1. Introduction
Recently, the DNA nanostructure has attracted a lot of attention because of its special properties, such as flexible size and shape, excellent biocompatibility, and increased possibilities for decorating precisely. Stabilized by strong hydrogen bonds, the DNA nanostructure can easily self-assemble into designed shapes, leading to its wide application in diagnosis, imaging or drug delivery [
1]. Among these nanoparticles, the DNA tetrahedron (Td), which has a high yield and rigid structure, is widely used [
2]. It has been reported that a Td can be internalized by a cell even without transfection reagent [
3], based on which scientists have designed various smart systems to deliver CpG motifs [
4], siRNA [
5] or small molecular drugs [
6], and to detect mRNA in living cells [
7]. However, there are still many problems that need to be explored for the further development of the Td, including factors influencing a Td’s assembly process and its stability during storage.
Previously published data showed that the types and concentrations of cations in the solution are the key factors affecting the stability of the DNA structure. Compared with monovalent cations, divalent cations, such as Mg
2+, have stronger interactions with DNA and a greater influence on DNA structure [
8]. There are two types of interactions between DNA and Mg
2+: electrostatic force (phosphate groups) and chemical bonds (like hydrogen bonds and coordinate bonds) [
9]. In a solvent, Mg
2+ has a coordination shell with six water molecules that can bind to the O6 of the DNA to form hydrogen bonds. Also, in some cases, coordinate water molecules are replaced by the N7 and O6 sites of the DNA guanine, forming coordination bonds between the DNA and Mg
2+. The effects of magnesium can be considered as ‘protective effects’ of DNA, not only decreasing the intramolecular repulsion in DNA but also making the DNA duplex more rigid [
10]. Importantly, it has been confirmed that the quantity of magnesium ions affects DNA structure. DNA is stabilized in the B conformation when Mg
2+ is at a modest concentration, but when the concentration is extremely high, the DNA structure exhibits a more compact form [
11].
Therefore, the influence of Mg
2+ on DNA nanostructure in applicable conditions has been studied. It was reported that a low concentration of Mg
2+ (≤1 mM) is detrimental to the stability of DNA nanostructures [
12]. However, Keller’s group proposed that high Mg
2+ concentrations are not necessary for maintaining stability if an optimal buffer is chosen based on the specific form of the DNA nanostructure [
13]. Furthermore, there are other ways to ensure DNA nanostructure integrity against the impacts of Mg
2+, such as oligolysine-coated protection [
14]. However, the role of Mg
2+ in DNA nanostructure preparation and storage (such as freezing-thawing and lyophilization) has not yet been investigated; such studies are important for improving experimental reproducibility or reducing batch variations through the preparation of one batch of Td used in multiple rounds of experiments for a longtime.
In this study, we synthesized Td at different Mg2+ concentrations and clarified the influence of Mg2+ on Td preparation and storage, which provides some enlightenment toward improving yields of other DNA nanostructures.
2. Results
Most previous studies ([
4], [
15], [
16], and [
17]) reported that a Td could be constructed in a Tris-Mg buffer (TM buffer) with 5 or 50 mM Mg
2+; therefore, we first assembled the Td at varied concentrations (1, 2, 5, 10, and 20 μM) in the common TM buffers containing 5 or 50 mM Mg
2+. The sequences of the DNA strands are presented in
Table S1. The yields and morphologies of the tetrahedrons were identified by electrophoresis and atomic force microscope (AFM), respectively. The results show that in the buffer containing 5 mM Mg
2+, Td was successfully assembled at all concentrations except for 20 μM. The band of the 20 μM DNA sample was stagnant in the well of gel, indicating the aggregation of DNA strands under this condition; these results correspond with the images observed by AFM (
Figure 1). Nevertheless, under the condition of 50 mM Mg
2+, the Td could only be formed in the first two DNA concentrations (1 and 2 μM), while the samples with the other concentrations (5, 10 and 20 μM) exhibited diameters that were much larger than normal (
Figure 1). These results indicate that the concentrations of Mg
2+ had an influence on the self-assembly process of the Td and that high Mg
2+ concentrations may be detrimental to the formation of high-concentration Td.
Next, we further clarified the role of Mg
2+ in the self-assembly process of Td by synthesizing Td in TM buffers containing diverse Mg
2+ concentrations (0.05, 0.5, 2, 10 and 25 mM MgCl
2). The electrophoresis results (
Figure 2A) show that different concentrations of Td were assembled in 2 mM MgCl
2. However, 1, 2 or 5 μM Td could be assembled in 25 mM Mg
2+, and aggregation appeared when the concentration of DNA increased to 10 or 20 μM. Nevertheless, the samples of 10 or 20 μM prepared in lower Mg
2+ density (0.5 and 0.05 mM) also showed poor yields, and bands with faster mobility were observed in the electrophoresis results, indicating the existence of free DNA strands. The dynamic light scattering (DLS) results further confirm that the successfully prepared Td had a hydrodynamic size of ~12.36 nm, and when aggregation occurred, the particle sizes increased with an increase of Mg
2+ concentration (
Figure 3). The polydispersity index (PDI) values of products are listed in
Table S2, indicating that greater particle heterogeneity as aggregation occurred. (Only particles larger than 3 nm can be measured in this apparatus; therefore, the DLS results of the Td prepared in 0.05 or 0.5 mM Mg
2+ were not available.)
