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. 2023 Jan 30;15(5):6274–6282. doi: 10.1021/acsami.2c15626

Secondary Structure of DNA Aptamer Influences Biomimetic Mineralization of Calcium Carbonate

Stephanie Colon 1, Alexander Paige 1, Rylie Bolarinho 1, Hailey Young 1, Aren E Gerdon 1,*
PMCID: PMC9924263  PMID: 36715729

Abstract

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Calcium materials, such as calcium carbonate, are produced in natural and industrial settings that range from oceanic to biomedical. An array of biological and biomimetic template molecules have been employed in controlling and understanding the mineralization reaction but have largely focused on small molecule additives or disordered polyelectrolytes. DNA aptamers are synthetic and programmable biomolecules with polyelectrolyte characteristics but with predictable and controllable secondary structure akin to native extracellular moieties. This work demonstrates for the first time the influence of DNA aptamers with known G-quadruplex structures on calcium carbonate mineralization. Aptamers demonstrate kinetic inhibition of mineral formation, sequence and pH-dependent uptake into the mineral, and morphological control of the primarily calcite material in controlled solution conditions. In reactions initiated from the complex matrix of ocean water, DNA aptamers demonstrated enhancement of mineralization kinetics and resulting amorphous material. This work provides new biomimetic tools to employ in controlled mineralization and demonstrates the influence that template secondary structure can have in material formation.

Keywords: mineralization, minerals, DNA, aptamers, biopolymers, ocean

Introduction

Calcium minerals, including calcium carbonate, calcium phosphate, and calcium oxalate are ubiquitously employed in natural, medical, and industrial settings due to the structural support and protection they provide.1−3 Calcium carbonate, in particular, has been widely studied due to the ease of chemical reaction and important relevance for numerous organisms found in nature, particularly ocean environments. Climate change and ocean acidification has brought the issue of calcium carbonate mineralization and demineralization to widespread attention.4−7 Recent studies have focused on mineralization models8,9 and biochemical pathways in corals, sea urchins, and other ocean organisms.4,10−14 Furthering our understanding of biological calcium carbonate mineralization could be beneficial in ameliorating mineral loss in ocean habitats. Additional emphasis has been placed on developing and using biomimetic templates to control mineralization in hopes of gaining new insight into mineralization models and producing new tools for use in medical, industrial, or environmental applications.1,3,15−18 In natural settings, chemical conditions such as pH, ionic strength, and ionic solubility interface with a host of extracellular proteins, such as amelogenin19,20 in enamel or asprich proteins5 in mollusks, that direct and control mineral formation. Biomimetic approaches to controlled mineralization have therefore focused on ion additives such as Mg2+,21 small molecules such as citrate or amino acids,22,23 and macromolecular polymers such as polyaspartic acid, polypeptides, cellulose derivatives, and more.15,18,24−26 These large polyelectrolytes are readily available, easy to work with, and do mimic the disordered state of naturally occurring proteins like amelogenin or asprich proteins. On the other hand, they are lacking in site-specific addressability, programmability, and tailorable secondary structure. Ruiz-Agudo and co-authors27 recently addressed this issue by using a designer ubiquitin protein with known secondary structure and with site-specific phosphorylation modifications to control crystallization. DNA and RNA aptamers are an additional class of biomimetic molecules that have been widely used in biosensor design and drug delivery,28−31 provide known and customizable secondary structure, convenient molecular modification, and addressability, and demonstrate control of mineralization.

