pH-dependent sedimentation of DNA in the presence of divalent, but not monovalent, metal ions

Abstract

Precipitation of DNA is performed frequently in molecular biology laboratories for the purpose of purification and concentration of samples and also for transfer of DNA into cells. Metal ions are used to facilitate these processes, though their precise functions are not well characterized. In the current study we have investigated the precipitation of double-stranded DNA by group 1 and group 2 metal ions. Double-stranded DNAs were not sedimented efficiently by metals alone, even at high concentrations. Increasing the pH to 11 or higher caused strong DNA precipitation in the presence of the divalent group 2 metals magnesium, calcium, strontium and barium, but not group 1 metals. Group 2 sedimentation profiles were distinctly different from that of the transition metal zinc, which caused precipitation at pH 8. Analysis of DNAs recovered from precipitates formed with calcium revealed that structural integrity was retained and that sedimentation efficiency was largely size-independent above 400 bp. Several tests supported a model whereby single-stranded DNA regions formed by denaturation at high pH became bound by the divalent metal cations. Neutralization of negative surface charges reduced the repulsive forces between molecules, leading to formation of insoluble aggregates that could be further stabilized by cation bridging (ionic crosslinking).

1. Introduction

Nucleic acids are routinely precipitated out of solutions, typically to purify DNA or RNA away from other biomolecules present in a cell extract or to concentrate them into a smaller volume [1–6]. Precipitation is also used to separate RNAs from DNA molecules [1,7–10]. Chromosomal and plasmid DNA purification protocols frequently involve sedimentation in the presence of alcohol, e.g., by adding 2.5-3 volumes of ethanol or 0.6-1.0 volume of isopropanol in the presence of 0.3 M sodium acetate [1,11–15]. Nucleic acids have also been precipitated out of solutions using organic chemicals such as spermine, spermidine and polyethylene glycol [15–21].

Precipitation can be accomplished by addition of metal salts to nucleic acids under specific conditions. For example, a common method for transfection of DNA into mammalian cells involves sedimentation of the DNA in the presence of calcium phosphate, followed by uptake of the metal:DNA complexes into cells via an endocytosis-like mechanism [22,23]. Calcium phosphate nanoparticles have also been employed for gene and siRNA delivery into cells [24–26]. Protocols have been developed for removal of RNAs from DNA solutions by selective sedimentation in the presence of lithium chloride, calcium chloride and other salts [1,9,27,28]. Nucleosome protein-bound DNA complexes have been precipitated using calcium and magnesium ions [29]. Plasmid DNA purification methods have also been developed that employed sedimentation of nucleic acids with calcium chloride [27,30]

In the current study we have investigated the abilities of group 1 and 2 metals to cause precipitation of DNAs out of aqueous solutions. Initial experiments characterized the spectrophotometric properties of the metals, with and without DNA present in the solutions. Strong DNA sedimentation was only induced by the divalent group 2 metals and only when the pH was raised to 11 or higher. Several experiments examined the impacts of different metal and buffer concentrations as well as the importance of the sizes of the DNAs. Results of tests investigating the potential mechanism of DNA sedimentation were most consistent with a model involving both charge neutralization and cation bridging. An important result of this work is the development of a simple approach for the sedimentation of DNAs out of aqueous solutions using group 2 metal chlorides.

2. Materials and Methods

2.1. Materials

All metal chlorides, formamide, and CAPS buffer were purchased from Sigma-Aldrich. Tris base was purchased from J. T. Baker and glacial acetic acid was from Mallinckrodt Chemicals. Ethylenediaminetetraacetic acid (EDTA)-disodium salt was obtained from EMD Chemicals, Inc. The 2-Log DNA ladder, also called 1 kb Plus DNA ladder, 1 kb Extend DNA ladder, bacteriophage lambda DNA and M13mp18 DNAs were purchased from New England Biolabs. E. coli chromosomal DNA (D2001) was obtained from Sigma-Aldrich. Agarose was from Gold Biotechnology.

