NA condensationlcoholexammine cobalt

a b s t r a c t

We performed systematic studies of �-DNA condensation on mica surfaces induced by alcohol andhexammine cobalt (III) [Co(NH3)6

3+] using atomic force microscopy (AFM). The critical condensationconcentration for [Co(NH3)6

3+] was found to be about 10 �M; the DNA molecules extended freely onmica when the concentration was below the critical value. The morphology of condensed DNA becamemore compact with increasing concentration. At about 500 �M [Co(NH3)6

3+] concentration, no conden-sation patterns could be observed due to charge inversion of the compact structures resulting in failureof adhesion to the positively charged surfaces. The critical concentration for alcohol was about 15% (v/v).At this concentration, a few intramolecular loops could be observed in the AFM images. With increasingethanol concentration the condensation pattern became more complicated ranging from flower-like to

lectrostatic interactionoroids

pancake-like. When the solution contained both alcohol and hexammine cobalt (III), DNA condensationpatterns could be observed even when the concentrations of the two condensation agents were lowerthan their critical values. We observed this phenomenon by adding mixtures of 10% alcohol and 8 �Mhexammine cobalt (III) to DNA solutions. The condensation patterns were more compact than those of thecondensation agents separately. Typical toroids were found at an appropriate alcohol and hexamminecobalt (III) concentration. The collaborative condensation phenomenon was analyzed by electrostatic

utral

interaction and charge ne

. Introduction

DNA condensation is the collapse of extended DNA chains intoompact, ordered particles containing only one or a few molecules.t can be induced by the addition of condensing agents and/or lig-nds (e.g., multivalent cations of valence three or greater, cationicolypeptides such as polylysine, basic proteins, alcohols, and neu-ral crowding polymers) [1]. Condensation and compactation ofNA have attracted in a large amount of interest due to its

mportance both in technological and biomedical applications, par-icularly in recent years as potential vehicles for gene delivery andene transfection [2–4]. By adding condensing agent to very diluteNA solution at low ionic strength, condensed toroids and rods cane observed [5]. With the increased DNA concentrations in solu-ions, some liquid crystal structures are also formed [6,7]. Given

he high negative surface charge, a strong repulsive energy barriers expected against compaction, surprisingly thus, DNA in solutionpontaneously undergoes a transition from an extended randomoil conformation to a condensed state upon addition of condens-

∗ Corresponding author. Tel.: +86 577 86689033.E-mail addresses: yanggc@wzu.edu.cn, yanggc77@gmail.com (G. Yang).

927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2010.10.040

ization.© 2010 Elsevier B.V. All rights reserved.

ing agents. DNA condensation may proceed as a collapse of singleDNA molecules or as intermolecular aggregation. The condensationmechanism by multivalent cations is likely a neutralization of thelarge negative charge of the DNA phosphates, thereby overcomingthe columbic repulsive forces to produce the compact conforma-tion. For neutral condensing agents like alcohols, the condensationmechanism is related to the solvent properties. In a good sol-vent, the monomers effectively repel each other, preferring to besurrounded by a solvent. This effect leads to a swollen coil confor-mation for flexible polymers in a good solvent. In a poor solvent,conversely, the monomers try to exclude the solvent and effectivelyattract one another. As a result, flexible chains form a compact glob-ule of roughly spherical shape to minimize the contacts betweenmonomers and solvent. The collapsed state configurations and thepathways to their formation are the results of the interplay betweentwo opposing forces: the bending force related to the chain stiffnessand the attractive force due to the poor solvency of the environ-ment. When the two different kinds of condensing agents exist in

solution, it is possible that a mixed mechanism drives the processof condensation.

Multivalent cations may cause localized bending or distortion ofthe DNA, which may facilitate condensation. Over the past severalyears, theorists have proposed attractive mechanisms that cause

62 Y. Wang et al. / Colloids and Surfaces B: Biointerfaces 83 (2011) 61–68

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ig. 1. Morphologires of condensed �-DNA at 15% (A), 20% (B) and 30% (C-1 and C-2, B and C-1 figures are rinsed with water solution and air-drying. The C-2 figure is

NA to condense in the presence of multivalent cations [8,9]. Theanning’s counterion condensing theory [10] explained that theNA condensing occurs in the presence of multivalent cations when90% of the negative charge of its backbone is neutralized [11].olyamines spermidine, spermine and multivalent cation hexam-ine cobalt (III) [Co(NH3)6

3+] are the most common cations useds condensing agents. Among of them, Co(NH3)6

3+ is a more potentondensing agent than spermidine as it produces more tightlyacked particles, and induces a high degree of bending [12,13].