DNA has been previously studied in calcium carbonate mineralization by Sommerdijk et al32 and by Lukeman et al33 with success, demonstrating mineralization inhibition and morphological control. DNA has more recently been used in relation to a number of other materials including noble metal nanoparticles,34,35 zinc oxide,36 silica,37−40 manganese pyrophosphate,41 calcium phosphate,41−46 and other metal cation phosphates.47−49 We have previously reported the SELEX identification of DNA aptamers with an ability to modulate calcium phosphate mineralization and to specifically label hydroxyapatite over amorphous calcium phosphate.50,51 The result of these two different SELEX protocols was a host of DNA sequences with a high prevalence of G nucleotides.50,51 Aptamer sequences from one SELEX method contained 43 ± 9% G nucleotides, and 84% of the sequences were capable of forming a G-quadruplex secondary structures.50 Aptamer G was identified from this pool as likely forming a G-quadruplex structure, according to computational methods and by showing an enhanced stability in the presence of potassium.50,52 A similar selected aptamer showed evidence of G-quadruplex formation in circular dichroism studies.51 Aptamer G– is the identical sequence to aptamer G but with four G nucleotides replaced with C nucleotides to disrupt the formation of the G-quadruplex. Both aptamer sequences showed destabilization in the presence of calcium cations, with aptamer G being sensitive to lower concentrations of calcium cation than aptamer G–, indicating a stronger interaction with the cation.52 The secondary structures present in these aptamers were demonstrated to play a role in calcium phosphate mineral formation. We hypothesize that DNA aptamers selected for calcium phosphate mineralization could be functional biomimetics in different buffer systems, including ocean water, with different mineralization anions, such as carbonate, and at different buffer pH values. Here, we report the stability of selected DNA aptamers in carbonate buffer at pH 7.4 and at pH 10.0. The aptamers were then employed in calcium carbonate mineralization experiments to explore their influence on mineralization kinetics, mineral crystallinity, and mineral morphology. No selected DNA aptamer, nor G-quadruplex secondary structure, to our knowledge, has ever been explored as a modulator of calcium carbonate mineralization at pH 7.4 or pH 10.0. Furthermore, biomimetic DNA aptamer influence on mineralization in ocean water has not previously been reported, making this work the first of its kind. This demonstrates an expanded use of DNA aptamers in biomimetic mineralization, adds mineralization templates with customizable secondary structure that are readily synthesized and functionalized, and sheds new light on biomimetic calcium carbonate mineralization.

Materials and Methods

Chemicals

Sodium carbonate (>99.5%), calcium chloride dihydrate (>99.5%), and sodium chloride (>99.5%) were purchased from Sigma Aldrich. Bogen’s Universal Indicator was purchased from Carolina Biological. Gel Star nucleic acid gel stain 10,000× was purchased from Lonza Rockland Inc. DNA strands were purchased from Integrated DNA Technologies and were reconstituted in 10 mM trizma hydrochloride buffer pH 7.4 before use. Synthesis and purity were confirmed by the vendor using mass spectrometry. UV absorbance was used to confirm concentration of DNA. The following ssDNA aptamers were used for analysis: (aptamer G) 5′-CAG GTG GGC GCG CTG TCG TGG GTG CTC GGG TGC GGT TGG G–3′; (aptamer G−) 5′-CAG GTG CGC GCG CTG TCG TGC GTG CTC GCG TGC GGT TGC G– 3′. All solutions were filtered, and fresh mineralization solutions were prepared each day.

Aptamer Secondary Structure Stability

Aptamer secondary structure stability dependence on pH in carbonate buffer was determined through titration with sodium hydroxide and measurement of fluorescence and circular dichroism (CD) spectra. DNA aptamers were prepared at 100 nM concentration for fluorescence or 10 μM concentration for CD in 2 mM pH 7.4 carbonate buffer. Fluorescence was measured (Horiba FluoroMax-3) with excitation at 493 nm and emission scanned from 500 to 600 nm with 2 nm slit widths after the addition of 1× Gel Star nucleic acid dye. Aliquots of 0.1 M NaOH were added, and fluorescence was remeasured. A blank sample with no DNA was titrated while measuring the pH with each aliquot addition to match pH with fluorescence intensity. CD was measured (Applied Photophysics Chirascan) in a 1 mm quartz cuvette (Hellma Analytics) from 340 to 210 nm. All spectra were blank subtracted and smoothed using a low-pass FFT filter with cutoff at 0.05 Hz in OriginLab Software. Sample pH was adjusted with aliquots of 0.1 M NaOH as above.