2.2. Spectrophotometric DNA sedimentation assays

Spectrophotometric assays employed UV-transparent micro- or minicuvettes in conjunction with either a Beckman-Coulter DU700 or Bio-Rad SmartSpec Plus spectrophotometer. Solutions to be analyzed by spectrophotometry were prepared using Eppendorf brand microcentrifuge tubes (VWR 20901-551); these tubes exhibited minimal leaching of UV light-absorbing chemicals from the polypropylene plastic in a previous study [31]. All metal chloride stocks were prepared as filtered 1 M solutions in deionized water except zinc chloride, which was aliquotted from 0.1 M stock solutions. Buffers (Tris and CAPS) were prepared as filtered 0.25 M solutions. Microcuvettes (BioRad TruView or Brand microcuvettes) and standard quartz cuvettes were used for spectroscopic assays and scans, respectively.

A typical 100/100 experiment (100 mM buffer + 100 mM metal chloride) was performed using 4 or 5 replicates of each sample in 150 μL volumes. Deionized water was mixed with sufficient DNA to give an A₂₆₀ reading of about 1.0 (equivalent to 50 ng/μL for dsDNA), followed by addition of 15 μL 1 M metal chloride. The reaction was then initiated by addition of 37.5 μL of 0.4 M buffer that had been set to a specific pH. For experiments performed at high pH without Tris or CAPS buffers, the pH was raised by addition of a small volume of 5 M sodium hydroxide. In assays involving 50 ng/μL double-stranded chromosomal and bacteriophage lambda DNAs with average size approximately 50,000 bp, the concentration of nucleotides/phosphates was 0.15 mM (based on molecular weight of 325 g/mol per DNA nucleotide).

After mixing, samples were incubated at RT for 5 min, centrifuged at 21,000g in an Eppendorf 5424 microcentrifuge for 10 min, and the top halves of the solutions immediately transferred to new tubes. Absorbances of the supernatants were then measured at 260 nm after blanking against solutions containing metal choride only. Averages and standard deviations were calculated and are presented in the figures.

2.3. Recovery and analysis of DNA from precipitated complexes

DNA was eluted out of pellets for subequent analysis using gel electrophoresis by removing the supernatant after centrifugation, rinsing the pellet twice with cold 50% ethanol, and resuspension in TE (10 mM Tris [pH 8.0] + 1 mM EDTA). Elution efficiency was improved by increasing the concentration of metal-chelating EDTA (10 mM and 50 mM) and by heating the samples at 42°C or 65°C for 5 min. Samples were mixed with purple loading dye (New England Biolabs) prior to electrophoresis.

Gel electrophoresis experiments were performed using 1.0% agarose gels run in 1 x TAE using Horizon gel systems (LabRepco) as described [32]. Gels were stained with ethidium bromide and photographed using an Alpha Innotech RED gel imagestation.

3. Results

3.1. Impact of group 1 and 2 metals on absorbance of ultraviolet light at 260 nm in aqueous solutions

The major goal of this study was to investigate the abilities of group 1 and 2 metal cations to cause aggregation and sedimentation of DNAs out of aqueous solutions. A long-term goal of the work is to develop new ways to selectively sediment nucleic acids out of solutions. Sedimentation was monitored using centrifugation assays, in which the absorbance of a solution at 260 nm becomes reduced after centrifugation because DNA-metal complexes are pelleted to the bottom of a microfuge tube. The approach is similar in concept to many nanoparticle:DNA binding assays that have been performed in our lab and elsewhere [33,34].