Alcohols and neutral or anionic polymers can also provoke DNAondensation. High concentrations of ethanol are commonly usedo precipitate DNA, but only under carefully controlled condi-ions, it can produce particles of well-defined morphologies [14,15].tomic force microscopy (AFM) experiments have examined theffect of alcohol on the structures of DNA confined to mica in theresence of Mg2+ [16]. At high alcohol concentration (>20%), variousigher order structures are formed, including flowers and toroids.esides, the DNA molecules adsorbed onto mica can be subse-uently condensed by a brief rinse with anhydrous alcohol [17,18].he majority of these surface-directed and ethanol-directed con-ensed structures are toroids, but a small fraction of rods can alsoe observed [19].

Both alcohol and multivalent cations can separately cause tran-itions in DNA helical structure. It might be anticipated that thewo agents would work together cooperatively [20]. Although0% ethanol is commonly used to precipitate DNA, as little as5–20% ethanol will also induce condensation if Co(NH3)6

3+ is alsodded to a solution at low ionic strength. In this paper, we sys-ematically investigate the �-DNA condensations on mica surfacenduced by alcohol and hexammine cobalt (III) [Co(NH3)6

3+] withtomic force microscopy (AFM). The independent critical conden-ation concentrations of the two condensing agents are measuredarefully. It is found that when both alcohol and Co(NH3)6

3+ coex-st in the solution, DNA condensation patterns can be observedven when the concentration of the two condensation agentsre lower than their critical values. The collaborative phenom-na can be interpreted by electrostatic interaction and solventffects.

. Experimental procedures

.1. Materials.

Bacteria �-phage DNA was purchased from New Englandiolabs and used without further purification. As received

rom the manufacturer, the �-phage DNA stock solution had

concentration of 500 ng/�L. The solvent is 1×TE buffer,

hich is composed of 10 mM Tris–HCl, pH 8.0, and 1 mMDTA. Anhydrous ethanol and methanol were purchasedrom Sinopharm Chemical Reagent, Beijing in China. Wateras deionized and purified by a Millipore system and had

nol. A very wide range of structures is seen at different ethanol concentration. Thensed with water and subsequently dried under ambient conditions.

a conductivity less than 1 × 10−6 �−1 cm−1. Mica was cutinto approximately 1 cm2 square pieces and mica surfaceswere always freshly cleaved before use. Hexammine cobalt(Co(NH3)6

3+) was purchased as chloride salts and dissolved inpure water before use. Other chemicals were all purchased fromSigma–Aldrich.

2.2. Sample preparation.

The protocols for sample preparation are as follows:Method 1: The stock solution of 500 ng/�l �-DNA was diluted

with 1× TE containing MgCl2. Experiments were performed bymixing ethanol solution of appropriate volume concentration toDNA solution. The final concentration of DNA is 2.5 ng/�l. Thefinal concentration of MgCl2 is 5 mM. Samples were incubated atroom temperature for different time. A 20 �l liquid of the mix-ture was deposited onto freshly cleaved mica and incubated for5 min. Following incubation, the mica surface was washed with100 � Milli-Q filtered water for 5 times (∼5 s) to remove the freeand excess molecules, and then rapidly blown dried using a gentlestream of nitrogen gas. As an alternative, the mica surface was notrinsed with water and subsequently dried under ambient condi-tions.

Method 2: The stock solution of 500 ng/�l �-DNA was dilutedto a final concentration of 5 ng/�l with 1× TE containing 10 mMMgCl2. Co(NH3)6

3+ solution of different concentration was mixedwith an equal volume of DNA solutions. The mixture was incubatedfor 1 h at 25 ◦C. Ten microliters of this solution was dropped imme-diately onto freshly cleaved mica surface. After air-drying for 3 min,the mica was gently rinsed with pure water in the same way asdescribed as above, then dried in air, and left sealed.

Method 3: The mixture solution of Co(NH3)63+ and alcohol were

added to an equal volume of 5 ng/�l DNA solutions containing10 mM MgCl2. The solution was incubated for 1 h at 25 ◦C. Tenmicroliters of this solution was deposited immediately onto freshlycleaved mica surface. After air-drying for 3 min, the mica was dealtwith in the same way as described as above.