After estimating the yields of Td in various environments by gray scanning (
Table S3 and
Figure 2B), we found that adequate Mg
2+ intensity is the first condition for constructing high-yielding Td; this observation was based on the fact that yields decreased dramatically to less than 20% in 0.05 mM Mg
2+, no matter what concentration of DNA nanostructures was prepared. Notably, DNA in higher concentrations is more vulnerable to the change in Mg
2+ density. As illustrated in
Table S3, when the concentration of Mg
2+ changed from 2 mM to 25 mM, the yield of 20 μM Td slumped from 55.4% to 7.8%, while that of the 1 μM sample decreased from 77.53% to 52.22% (
Table S3).
As for the Td storage, we examined the effect of Mg
2+ during low-temperature freezing and vacuum drying. In this part, we firstly prepared Td in TM buffer containing 5 or 2 mM Mg
2+, and then carried out either the process of freeze-thawing three times or the process of lyophilization treatment. As a result, after freeze-thawing treatment, irrespectively of time, the bands of Td prepared under both 5 and 2 mM Mg
2+ conditions had an identical mobile speed, and the gels showed no difference to that of freshly prepared ones (
Figure 4). These results indicate that iterative freeze-thawing processing had no influence on the structure of the Td. However, when dissolving the freeze-dried powder of the tetrahedrons in an equivalent volume of deionized water, the tetrahedrons prepared in 5 mM Mg
2+ mostly stayed in wells and formed disordered aggregations, as observed on the AFM images. Differently, tetrahedrons at all concentrations prepared in 2 mM Mg
2+ mostly maintained their previous structures (
Figure 5). These results indicate that the stability of the Td structure was more sensitive to the process of vacuum drying than to that of low-temperature freezing. To further verify the influence of Mg
2+ in the process of lyophilization, we decreased its concentration to 0.5 mM during preparation, repeated the experiment, and found that the Td after the freeze-drying in this condition produced little aggregation (
Figure 5). All these data imply that lower Mg
2+ in the initial prepared buffer benefits the structure stability of the Td in the process of lyophilization.
Because PBS, Mueller-Hinton broth (MHB) and Dulbecco’s modified eagle’s medium (DMEM) are commonly-used biological buffers for cellular or bacterial culture, we investigated whether lyophilizated powder of Td could be directly dissolved in these buffers. In this study, freeze-dried powders of 1 or 5 μM Td, prepared in TM buffer containing 5, 2, or 0.5 mM Mg
2+, were dissolved in different solutions. We found that aggregation was more pronounced in the samples dissolved in PBS, MHB and DMEM than the sample dissolved in water (
Figure 6A). The results indicate that any solvent containing more complex components than water resulted in the aggregation of Td powder after re-dissolving. Furthermore, we investigated the feasibility of concentrating Td (prepared in 2 mM Mg
2+) by dissolving its freeze-dried powder in different volumes of deionized water, which were 3/4, 1/2 or 1/4 of the initial volume of prepared buffer. As demonstrated by the electrophoresis results (
Figure 6B), the Td powders were successfully re-dissolved and mostly retained their original structures at the above volumes, indicating that it is a practicable way to concentrate Td prepared in an appropriate Mg
2+ intensity (2 mM Mg
2+).
3. Discussion
These results imply that Mg
2+ concentration plays an important role when synthesizing Td, and that relatively low concentrations of Mg
2+ are more helpful in producing Td with a high concentration; however, the formation will not succeed without enough Mg
2+. In addition, the yield declined visibly when the Td concentration increased to 20 μM (
Figure 7A). The exact quantitative balance of DNA and Mg
2+ and the underling mechanism are still unclear. It would be meaningful to research the exact interaction between DNA and Mg
2+ in the process of assembling tetrahedrons, which will guide further studies to meet the requirements of diverse DNA nanostructures. Furthermore, we found that vacuum drying exerts a more conspicuous effect on the Td structure than freeze-thawing. Notably, the Mg
2+ concentration during Td preparation has a great influence on this process (
Figure 7B). In addition, considering the complexity of the lyophilization process and for avoiding the interference of other components, water as a re-dissolving solution is the best choice. It has been reported that high Mg
2+ can cause condensation between DNA double helixes by ion-bridging, and this process varies according to different kinds of DNA configuration [
18], which may explain our observed results that more Mg
2+ tends to result in unexpected aggregation during the preparation and lyophilization. When preparing the Td, the unexpected entanglement between DNA strands is more likely to occur due to high DNA concentration, which may lead to DNA aggregation. However, the prepared Td remains its structure during lyophilization only if the Mg
2+ concentration in the prepared buffer is appropriate because hydrogen bonds in the Td are beneficial for maintaining the structure. As for the influence of the buffer when re-dissolving the freeze-dried powder, we speculate that the bond angle is distorted due to the special structure of Td, which is highly vulnerable to complicated elements in PBS, MHB or DMEM when dissolved. Importantly, our results suggest that freeze-dried dissolving is a practicable way to concentrate Td if it is prepared in initial solutions with less than 2 mM Mg
2+. Nevertheless, the reaction mechanism during this process is not clear. It would be valuable to further study the relationship between Mg
2+ and DNA and to seek the conformations of the crystal structure of the Td during the process.