Initial tests established that group 1 (Li⁺, Na⁺, K⁺, Rb⁺ and Cs⁺) and group 2 metal ions (Mg²⁺, Ca²⁺, Sr²⁺ and Ba²⁺) do not absorb or scatter light at 260 nm over the range of concentrations used for these experiments. Using metal chloride salts dissolved in sterile deionized water, we observed that A₂₆₀ readings remained close to zero at metal concentrations up to 1 M (Supplementary Figure 1A and 1B). Next, we assessed the effects of the metal ions on the absorbance of UV light by DNA. Linear, double-stranded E. coli chromosomal DNA was mixed with water to give an A₂₆₀ of 1.0, resulting in an approximate final concentration of 50 ng/μL. The average sizes of the fragments in this DNA preparation were 50,000 bp (Figure 1A) and the solutions were approximately 160 μM with respect to bases and phosphates. The absorbances of the solutions were measured after addition of group 1 and 2 metals at concentrations ranging from 1 mM to 500 mM.

Fig. 1.

DNA is not sedimented efficiently by simple addition of large amounts of group 1 or 2 metal chlorides to aqueous solutions. (A) Initial experiments utilized purified linear fragments of E. coli chromosomal DNA with approximate average sizes of 40,000 bp (lane 2). (B) The A₂₆₀ values of solutions containing DNA plus metal chloride are shown before and after centrifugation. After mixing, solutions to be spun were centrifuged at 21,000g for 10 min at RT and the upper half transferred to a new tube for measurement of absorbance. Error bars indicate standard deviations.

Absorbance of the DNA was reduced modestly by all metals tested (Supplementary Figure 2). A saturation effect was observed, whereby absorbances were reduced at 1 mM but then plateaued and did not decrease further as concentrations were increased up to 500 mM. This phenomenon was examined in more detail by mixing DNA with different concentrations of the group 2 metal calcium and performing absorbance scans from 225 to 350 nm. Association with Ca²⁺ ions reduced the maximum absorbance at 260 nm similarly at all concentrations tested (Supplementary Figure 3). The scans also revealed that the shape of the absorbance peak at 260 nm was not altered substantially by the presence of the metal ions. These results indicated that the centrifugation experiments described below, which employed metal concentrations ranging from 50-500 mM, should always include a control solution containing metal plus DNA to factor in this absorbance-lowering effect.

3.2. Group 2 metals induce precipitation of DNAs at high pH

The ability of metals to sediment DNA in aqueous solution was tested by measuring the A₂₆₀ of metal+DNA solutions before and after centrifugation at 21,000g for 10 min at RT. This resulted in modest reductions of 10-15% in A₂₆₀ values when 500 mM metal, the highest concentration tested, was used (Figure 1B). This result indicates that a small fraction of the DNA molecules became complexed with metal ions and could be sedimented upon centrifugation. The major conclusion of these experiments was that the DNA could not be precipitated efficiently by simply adding large amounts of either group 1 or group 2 metals.

The impact of increasing the pH on metal-induced DNA sedimentation was monitored by mixing DNAs and 100 mM metal ions together in Tris buffer solutions set to pH values ranging from 6 to 12, followed by centrifugation at 21,000g for 10 min. The approach used here is analogous to that described by Kejnovsky and Kypr [35]. The A₂₆₀’s of DNA solutions containing group 1 metals did not change appreciably after centrifugation regardless of pH (Figure 2A). Similar results were seen with both 100 mM and 500 mM metal solutions. By contrast, all group 2 metal solutions displayed strong reductions in the A₂₆₀’s of the supernatants when the pH was raised from 6 to 12 (Figure 2B). These results differ from those of the previous study [35], that analyzed sedimentation of DNA in the presence of zinc ions and Tris buffer. This difference was confirmed by mixing chromosomal DNA with 10 mM ZnCl₂ and Tris set to different pH values (Figure 2C). Strong sedimentation was observed at pH 8.0, in accord with the results of the earlier study.

Fig. 2.