Mineralization Kinetics Measurement with Absorbance

Sodium carbonate and calcium chloride stock solutions were prepared daily and filtered. Mineralization samples were prepared by diluting sodium carbonate to either 10.0 mM at pH 7.4 or 2.0 mM at pH 10.0, diluting sodium chloride to either 25.0 mM at pH 7.4 or 10.0 mM at pH 10.0, adding a universal indicator at a 1:200 dilution, and adding a DNA aptamer when appropriate at either 25 or 500 nM. DNA aptamers were annealed to 95 °C for 2 min using a BioRad T100 thermocycler in sodium carbonate buffer at the appropriate pH prior to dilution. This solution was vortexed and added to a 96-well plate with a minimum of three replicates. The experiment was initiated by the addition of calcium chloride to a final concentration of either 10.0 mM at pH 7.4 or 2.0 mM at pH 10.0. Absorbance was measured with a SpectraMax 190 plate reader maintaining 23 °C and either 420 nm for pH 7.4 or 520 nm for pH 10.0, collecting a data point every 10 min for 4 h. Student’s t-tests were used to demonstrate statistical differences between samples of interest. The standard error is reported as the standard deviation (s) divided by the square root of n measurements.

DNA Aptamer Affinity to Calcium Carbonate

Calcium carbonate was prepared by stirring a solution containing 5.0 mM calcium chloride and 5.0 mM sodium carbonate at pH 10.0 for 24 h. The precipitate was collected via vacuum filtration and washed with DI water. After drying in air, the sample was confirmed to be calcite using a Thermo Nicolet iS10 FT-IR with ATR. Aptamer affinity was analyzed by combining the aptamer with calcium carbonate in the appropriate buffer for 30 min. The precipitate was collected via centrifugation, and the supernatant was analyzed by absorbance at 260 and 400 nm with an Agilent 8453 spectrophotometer. Absorbance at 400 nm was subtracted from absorbance at 260 nm to account for baseline variations. Sodium carbonate buffers were used at 2.0 mM with 10 mM sodium chloride and pH 10.0 and at 10.0 mM with 25 mM sodium chloride and pH 7.4. DNA aptamers were annealed to 95 °C in the respective buffer prior to use and were used at concentrations ranging from 25 to 2500 nM. Calcium carbonate was added to each sample to a final concentration of 1.67 mg/mL. Controls were included with a DNA aptamer but no calcium carbonate.

DNA Aptamer Uptake during Mineralization

Mineralization experiments were prepared as above at both pH 10.0 and pH 7.4 and with DNA aptamers at 25 and 500 nM. Samples were prepared at 500 μL of final volume each in sextuplet and were mixed by vortexing and rotation. Duplicate samples were removed after 10, 20, 30, and 60 min, 4 h, and 24 h and centrifuged to pellet the mineral. The supernatant was analyzed directly with absorbance at 260 and 400 nm as described above.

Analysis of Mineral Crystallinity

Mineralization experiments were prepared as above at both pH 10.0 and pH 7.4 and with DNA aptamers at 25 and 500 nM. Samples were prepared at larger 10.0 mL volumes in order to collect enough mineral for analysis by FT-IR. Reactions proceeded for 4 or 24 h, and mineral was collected by centrifugation at 10,000 rpm for 5 min with DI water rinsing. The mineral was suspended in ethanol, cast onto a glass microscope slide, and dried overnight. The sample was collected and analyzed on a Thermo Nicolet iS10 FT-IR with ATR.

Analysis of Mineral Morphology

Mineralization experiments were prepared as above. After 0.5, 4, or 24 h, the reacting sample was vortexed and 5 μL of solution was added to a Cu Formvar TEM grid (Ted Pella) for 2 min. The solution was removed with filter paper and dried in air before analysis with a JEOL 1200EX-80 KV instrument.