2.3. Atomic force microscopy measurements

The imaging was performed in air with a multi-mode AFM withnanoscope controller (SPM-9600, Shimadzu, Kyoto, Japan) in thetapping-mode. All AFM images shown are height images with scanspeeds of ∼2 Hz and data collection at 512 × 512 pixels. The length,height, and width of the DNA in AFM imaging were measured man-ually using the off-line analysis software with AFM.

All the experiments were repeated at least three times to ensureconsistent results. The experimental error in critical concentrationfor hexammine cobalt (III) measurement is ±1 �M and the exper-imental error in critical concentration for ethanol measurement is±3% (v/v). Typical data was presented in this paper.

aces B: Biointerfaces 83 (2011) 61–68 63

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Y. Wang et al. / Colloids and Surf

. Results and discussion

.1. The DNA condensation induced by ethanol

In the first protocol, DNA solutions were incubated with dif-erent concentrations of ethanol, followed by a rinse with waterolution and then rapidly blown dried using a gentle stream ofitrogen gas as shown in Fig. 1A, B and C-1. Alternatively, the spec-

mens are not rinsed with water and subsequently dried undermbient conditions as shown in Fig. 1C-2.

The critical concentration for alcohol induced DNA condensa-ion is about 15% (v/v). When the alcohol concentration in DNAolutions decreased to 15%, there are few intermolecular con-acts, but individual molecules have a few intramolecular loops.t ethanol >15%, multi-molecular complexes appear, including

hose with flower-like morphologies. As the ethanol concentrations increased to 30%, a range of more complex flowers structuresppears as in Fig. 1C-1. A typical flower pattern has a core in theentre and DNA wraps around it at high density. There is a gen-ral tendency for the structures to become more complex withncreasing ethanol concentration. Ethanol can alter DNA conforma-ion from B-form to A- or C-form, depending on the nature of theounterion [21–23]. It has been shown that 80% ethanol is requiredor a complete B-to-A transition of a DNA in the presence of cationsuch as Na+, K+ or Cs+ [22]. In contrast, the presence of the stronglyydrated counterion Mg2+ is found to prevent the formation of the-form, as ethanol concentration increases [23]. Divalent metalations do not provoke condensation in water at room tempera-ures, but an opposite effect may be observed at somewhat elevatedemperatures or in water–alcohol mixtures. MgCl2 can also induceNA condensation in alcohol–water mixtures [24,25]. The rinseith water was performed without excess Mg2+ or free DNA torevent the formation of salt crystals or condensation inducedy magnesium cations on the mica surface. In Fig. 1C-2, the AFM

maging reveals morphologies which are not flowers. However, theorphologies of rods are seen on mica surface. The surface of mica

as to be rinsed with water, so that there are no salt crystals orther condensation dots on it. Certainly, DNA–DNA attractions dueo alcohol exclusion would be very easily reversed with a waterinse. In our experiments, as the time of depositing on mica is longnough, the interaction between the DNA and mica is strong. As aesult, the decondensation on mica surface does not occur clearly.

At low ethanol concentration, the condensation morphologiesf flowers could be expected. On the other hand, at high concen-ration of ethanol with carefully controlled conditions particles ofell defined morphology can be obtained, and can be used to pre-

ipitate DNA. As the ethanol concentration is increased to >80% inur experiments, a range of more complex pancakes and snowflaketructures appear, as shown in Fig. 2. There the morphologiesre replaced by more extensively aggregated structures. It is wellnown that high concentrations of ethanol affect the solubility andonformation of DNA.

.2. The DNA condensation induced by hexammine cobalt

In the second condensation protocol, the stock solution of00 ng/�l �-DNA was diluted to a final concentration of 5 ng/�lith 1× TE containing 10 mM MgCl2. Co(NH3)6

3+ at different con-entration was mixed with an equal volume of DNA solutions.he final concentrations of Co(NH3)6

3+ are 10 �M, 20 �M, 40 �M,0 �M, 100 �M, 200 �M, 300 �M and 500 �M. A striking feature of

NA condensation is that the dimensions and morphology of con-ensed DNA particles are largely independent of the size of the DNA.NA fragments shorter than about 400 base pairs will not condense

nto orderly, discrete particles. For example, 150 bp mononucleo-omal DNA cannot be condensed from dilute solution into discrete

Fig. 2. Range of intermediates of condensed �-DNA at 80% ethanol and the time ofincubation is about 10 h. A range of more complex pancakes and snowflake struc-tures appears.

particles of orderly morphology [26,27]. At least several hundredbase pairs must interact, either intramolecularly or intermolecu-larly, to form a stably condensed particle. We use the �-DNA whichhas 48,502 bp in our experiment serving in favor of condensation.Samples prepared by pre-mixing DNA and Co(NH3)6

3+ in tube areimaged and presented in Fig. 3. Fig. 3A–H represents the morpholo-gies of DNA on mica surfaces with the Co(NH3)6

3+ concentrationsof 10, 20, 40, 60, 100, 200, 300 and 500 �M, respectively.