Elevation of pH causes sedimentation of DNA in the presence of group 2 but not group 1 metals. Percentage change in A₂₆₀ after centrifugation at 21,000g for 10 min at RT and recovery of the supernatant (top half) from each solution for (A) group 1 and (B) group 2 metal chlorides. (C) Supernatant absorbances at pH 6 - 12 for DNA solutions containing 10 mM zinc chloride. For this experiment, the absorbance at pH 6.0 was set at 100%. All experiments employed Tris buffer to set solution pH values.

The results in Figure 2 indicated that DNAs could be sedimented strongly by all of the group 2 metals when the pH was raised above 10. However, Tris (pKa 8.1) does not buffer efficiently at such high pH values and therefore it was replaced with CAPS buffer (pKa 10.4) for subsequent experiments. Spectrophotometry tests showed that CAPS does not absorb light at 260 nm at pH values ranging from 6 to 12 (Figure 3A) and that DNA is precipitated by all group 2 metal ions in this buffer at pH values above 10, similar to the results obtained with Tris (Figure 3B).

Fig. 3.

Substitution of CAPS buffer (pKa 10.4) to improve buffering capacity at the high pH values where sedimentation occurs. (A) CAPS solutions (100 mM) set to pH 6, 8, 10 or 12 do not absorb light at 260 nm. Inset picture: chemical structure of CAPS. (B) Group 2 metals sediment DNA at pH 12 in the presence of 100 mM CAPS, similar to previous results with Tris. (C) Experiment combining CaCl₂ with CAPS demonstrates that sedimentation occurs at both pH 11 and 12.

To explore the phenomenon further, several new experiments were performed using calcium. This group 2 metal was chosen because strontium and barium are not natural to living cells. Also, magnesium may be less useful for potential future applications since it is a cofactor for many DNA nucleases that may be present in partially purified DNA preparations. Using CAPS and CaCl₂ mixed with linear chromosomal DNA fragments, the sedimentation effect was refined and found to be strongest at pH values of 11 and 12 (Figure 3C) using 100 mM CAPS and 500 mM CaCl₂.

In order to better understand the influence of buffer and metal ion concentrations, precipitation studies were performed with varying amounts of each component. When the concentration of Ca²⁺ was varied from 50 mM to 500 mM in 100 mM CAPS buffer, a minimum of 200 mM was found to be required for strong sedimentation at pH 11 and 12 (Figure 4A). Setting the Ca²⁺ to 200 mM and varying the amount of buffer revealed that 50 to 100 mM CAPS produced the best results (Figure 4C).

Fig. 4.

Impact of varying CAPS and calcium concentrations on DNA sedimentation. (A) 100 mM CAPS combined with 50 - 500 mM CaCl₂. (B) 10 - 100 mM CAPS combined with 200 mM CaCl₂.

3.3. Impact of calcium sedimentation on DNA integrity

Potential changes in the integrity of DNA produced by incubation with Ca ions at high pH were assessed using purified bacteriophage lambda DNA. Molecules of this phage DNA are all the same size (48,502 bp), in contrast to the random fragments of chromosomal DNA with average size approximately 50,000 bp that were used in the previous experiments. Lambda DNA was mixed with 200 mM CaCl₂ and 100 mM CAPS (pH 12) and centrifuged as before. This time the pellets formed after centrifugation were resuspended in TE (10 mM Tris [pH 8.0] 1 mM EDTA) in an attempt to recover the precipitated DNA for analysis by agarose gel electrophoresis. Very little DNA could be recovered out of the pellets initially (Figure 5A). Increasing the amount of the metal chelator EDTA in the resuspension solutions from 1 mM to 10 and 50 mM improved recovery, and heating the resuspended pellets at 65°C also strongly enhanced recovery (Figure 5B). These experiments demonstrated that the treatment did not produce gross degradation of the DNA.

Fig. 5.