Mineralization Analysis in Ocean Water

Water was collected from the Cape Cod Bay, specifically from Grays Beach Park and Duxbury Beach Reservation on November 5 and 6, 2022. Initial pH measurements were made, and a 10 mL portion was titrated to pH 10.0 with 1.0 M NaOH to determine the amount of base required to achieve that pH and to estimate the concentration of carbonate present. Atomic emission spectroscopy was used with standard addition of calcium to determine the concentration of calcium in the ocean water sample. Briefly, 50 μL of ocean water, 1 mL of 1.0 M nitric acid, and various additions of 1.74 mM calcium chloride were added to 10 mL volumetric flasks and analyzed on an Agilent MP4100 ICP-AES instrument. For mineralization, samples were filtered and combined with a universal indicator to a 1:200 dilution, DNA aptamer to 500 nM, and in some cases, additional carbonate buffer to 4 mM. In all cases, whole ocean water comprised 90% or more of the reaction volume. Mineralization experiments were initiated with NaOH to bring the solution pH to 10.0 and absorbance measured as above. For TEM analysis, samples were prepared in the same way, but without the universal indicator, and measured as above.

Results and Discussion

DNA aptamers used in this study were previously identified in a Precipitation SELEX50 experiment with calcium phosphate mineralization, but have not been previously reported with carbonate anions found in geologic and oceanic calcium carbonate mineralization. The two aptamers were therefore analyzed for their stability in carbonate buffer at pH 7.4 and pH 10.0. A pH 7.4 carbonate buffer was used with respect to human physiology and in analogy to pH 7.4 tris and phosphate buffers used in previous experiments.50,52 A pH 10.0 carbonate buffer was used in comparison to a wide range of previously published works on calcium carbonate mineralization at elevated pH.23,27,32,53 A pH of 10.0 is also relevant to ocean-dwelling organisms that control mineralization of an exoskeleton by controlling the pH within vesicles that produce and deposit mineral for a growing shell.4 Given the two pKa values54 of carbonate, 6.35 and 10.32, the anion is highly deprotonated at pH 10.0 and deprotonates fully in precipitation with calcium. Available algorithms, such as Mfold55 and QGRS Mapper56 (Supporting Information, Figure S1), give insight into possible aptamer folding and provide a starting point for discussion but do not account for changes in pH. Aptamer stability was therefore assessed with circular dichroism measurements and with fluorescence of a Gel Star dye with and without aptamer G and aptamer G–, beginning at pH 7.4. This dye exhibits a higher fluorescence intensity in the presence of double-stranded DNA or single-stranded DNA with significant secondary structure or internal hybridization. Upon destabilization of the DNA structure, the dye unbinds, leading to lower fluorescence intensity. Figure 1A shows that at pH 7.4, samples with DNA aptamer exhibit strong fluorescence compared to the control with no DNA. As the samples are titrated with hydroxide, fluorescence decreases for both aptamers, though at significantly different rates. At pH 10.0, aptamer G had lost 22.1% fluorescence intensity compared to 11.3% intensity loss for aptamer G–. Gel Star dye is recommended for use within a pH range of 7–8.5, according to the manufacturer (Lonza). Therefore, the results at a higher pH may not be relied on quantitatively, but the observed differences in fluorescence between the two DNA aptamers remains qualitatively. This result does not identify a specific secondary structure for these aptamers, but does suggest that aptamer G is more affected by the increase in pH than aptamer G–. Circular dichroism (CD) experiments were similarly conducted at a range of pH values. Figure 1B shows significant differences between aptamer G and aptamer G– secondary structures at pH 7.4, with aptamer G spectra matching a typical hybrid or 3 + 1 G-quadruplex structure57 and aptamer G– spectra matching a typical hairpin structure.58 Increasing the pH to near 10 produced no significant change in the aptamer G– spectra, suggesting that it maintains its folded structure at this pH. Aptamer G shows a modest shift in CD spectra at 260 nm, indicating a greater influence of pH on the G-quadruplex structure compared to the hairpin structure. The Watson–Crick-type base pairing found in aptamer G– is likely more intact than the G-quadruplex structure in aptamer G at high pH. This is generally in agreement with previous studies52 showing that aptamer G was destabilized to a greater extent than aptamer G– in the presence of calcium cation, indicating a stronger interaction between aptamer G and calcium cation. Additional CD spectra of both aptamers at a range of pH values and in the presence of calcium or potassium support this conclusion (Supporting Information, Figure S2).