When the concentration of Co(NH3)63+ is less than 10 �M, no

condensation is observed and DNA is freely extended on mica, con-sistent with the existing results [28]. However, when it crosses overthe critical concentration, the condensation grows gradually. Thecondensation emerges very explicitly at 20 �M as we can see inFig. 3B. When the concentration of the condensing agent increasesfrom 20 to 200 �M, we can see similar patterns but with increas-

ing clustering as shown in Fig. 3C–F. In these cases, DNA segmentswrap around the core to form a flat disk, or in some circumstances,wrap in a way like the petals around the core of a flower suchas in Fig. 3C. Most of these condensates are single layered, con-

64 Y. Wang et al. / Colloids and Surfaces B: Biointerfaces 83 (2011) 61–68

Fig. 3. The condensed �-DNA in hexammine cobalt at different concentration. A–H shows respectively the condensation patterns under 10, 20, 40, 60, 100, 200, 300 and500 �M hexammine cobalt.

Fig. 4. (A) The condensed �-DNA in hexammine cobalt at concentration of 20 �M. The structures like toroids and flowers could be observed on the mica surfaces. (B). It is amagnification of toroid of (A). Scale bar represent 100 nm. (C) Cross-sectional profile of the toroid in (B) along the marked line.

Y. Wang et al. / Colloids and Surfaces B: Biointerfaces 83 (2011) 61–68 65

F A solu(

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eDeclrabD

tcrc

F(

ig. 5. The condensed �-DNA by adding mixtures of alcohols and Co(NH3)63+ to DN

C) ethanol concentration to the condensed �-DNA in 10 �M hexammine cobalt.

aining more than one DNA molecules, but still rather flat. Wheno(NH3)6

3+ concentration increases to 300 �M, the flower patternsisappear and other patterns like flat pancakes emerge. Therefore,ultivalent cations not only profoundly affect the structure of DNA,

oth by condensing it and by modifying its local structure, but alsonfluence their adsorption to surface. When the Co(NH3)6

3+ concen-ration increases further to 500 �M, the results become remarkablyifferent with hardly any pattern found (Fig. 3H). We tried to imagehe condensates at much higher concentration up to 5 mM, and theimilar results were obtained. Condensation of DNA by multiva-ent cations generally forms particles of regular shape, rather thanhe randomly entangled aggregates that might be naively antici-ated. In fact, coiled DNA formed in the solution may be attachedy Co(NH3)6

3+ onto mica surfaces and form flat patterns. On thether hand, for the compact morphologies formed in the solution,t is difficult to get adhered to the charged surfaces, and to sustainhe rinsing and nitrogen blowing.

The structures observed here reflect a compromise between theffects of condensing agents and DNA interactions with mica. WhenNA binds to mica, the Co3+/Mg2+ ions on it must be released. Thenergy lost in releasing ions increases with increasing salt con-entration. At higher Co3+ concentrations, mica binding tends toost since the energy gained in mica is less than the energy lost ineleasing Co3+/Mg2+. On the other hand, the flower-like structuresre observed rather than the more compact toroids or globulesecause of release of Co3+ that mediates attraction to gain strongerNA–mica interactions [29].

In our experiments, the most common structures observed arehe flat patterns of DNA, while the regular structures like toroidsould hardly be observed on the mica surfaces. We still found someegular structures as typically shown in Fig. 4A, where a DNA toroidoexists with a neighboring flower pattern. The toroidal confor-

ig. 6. The condensed �-DNA by adding mixtures of alcohols and Co(NH3)63+ to DNA solu

C) methanol concentration to the condensed �-DNA in 10 �M hexammine cobalt.

tions. (A)–(C) Shows the condensation morphologies adding 10% (A), 20% (B), 30%

mation of the condensed DNA, with a well-defined central holesurrounded by circumferentially wound double helical strands, isone of the most striking aspects of the condensation phenomenon.Fig. 4B is a typical toroid structure of �-DNA induced by 20 �Mhexammine cobalt. It is measured by the online analyzing softwarewith the AFM and the result is shown in Fig. 4C. The outer diameterof the toroid in Fig. 4B is about 146.56 nm while the inner diam-eter is about 44.16 nm. We measured all the toroids obtained andtheir dimensions range from 120 nm to 200 nm, which is consis-tent with the results obtained by electron microscopy [20]. SinceDNA is a semiflexible polymer molecule and the attractive forcesbetween DNA segments are rather weak, the DNA molecules in poorsolute tend to form compact toroidal structure in solution due tothe equilibrium between the exclusion and bending energy whenthe concentration of condensing agent is low. They can be observedless often in AFM imaging than electron microscopy, because thecompact toroids are difficult to adsorb to mica surface after with-standing of water rinsing and nitrogen blowing during samplepreparation.