Recovery of DNA from CaCl₂+DNA pellets formed at pH 11 and 12. (A) Bacteriophage lambda DNA (48.5 kb) could be recovered from pH 12 sediments by incubation with 10 and 50 mM EDTA. (B) Use of 10 mM EDTA and heating at 42°C or 65°C improved DNA recovery. (C) Large DNA fragments (0.5 - 48 kb) were strongly sedimented at pH 11 and 12. No DNA could be recovered from the supernatants. (D) Recovery of DNA fragments smaller than 500 bp was less efficient than with larger fragments.

3.4. Impact of size on DNA sedimentation efficiency at high pH

Using TE containing 10 mM EDTA along with a 65°C heating step to resuspend pellets, the possibility that the efficiency of Ca²⁺-induced sedimentation at high pH might exhibit size-specificity was tested. A high molecular weight DNA ladder (New England Biolabs 1 kb Extend DNA ladder) was precipitated at pH 11 and 12 and the pellets and supernatants were examined by gel electrophoresis. All DNA ladder fragments (ranging from 500 bp to 48,000 bp) were found in the pellet (Figure 5C). To recover DNA remaining in the supernatants, three volumes of cold ethanol was added to them, followed by mixing and centrifugation at 21,000g for 15 min and resuspension of the potential DNA pellets in TE. No DNA could be detected after this procedure (Figure 5C, lanes labelled Supernatant), indicating that essentially all DNA was sedimented in the initial centrifugation step. Sedimentation of smaller DNAs was evaluated using a low molecular weight ladder (New England Biolabs 1 Kb Plus). Yields of fragments smaller than 500 bp were modestly reduced in the pellets (Figure 5D).

3.5. DNA becomes partially single-stranded at high pH, producing structures that are sedimented more efficiently than double-stranded DNA

The precise mechanism by which group 2 metals cause precipitation of DNAs at pH 11 and 12 is unknown. Previous work in our lab suggested that single-stranded DNAs (ssDNAs), but not double-stranded DNAs (dsDNAs), bind to nanoclay particles via charge neutralization and metal cation bridging processes very efficiently [33,34]. Those experiments also demonstrated that group 2 divalent metals such as calcium and magnesium could increase DNA binding to clays more strongly than group 1 metals. Double-stranded DNA becomes denatured and partially single-stranded between pH 11 and 12, the pH range where sedimentation is observed in the current study, due to disruption of hydrogen bonding and base-stacking interactions by hydroxide ions [36,37]. It is possible that Ca²⁺ ions may be joining single-stranded regions of pH-denatured DNA molecules together via charge neutralization and subsequent cation bridging effects to generate insoluble aggregates.

To test this hypothesis, we first measured the level of denaturation of chromosomal DNA at high pH. Complete conversion of dsDNA to ssDNA causes the absorbance of UV light at 260 nm to increase by 35%, according to published extinction coefficients for each form of DNA [1]. A₂₆₀ readings were obtained of dsDNA before and after mixing it with the chemical denaturant formamide (50%) and heating at 95°C for 3 min to induce denaturation. Absorbance increased by 34.5%, in agreement with the theoretical prediction and indicating nearly complete denaturation (Figure 6A). Next, absorbance of the DNA was measured in (i) aqueous solution set to pH 12.0 using sodium hydroxide (no CAPS buffer and no Ca²⁺) and (ii) 100 mM CAPS buffer set to pH 8 versus pH 12, also without Ca²⁺). Raising the pH induced a 14.8% increase in absorbance without CAPS and a 10.1% increase with CAPS present (Figure 6A). If ssDNA formation is proportional to increased absorbance, then these results suggest that 42.3% (14.8/35) and 28.9% (10.1/35) of the DNA became single-stranded at pH 12. Thus, a large proportion of the DNA is converted to single strands at the higher pH.

Fig. 6.

Evidence that ssDNA formation plays an important role in calcium-induced sedimentation at high pH. (A) Increased absorbance at high pH, with or without CAPS present, indicated that the DNA became partially denatured. (B - D) Pure single-stranded M13mp18 DNA was strongly sedimented in the presence of 100 mM CaCl₂, without raising the pH. By contrast, M13mp18 dsDNA was only modestly sedimented. (E) Conversion of dsDNA to ssDNA by heating at 95°C for 3 min in the presence of 100 mM CaCl₂ caused strong DNA precipitation.