Figure 1.

Figure 1

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Analysis of the DNA aptamer structure and stability at different pH values. (A) Fluorescence measurements of Gel Star dye labeled aptamer G and aptamer G– in carbonate buffer with pH from 7.4 to greater than 10.5; (B) circular dichroism measurements of aptamer G and aptamer G– in carbonate buffer at pH 7.4 and pH 9.9.

The influence of the two aptamers on mineralization kinetics was analyzed using previously described absorbance measurements.50,52 Briefly, bicarbonate anions deprotonate to carbonate during the mineralization process, decreasing the solution pH. This pH change can be measured via the change in absorbance of a pH indicator at low concentrations in solution. Change in absorbance, therefore, correlates to change in pH and mineral formation. Mineralization solution conditions were chosen based on calculated supersaturation conditions and experimentation at a range of ion concentrations (data not shown) to provide the best analytical sensitivity in the assay. At excessively high supersaturation conditions, mineralization would occur quickly regardless of any organic template, and at extremely low supersaturation conditions, absorbance changes were too small for measurement. The final ion concentrations used in the experiments reported here are within ranges relevant to biomedical and ocean chemistry conditions. At pH 7.4, in the absence of an aptamer, mineralization is measurable after approximately 30 min and plateaus near 240 min (Figure 2A,B). When low 25 nM concentrations of each aptamer are included, mineralization appears to be slightly delayed, particularly at the 100 min timepoint, but shows no statistical difference from the control (P > 0.05) at 200 min. When higher 500 nM concentrations of the aptamer are used, inhibition of mineralization is observed at early timepoints, suggesting that aptamers may be influencing nucleation. Aptamer G continues to delay nucleation past 100 min and shows a significant difference from the control (P < 0.0005) at 200 min. Aptamer G– shows reduced mineralization such that it is not statistically different from the control (P > 0.05). Reduced and delayed mineralization may be due to aptamers interacting with calcium cations or with calcium carbonate prenucleation clusters, stabilizing those clusters, inhibiting bulk nucleation and growth. At this pH, aptamers are expected to maintain their secondary structures, which may play a role in their ability to interact with calcium cations and forming mineral.50,52 In a previous work with calcium phosphate mineralization,52 both aptamers also demonstrated concentration-dependent influence on kinetics but demonstrated enhancement and inhibition at different stages of mineralization. Only inhibition was observed here (Figure 2), which might suggest that free phosphate with a larger negative charge is more likely than free carbonate with a smaller negative charge to mineralize with calcium that is already potentially bound in a DNA aptamer-folded secondary structure.

Figure 2.

Figure 2

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Mineralization kinetics with and without DNA aptamers G and G– at two concentrations, measured by change in absorbance of a pH indicator. Error bars represent standard error from replicate measurements. In (A) and (B), 10.0 mM calcium, 10.0 mM carbonate, 25.0 mM sodium chloride, and pH 7.4 were used. In (C) and (D), 2.0 mM calcium, 2.0 mM carbonate, 10.0 mM sodium chloride, and pH 10.0 were used. *(P > 0.05), **(P < 0.0005).

At pH 10.0, the control with no aptamer shows faster initial kinetics and an earlier plateau near 150 min compared to the pH 7.4 experiments (Figure 2C,D). This is expected due to a higher supersaturation ratio in solution with respect to calcium carbonate. Unexpectedly, both aptamers at both higher and lower concentrations show significant mineralization inhibition, although it is possible that some amount of mineral is forming due to the small increases in absorbance observed from 100 to 200 min. Both aptamers were shown to have some perturbation of secondary structure at this pH (Figure 1) and may be interacting with calcium cations or calcium carbonate prenucleation clusters differently than at pH 7.4. The interaction appears to be strong due to the strong mineralization inhibition.