3.3. The DNA condensation induced by the mixture of alcohol andhexammine cobalt

In the third condensation protocol, the stock solution of500 ng/�L �-DNA was diluted to a final concentration of 5 ng/�Lwith 1× TE containing 10 mM MgCl2. We explored the condensa-tion by adding mixtures of different concentrations of alcohols and

Co(NH3)6

3+ to an equal volume of DNA solutions. The condensa-tion patterns are more compact than those by the correspondingcondensation agents separately.

For a specific condensing agent, condensation occurs only abovethe critical concentration for given DNA and salt concentrations.

tions. (A)–(C) Shows the condensation morphologies adding 10% (A), 20% (B), 30%

66 Y. Wang et al. / Colloids and Surfaces B: Biointerfaces 83 (2011) 61–68

Fig. 7. The condensed �-DNA by adding mixtures of alcohols and Co(NH3)63+ to

Dcp

ICtoaDcniiatoaiwct

NA solutions. (A) Shows the condensation morphologies adding 5% ethanol to theondensed �-DNA in 5 �M hexammine cobalt. (B) Shows the condensation mor-hologies adding 10% ethanol to the condensed �-DNA in 8 �M hexammine cobalt.

n our experiment, we measured the critical concentration ofo(NH3)6

3+ and ethanol, which are 10 �M and 15% (v/v), respec-ively. The extent of condensation near criticality is a linear functionf excess [Co(NH3)6

3+]. When the concentration of a condensinggent is decreased to below its critical value, the condensation ofNA cannot be observed in AFM imaging. When more than oneondensing agents exist in solution, behavior of DNA condensationeeds further investigation. Some results by dynamic light scatter-

ng and electron microscopy can be found in Ref. [20]. We exploren this direction by using Co(NH3)6

3+ and alcohol with AFM. Wettempted to use ethanol and methanol, respectively and obtainedhe similar results. The samples were prepared by adding mixturesf 10%, 20%, 30% alcohols and 10 �M Co(NH3)6

3+ to DNA solutionsnd incubating for an hour, where hexamine cobalt is at the crit-

cal value. The resultant AFM images are shown in Figs. 5 and 6,

hich correspond to ethanol and methanol respectively. We canompare the cases by 20% ethanol, 10 �M hexamine cobalt andheir combination, which are shown in Figs. 1B, 3A and 5B. It can

Fig. 8. (A) The condensed �-DNA in hexammine cobalt (100 �M) and 30% ethanolsolution. (B) The toriodal morphologies of �-DNA in hexammine cobalt (100 �M)and 5% ethanol solution.

be seen that the condensation patterns become much more appar-ent and more compact than those induced by the correspondingcondensing agents separately. We may explain the observation byconsidering the electrostatic interaction. The addition of alcohollowers the dielectric constants of the solution. The low dielec-tric solute (alcohol in the case) may also have lower affinity forbimolecular surfaces, when compared to water. The rate as wellas the extent of condensation increases as the dielectric constantis lowered. We observed the similar phenomena for ethanol andmethanol. Theoretically, ethanol may induce stronger condensa-tion than methanol because of a lower dielectric constant (25 vs.30). But the resultant difference is hard to distinguish in the cor-responding AFM images (Figs. 5 and 6). Based on the observation,we can conclude that alcohols like methanol and ethanol enhanceDNA condensation via changing the dielectric force.

Interestingly, when both alcohol and Co(NH3)63+ exist in the

DNA solution, DNA condensation patterns can appear even whenboth the concentrations are lower than their critical values, shownin Fig. 7. In Fig. 7A, we can see an initial stage of condensation of

Y. Wang et al. / Colloids and Surfaces B: Biointerfaces 83 (2011) 61–68 67

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ig. 9. Structures of DNA condensation induced by 10% alcohol and 15 �M Co(NH3

en times and twenty times.