To test the model further, we examined the sedimentation of different forms of bacteriophage M13 DNA in the presence of calcium ions. This nucleic acid, which is 7,249 bp long, can be obtained commercially as either pure double-stranded or pure single-stranded circular DNA (Figure 6B). The different forms were mixed separately with 100 mM CaCl₂ in water without pH adjustment and the A₂₆₀ readings were recorded before and after centrifugation at 21,000g (performed as done for the previous centrifugation binding assays). The A₂₆₀ of the ssDNA solution was reduced by 97%, while that of the dsDNA was only reduced by 51% (Figure 6C and 6D). This is consistent with the idea that the ssDNAs aggregated strongly in the presence of Ca²⁺ ions and became insoluble, while the dsDNAs aggregated much less efficiently.

As a final test, we mixed linear chromosomal dsDNA (the same DNA used for experiments shown in Figures 1–4) with 100 mM CaCl₂ and centrifuged at 21,000g either (a) without heating or (b) immediately after heating at 95°C for 3 minutes to convert most of the dsDNA to ssDNA. Without heating, only 14.3% of the dsDNA was sedimented by calcium (Figure 6E). By contrast, conversion to ssDNA by heat denaturation in the presence of the Ca²⁺ ions followed by centrifugation caused a 99% reduction in the A₂₆₀ of the supernatant.

3.6. A model involving precipitation of DNA via formation of insoluble metal hydroxides is not supported

The aggregate results in Figure 6 support a model involving denaturation to form ssDNA regions, followed by charge neutralization and calcium bridging (ssDNA-Ca²⁺-ssDNA) for the aggregation and precipitation of DNAs at high pH. However, it is well established that many divalent metals such as calcium and magnesium form weakly soluble metal hydroxides in the form of M(OH)₂ at high pH, which could potentially affect the solubility of DNA associated with them. Such a model was suggested for precipitation of DNA by zinc [35]. To investigate this possibility, we calculated the relative proportions of the three major species of calcium (Ca²⁺, CaOH⁺ and Ca(OH)₂) at different pH values in 0.1 M solutions using published dissociation constants for Ca(OH)₂ and CaOH⁺ (Suppl. Fig. 4). As shown in Figure 7A, almost all of the metal is in the form of Ca²⁺ ions below pH 10.0, but it is increasingly converted to Ca(OH)₂ as the pH is increased to 12.0. Only a small proportion of the metal accumulates as the monohydroxide form (Figure 7A).

Fig. 7.

Assessment of the role of metal hydroxide formation in precipitation of DNA at high pH. (A) Weakly soluble Ca(OH)₂ forms when the pH is raised above 10 in 100 mM CaCl₂ solutions (see supplementary Figure S4). (B) Precipitation-induced light scattering at 600 nm was observed in 100 mM MgCl₂ and CaCl₂ solutions at pH 12 due to accumulation of metal hydroxides. Note that there was no DNA or CAPS present in the mixtures. (C and D) Modest light scattering was observed at pH 12 in 10 mM MgCl₂ solutions but not CaCl₂ solutions. (E) Metal hydroxide-induced light scattering was not detected in 10 mM CaCl₂ solutions at pH 12, but mixing with DNA and centrifugation produced strong sedimentation. By contrast, MgCl₂ solutions exhibited strong scattering but could only precipitate a small fraction of DNA.