Further exploring the interaction of the aptamer with the forming mineral, two additional experiments were conducted. The first involved calcium carbonate that was prepared independently at high supersaturation ratios, collected, washed in DI water, and analyzed by FT-IR spectroscopy. This mineral was used with DNA aptamers in binding experiments to determine if the two aptamers interact with preformed mineral with a different affinity. Neither aptamer showed significant affinity to this preformed mineral (Supporting Information, Figure S3) at either pH, suggesting that the aptamers may be highly involved in the nucleation process but less so in interacting with or directing the growth of larger mineral particles. The second experiment measured aptamer uptake by the forming mineral over a 24 h period by measuring the amount of aptamer remaining in the supernatant once the mineral was removed by centrifugation. Figure 3 shows that at both pH 7.4 and 10.0, a greater percent of aptamer G was taken up compared to aptamer G–. Uptake of aptamer G approached 100% at pH 7.4 and followed a linear trend with time. Far less aptamer G– was taken up by the mineral at pH 7.4 and appeared non-linear, reaching a plateau at later time points. This appears to agree with kinetic studies where aptamer G– had less of an effect on mineralization at this pH. Greater mineralization inhibition was observed with aptamer G, indicating a stronger interaction or the interaction of more aptamer with less mineral. At pH 10.0, more aptamer G is taken up compared to aptamer G– but through a very different process than that seen at pH 7.4. The aptamer is taken up very quickly, within the first 30 min, then plateaus, and even decreases to a steady-state. It appears that a much smaller amount of aptamer is required to influence mineralization. Since the aptamer secondary structure is perturbed at this pH (Figure 1), the DNA may be in a less compact structure where multivalency interactions have a greater effect. Aptamer G is destabilized to a greater extent than aptamer G– (Figure 1) and so may be taken up at a higher rate. Similarly, aggregation of particles in solution may exclude or displace surface-bound DNA at this pH as mineral surface area decreases.

Figure 3.

Figure 3

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Rate and amount of aptamer uptake into the forming mineral over 24 h (1440 min) at pH 7.4 and pH 10.0. Regression analysis shows a strong linear correlation (R2 = 0.99) for aptamer G pH 7.4 but not for aptamer G– pH 7.4 (R2 = 0.86), which appears to plateau at a lower uptake percent. Error bars represent standard deviation of triplicate trials.

Aptamer influence on the crystallinity and morphology of the forming mineral was explored using FT-IR spectroscopy, TEM, and SEM imaging. Figure 4 shows FT-IR spectroscopy of mineral formed at pH 7.4 and pH 10.0 with and without DNA aptamer. No significant difference was observed between mineral formed at pH 7.4 and pH 10, nor between aptamer G and G–. All spectra show major peaks at 711–712, 869–870, and 1375–1390 cm–1, indicative of primarily calcite.54 Control experiments containing no DNA aptamer are also indicative of calcite, but show an additional peak at 743–745 cm–1 pointing to the presence of vaterite.54 Kinetic analysis discussed above indicates that aptamers may influence nucleation and prenucleation clusters, but when crystalline material forms, it appears to be maintained as primarily calcite under these conditions. This may be expected, given that calcite is the thermodynamically favorable form of calcium carbonate,59 but it is unlike previous results using calcium phosphate where aptamers with a G-quadruplex maintained mineral in an amorphous phase, while aptamers without a G-quadruplex appeared to promote a crystalline phase.52 One hypothesis for the control of crystallinity in the case of calcium phosphate was that aptamers with a G-quadruplex demonstrated a higher affinity for the mineral surface, which could influence the transformation of amorphous calcium phosphate into hydroxyapatite. Aptamer G and aptamer G– did not demonstrate affinity to a calcium carbonate surface (Supporting Information, Figure S3) and did not demonstrate an influence on crystallinity.

Figure 4.

Figure 4

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FT-IR spectroscopy of mineral formed after 24 h at (A) pH 7.4 and (B) pH 10.0 and with and without DNA aptamer. Major peaks marked with gray dashed lines indicate primarily calcite in all cases, except in the control with no aptamer where a small amount of vaterite may persist.