NA by adding mixtures of 5% alcohols and 5 �M Co(NH3)63+ to

NA solutions. These concentrations are much lower than theirritical values. In Fig. 7B the condensation becomes very apparentnd quite compact in the core, while the concentrations of alcohol10%) and Co(NH3)6

3+ (8 �M) are still below their critical ones. Thiss an interesting collaborative phenomenon.

We may ascribe the observation to the electrostatic interactionn solution. Alcohols are poor solutes for DNA solution, and are typ-cally excluded from the DNA phase, which could be said to “crowd”he solution, so as to exert a force on it and force DNA together andnally push DNA toward more compact states [30]. We considerhat the observation is a collaborative phenomena rather than andditive one. As we can see in Fig. 1B (20% ethanol) and Fig. 3A10 �M hexammine cobalt), where the condensation is quite looseo as hard to judge. However when these two are mixed together, auch more apparent and compact condensation is obtained, e.g. in

ig. 5B. Changes in the morphologies of DNA complex in the pres-nce of ethanol may well indicate the crucial role of the electrostaticorce in causing DNA condensation. The electrostatic interactionsecome stronger as the dielectric constant ε decreases. Loweringlowers the effective phosphate charge by increasing counterion

ondensation. This should facilitate DNA condensation. The coop-ration between the two agents amplifies the condensing effectignificantly. This cooperation is worth to investigate further theo-etically in the future.

At an appropriate ethanol and Co(NH3)63+ concentration, we

an see that the DNA condenses into three-dimensional toroids onurfaces, which are shown in Fig. 8. In our experiment, the toroidsre more easily formed at a lower ethanol concentration since theondensation undergoes at a lower rate. At high ethanol concentra-ion, the compact DNA condensates induced by Co(NH3)6

3+ form soapidly that it is difficult for bound cations to have time to diffuselong the duplex backbone to their favorable binding sites to formregular structure.

In sample preparation, water rinse may induce the decon-ensation of DNA and influence its observation on mica surface.pecifically, DNA–DNA attractions due to alcohol exclusion may beeversed with a water rinse. We tested this water rinse effect, ashown in Fig. 9. In the study of mixed condensating agents (10%lcohol and 15 �M Co(NH3)6

3+), we made the comparisons of theFM images rinsed with water for different duration, which arehown in Fig. 8. We can see that there is nearly no change of theondensation structures. It seems that the interaction between theNA and mica is strong enough to sustain the water rinse because ofo(NH3)6

3+ binding to DNA, which is different from alcohol exclu-ion.

. Conclusions

Compaction of DNA by condensing agents can provide insightsnto DNA assembly processes, which relate closely to the essencef gene transfection and gene therapy. We have studied system-

[[[[[[

he comparisons of the AFM imagines rinsed with water for two times, four times,

atically the DNA condensations confined to a mica surface in thepresence of varying concentrations of hexammine cobalt and alco-hols, independently. The critical condensation concentration forCo(NH3)6

3+ is about 10 �M, while for ethanol it is 15% (v/v). TheDNA molecules extend freely on mica when the concentration isbelow the critical values. The morphologies of DNA condensationbecome more compact with the increasing concentration. Whenboth alcohol and Co(NH3)6

3+ exist in the solution, DNA condensa-tion patterns can be observed even when the concentration of eachof the two condensation agents are lower than their own individualcritical values. Addition of hexammine cobalt and alcohol leads toform flat and single-layered DNA patterns on mica surfaces. Theirshapes are nearly circular with a core in the centre, and the DNAstrands wrap around the core in a compact and relatively orderedmanner.

Through the comparison of DNA–hexammine cobalt conden-sation and DNA–alcohol condensation, we have found that thecondensation of DNA in three-dimensional toroids on surfaces isdifficult because of the rather strong attractive forces between DNAsegments. It is difficult for bound cations to have sufficient time todiffuse along the duplex backbone to their favorable binding sites.Due to such restrictions on forming 3D structures, DNA wraps toform a rather flat and compact patterns, like flowers, pancakes andsnowflakes. When a mixture of alcohol and Co(NH3)6

3+ is used, theaddition of alcohol tends to produce more condensed structureswhen the condensation is provoked by [Co(NH3)6

3+] resulting amuch stronger condensation.

Acknowledgements

This work is partially supported by the National Key BasicResearch Project of China (Grant No. 2007CB935900), the NationalNatural Science Foundation of China (Grant No. 10974146 and20934004) and Zhejiang Provincial Natural Science Foundation(Grant No. Y6090222).

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