Formation of a precipitate induces turbidity in solutions that can be measured as an increase in light scattering, and we therefore used this technique to assess the role of solubility. Scattering of light by 100 mM and 10 mM MgCl₂ and CaCl₂ solutions, without DNA or CAPS buffer, was measured at 600 nm. These group 2 metals exhibited similar sedimentation profiles in our previous experiments (Figures 2 and 3). No scattering was detected at pH 8.0 for any of the solutions (not shown). However, both of the 100 mM solutions showed increased OD₆₀₀ readings at pH 12.0, with Mg solutions visibly cloudy and showing the highest readings (3.1 vs 0.08; Figure 7B). The elevated OD₆₀₀ for magnesium indicates that this metal was more strongly precipitated at the higher pH and is consistent with the low Ksp for Mg(OH)₂ of 5.6 x 10⁻¹² versus that of Ca(OH)₂, which is 5 x 10⁻⁶ [38]. At the lower concentration of 10 mM, the OD₆₀₀ was 0.19 for MgCl₂ and was essentially undetectable in the CaCl₂ solution (Figure 7C and 7D). Next, chromosomal DNA was mixed with 10 mM of each metal chloride, without CAPS buffer again, and the pH left unchanged or raised to 12.0 before centrifugation at 21,000g. Although the 10 mM CaCl₂ solution showed no detectable precipitate-induced light scattering (Figure 7D), the metal was still capable of sedimenting 62% of the DNA (Figure 7E). Furthermore, 10 mM Mg solutions showed strong Mg(OH)₂ precipitation at pH 12.0, but when combined with DNA and centrifuged, only 23% of the DNA was sedimented (Figure 7D). These results demonstrate a lack of correlation between metal hydroxide precipitation and DNA sedimentation, which is inconsistent with the insolubility model.

Discussion

In the current study we have assessed the abilities of group 1 and 2 metals to precipitate DNA out of aqueous solutions. Initial tests revealed that simply adding large amounts of each metal chloride (up to 1 M), followed by centrifugation at high speed, was not sufficient to sediment linear dsDNAs efficiently. When the pH of metal+DNA solutions was increased using either Tris or CAPS buffer, however, the DNAs were strongly sedimented by the group 2 metals Mg, Ca, Sr and Ba. The monovalent alkali metals Li, Na, K, Rb and Cs were ineffective at all pH values, possibly due to a reduced ability to neutralize surface charges and participate in cation bridging interactions (described below) and because they are primarily outer sphere ions that do not have high affinity for DNA [39–43].

The pH profiles of the group 2 metals were similar to each other, displaying strong sedimentation at pH 11-12 in the presence of 100 mM and 500 mM metal chlorides. The profiles were distinctly different from that of zinc, which caused strongest sedimentation at pH 8. The result with zinc is in accord with the results of a past study of zinc-induced precipitation of plasmid DNA that also observed precipitation between pH 7.5 and 9 [35]. Thus, precipitation with zinc doesn’t require conversion of the dsDNA to ssDNA at pH 11-12. A previous study by Nejdl et al. [44] indicated that association with zinc can lead to local unwinding of DNA, forming regions that are partially single-stranded. Such regions could potentially associate with other Zn²⁺-bound ssDNA segments, leading to aggregation and sedimentation. Additional support for zinc binding distinctly differently comes from studies showing differences in the interactions of calcium and zinc ions with DNA. For example, group 2 metals bind primarily to phosphate groups, usually indirectly through water molecules via outer sphere interactions. By contrast, zinc and other transition metals show association with both phosphate groups and bases, they are more likely to coordinate directly with the nucleic acid rather than through water molecules, and they may participate in interstrand crosslinking [39,40,45]. In addition, transition metals are more likely to induce detectable changes in DNA helix structure and in the level of compaction of the DNA [46,47]. Finally, zinc has also been shown to bind more strongly to nucleoside mono- and triphosphates in in vitro studies [48].

Calcium was used to characterize the sedimentation phenomenon in more detail. Titration experiments using different amounts of calcium and CAPS buffer suggested that the concentration of the metal was most important, with 200 mM being sufficient to achieve strong sedimentation at pH 11 and 12. Recovery of DNA out of the precipitates followed by analysis via gel electrophoresis indicated that the DNA strands were not broken by the treatment and that DNAs that were 0.5 kb or larger were sedimented most efficiently.