While crystallinity was consistent at different pH values and with different aptamer templates, mineral morphology showed marked differences under these conditions. Figure 5 shows TEM images of mineral produced at pH 7.4 and pH 10.0, with and without aptamer at 500 nM, and at 4 and 24 h. Differences in morphology are apparent and show an interesting impact of the DNA aptamer. The control experiment at pH 7.4 shows small ∼33 nm clusters growing into a larger ∼133 nm particle after 4 h and slightly larger 46 ± 16 nm clusters in loose networks after 24 h. When aptamer G– was present in solution at pH 7.4, a somewhat similar material is formed at 4 h with 31 ± 14 nm particles, some of which were beginning to adopt a quasi-cubic structure. After 24 h, larger structures formed with 250–500 nm features, but no distinct morphology. Aptamer G formed uniform 141 ± 8 nm spherical structures that appeared perhaps in a polymer, viscous, or hydrated network and as independent particles, correlating to a polymer-induced liquid precursor (PILP) model. After 24 h, much larger 3 ± 1 μm particles emerged with oblong or football shapes. Aptamer G, while being taken up by the mineral, does appear to influence the morphology, promoting spherical or rounded features. This matches previous results with calcium phosphate where aptamer G had a similar influence, maintaining aggregated and disordered particles.50,52

Figure 5.

Figure 5

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TEM images of mineral formed after 4 and 24 h at pH 7.4 and pH 10.0 and with and without DNA aptamer. Morphological differences are observed, showing the influence of the DNA aptamer.

At pH 10.0, a different morphology trend is observed. In the control experiment, 141 ± 43 nm aggregate particles are observed after 4 h and much larger 3.8 μm rhombohedral structures, as expected for calcite, are found at 24 h. When aptamer G– was included in the reaction, large 5 μm rhombohedral structures were observed at the 4 h timepoint, which grew to greater than 19 μm at 24 h. This is supported by SEM images taken of the same 24 h sample (Supporting Information, S3). Conversely, aptamer G continued to maintain the mineral in spherical shapes at 4 h with 111 ± 78 nm diameters and 24 h with 515 ± 217 nm diameters. This is also supported by SEM images taken of the same 24 h sample (Supporting Information, Figure S4). It is interesting to note that aptamer G influences morphology of mineralization consistently at both solution pH conditions. Mineral appears spherical or spheroid in shape with aptamer G at both pH 7.4 and pH 10.0. Aptamer G– does not exhibit this consistency of control where large rhombohedral structures were only found at pH 10.0.

A primary motivation of this work is in applying an understanding of templated mineralization gained from controlled solution experiments to more complex natural environments such as the ocean. Bulk precipitation of calcium carbonate in ocean water is generally inhibited by high ionic strength conditions, prevalence of competing divalent cations such as magnesium, and a solution pH well below the second pKa of carbonate.60,61 It has been hypothesized that ocean organisms, such as coral polyps, endocytose ocean water as a source of calcium but increase pH and carbonate concentrations in vivo to promote desired mineralization.4,7 In analogy to this, mineralization in whole ocean water was analyzed with DNA aptamers, with added sodium carbonate and with pH elevated to 10.0. Ocean water was collected from the Cape Cod Bay and was found to contain 11.0 ± 0.2 mM calcium cation, approximately 7.3 mM bicarbonate, and an initial pH of 7.87, which represents lower mineralization supersaturation conditions than those used in experiments above in Figure 2. Figure 6 shows the kinetics of mineralization when carbonate concentration in solution was increased by 4 mM and pH was raised to 10.0. A pH equilibration period occurs in the first 20 min of the experiment, followed by a slow increase in mineralization over 4 h when either aptamer G and aptamer G– were present when compared to the control. In Figure 2, both aptamers demonstrated consistent inhibition of mineralization kinetics but control of mineral morphology. In complex ocean water, the DNA aptamers appear to promote mineralization kinetics and result in a mineral that is primarily amorphous in all cases. TEM images of the resulting mineral can be found in the Supporting Information, Figure S5. Amorphous carbonate materials would be expected under these conditions due to the presence of magnesium, which has been found to play an important role in slowing conversion to crystalline material in ocean organisms.62,63 These results demonstrate the important applications of DNA aptamers and other biomimetic molecules to mineralization in natural settings and point to numerous future experiments where ocean water could be collected from various global locations at different times of the year and where ocean chemistry could be systematically altered, such as in the addition of exogenous carbonate, to further clarify significant ocean chemistry components.

Figure 6.

Figure 6

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Mineralization kinetics in ocean water with 4 mM added carbonate, adjusted to pH 10.0, and with and without DNA aptamers G and G– at 500 nM, measured by change in absorbance of a pH indicator.

Conclusions

The summary of these results demonstrates the influence of DNA aptamers and pH on the biomimetic mineralization of calcium carbonate. The aptamer G sequence, with a G-quadruplex structure, shows greater inhibition of mineralization, greater uptake into forming mineral, and more pronounced control of mineral morphology in controlled solution experiments. This correlates to previous results with calcium phosphate materials where aptamer G was able to maintain mineral in disordered aggregates rather than ordered bundles.52 DiMasi et al.64 proposed in 2007 that a fully flexible template would not be able to control mineralization, while a rigid template that aligns perfectly with crystal faces had not yet been demonstrated but that an adaptable and flexible template would be able to recapitulate the needed structure. DNA molecules in general appear to have an ability to interact with calcium cations and possibly with small prenucleation clusters, but we hypothesize that the double-stranded and hairpin structure found in aptamer G– may be too rigid to sustain modulation of mineral morphology. The G-quadruplex structure found in aptamer G, on the other hand, provides the polyanionic primary structure in what we hypothesize to be a quasi-stable, adaptable secondary structure that can conform to the growing particle, maintain contact with the particle, and modulate morphology. The generalized DNA-templated mineralization model previously proposed in relation to calcium phosphate52 stated that calcium cations interact with and destabilize the G-quadruplex structure, aptamers influence mineralization kinetics in a concentration-dependent manner, DNA aptamers with a G-quadruplex have a higher affinity for calcium mineral, and DNA aptamers have secondary structure-dependent influence on both crystallinity and morphology. This model appears to largely apply to the mineralization of calcium carbonate with two key differences: (1) aptamers showed different influence on crystallinity in calcium phosphate but did not in calcium carbonate and (2) differences in equilibrium affinity to calcium phosphate mineral is apparent between the two aptamers but was not replicated in the case of calcium carbonate; instead, the kinetic rate of uptake showed marked differences between the two aptamers and at each pH. We therefore anticipate that G-quadruplex structures, similar DNA secondary structures, or switchable aptamer structures could prove to be uniquely beneficial in templated mineralization and should be explored further. At present, a benefit of this work is the introduction of biomimetic, synthetic, molecular, and programmable tools that can modulate mineral properties at different pH values producing different morphologies. Programmability of DNA aptamers and DNA technology,28−31,51 such as placement of DNA aptamers into larger structures, placement of functional tags point-by-point in an aptamer sequence, or point-by-point mutation to modulate sequence and activity, has been widely demonstrated and should be relevant to the application of DNA aptamers to materials chemistry. We have demonstrated the utility of studying these aptamers in ocean-relevant conditions, and future work should include a full study of the influence of these aptamers in whole ocean water at various levels of acidification or where ocean water is supplemented with exogenous carbonate analogous to ocean organism systems.

Acknowledgments

The authors would like to thank the Harvard Medical School Electron Microscopy Facility for access, training, and discussion of instrumental methods.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsami.2c15626.

  • Aptamer Mfold and QGRS structures, circular dichroism spectra, aptamer affinity binding, SEM images of mineral, and TEM images of mineral produced from ocean water samples (PDF)

Author Contributions

‡ S. C. and A. P. contributed equally.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. All figures and images were created by the authors of this manuscript.

Funding for this work was provided by Emmanuel College, the National Science Foundation DMR 1306117, and the National Science Foundation DMR 1904460.

The authors declare no competing financial interest.

Supplementary Material

am2c15626_si_001.pdf (712KB, pdf)

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