Two separate phenomena are known to occur in solutions raised to the critical pH range of 11-12: the increased relative number of hydroxide ions causes dsDNA to denature to form regions that are single-stranded, and group 2 metal ions form weakly soluble dihydroxides. Each of these changes suggested a different mechanism for DNA aggregation and sedimentation. We confirmed that the dsDNA used for most experiments in the study became partially denatured to form ssDNA at high pH. Subsequent experiments demonstrated that simply adding CaCl₂ to pure single-stranded M13 DNA, without changing the pH, resulted in sedimentation of 97% of the DNA after centrifugation. By contrast, a much smaller fraction of double-stranded M13 DNA was sedimented under the same conditions. In addition, heating dsDNA to 95°C to convert it to ssDNA while in the presence of calcium ions caused 99% of the DNA to sediment after centrifugation. This series of experiments also revealed that the buffer was unnecessary; simply adjusting the pH to 12 produced effects that were similar to those seen when setting the pH with CAPS buffer.

The findings described above did not exclude potential contributions due to the increasing insolubility of metal hydroxides at high pH. To assess this contribution, we compared metal hydroxide precipitate-induced turbidity (measured via light scattering assays) versus DNA sedimentation efficiency for both MgCl₂ and CaCl₂ at pH 12. At 10 mM, CaCl₂ produced no detectable Ca(OH)₂ precipitation, but caused strong sedimentation of the DNA (62%). MgCl₂ solutions exhibited strong magnesium hydroxide precipitate formation but did not sediment DNA efficiently. Thus, these data indicate that there is no correlation between the level of insoluble metal hydroxides in solution and the efficiency of DNA sedimentation.

The results of these experiments are most compatible with a model in which dsDNAs become partially denatured at high pH, creating localized segments of single-stranded DNA. These regions may manifest as frayed single-stranded ends and/or as internal regions of ssDNA [49]. The negatively charged phosphates and possibly the bases within the ssDNA become bound by divalent metal ions, reducing the surface charge on each strand. This neutralization of negative charges, combined with the overall charge screening effect caused by having dissolved M²⁺ ions present throughout the solution, eliminates repulsive forces between the DNAs and leads to aggregation into large complexes (Figure 8). These colloidal aggregates are unstable in solution and subsequent centrifugation causes them to form a pellet on the bottom of a tube. Within the precipitates the single-stranded regions of DNA are likely held together tightly through cation bridging (ionic crosslinking) effects. Such a mechanism would be consistent with past studies of the bridging interactions of divalent metals with nanoclays and other molecules [33,34,50,51]. We note that additional changes in molecular interactions may also be involved. For example, there may be changes in the interactions of the metal ions with bases and phosphates of DNA at high pH, potentially caused by deprotonaton of base nitrogens (39,40,52).

Fig. 8.

Model for sedimentation of DNA by group 2 metals at high pH. Linear double-stranded DNAs become partially denatured at high pH and association of ssDNAs with metal ions neutralizes charges and reduces the repulsive forces keeping strands apart, leading to aggregation. Multi-strand complexes are formed that can no longer remain solvated. Preferential fraying of the ends due to decreased base-stacking is suggested in the figure, but ssDNA regions might also form internally [49].

Highlights (England et al.).

• Divalent metal precipitation of DNA is pH-dependent

• Monovalent metals are poor inducers of DNA sedimentation

• Group 1 and group 2 metals reduce absorbance of DNA at 260 nm

• Metal-induced DNA sedimentation involves charge neutralization and cation bridging

• Group 2 metal sedimentation of DNA does not involve insoluble metal hydroxides

Abbreviations:

nt

nucleotides

bp

base-pairs

dsDNA

double-stranded DNA

ssDNA

single-stranded DNA