Preservation of DNA nanostructure carriers: Effects of freeze-thawing

Letter

Preservation of DNA nanostructure carriers: Effects of freezethawing and ionic strength during lyophilization and storage Bing Zhu, Yan Zhao, Jiangbing Dai, Jianbang Wang, Shu Xing, Linjie Guo, Nan Chen, Xiangmeng Qu, Li Li, Juwen Shen, Jiye Shi, Jiang Li, and Lihua Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 26 May 2017 Downloaded from http://pubs.acs.org on May 26, 2017

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ACS Applied Materials & Interfaces

Preservation of DNA nanostructure carriers: Effects of freeze-thawing and ionic strength during lyophilization and storage Bing Zhu, 1,2 Yan Zhao,1,2 Jiangbing Dai,1,2 Jianbang Wang,1,2 Shu Xing,1 Linjie Guo,1 Nan Chen,1 Xiangmeng Qu,3 Li Li,3 Juwen Shen,3 Jiye Shi,4 Jiang Li,1* Lihua Wang1* 1 Division of Physical Biology and Bioimaging Center, Shanghai Synchrotron Radiation Facility; CAS Key Laboratory of Interfacial Physics and Technology; Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China 2 University of Chinese Academy of Sciences, Beijing 10049, China 3 Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241, China 4 UCB Pharma, 208 Bath Road, Slough SL1 3WE, United Kingdom KEYWORDS: lyophilization, DNA nanostructures, structure-friendly, ionic strength, storage

ABSTRACT: DNA nanostructures have attracted wide interest in biomedical applications, especially as nanocarriers for drug delivery. Therefore, it is important to ensure the structural integrity of DNA nanostructures under ambient temperature storage. In this study, we examined

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lyophilization-based preservation of DNA nanostructures by investigating the structural integrity of different DNA nanostructures reconstituted from lyophilization. We demonstrated that lyophilization under appropriate ionic strength is amenable to the storage of DNA nanostructures. Compared with that stored in liquid solution, DNA nanostructure carriers reconstituted from lyophilization showed significantly better structural integrity after an accelerated aging test equivalent to 100-day room-temperature storage.

In recent years, DNA technology has shown great promise in biomedical applications such as molecular diagnosis and drug delivery due to their excellent programmability, biocompatibility and biodegradability.1-2 Particularly, DNA nanostructures as nanocarriers have been exploited for intracellular delivery of therapeutic molecules including chemical drugs,3 functional nucleic acids, and proteins.4 Strategies of targeted delivery,5-6 controlled drug release7 and even intelligent theranostics8 have also been realized based on DNA nanostructures. However, as a promising class of biomedical reagents, DNA molecules are known vulnerable to many common environmental factors like oxidation,9 nucleases,10 unbefitting pH11 or ionic strength,12 which largely affects their structural stability in storage. Therefore, in laboratory studies, DNA nanostructures are usually freshly prepared before use, arousing problem in batch-to-batch consistency, and hampering their practical applications in biomedicine.13-14 Freeze-drying, also termed lyophilization, has long been used for storage of biological samples (e.g. antibodies,15 bacterial strains,16 plasmids17 or even live mammalian cells18). Previous studies have revealed that the genetic functions of natural DNA molecules or vectors in these samples can be well reconstituted from lyophilization,19-20 during which the addition of


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stabilizers (e.g. disaccharides) is a critical step to maintain biological and structural integrity of DNA.21-22 However, different with natural linear or circuilar DNA molecules, DNA helices in synthetic DNA nanostructures are more densely packed, leading to rigid fixed-shaped structures, which show different structural stability.6, 10 The effects of lyophilization on these synthetic DNA nanostructures have yet to be demonstrated. The feasibility of storing them this way is unknown. Therefore, in this study, we propose a lyophilization-based strategy for preservation of DNA nanostructures (shown in Figure 1). We investigate the structural integrity of tetrahedral DNA nanostructures (TDNs) and DNA origami structures reconstituted from lyophilization under different Mg2+ concentrations. We also evaluated the stability of the freeze-dried DNA nanostructures via an accelerated aging test.

Figure 1. Schematics of the lyophilization-based preservation and reconstitution of DNA nanostructures.


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Figure 2. Gel electrophoresis and AFM images of different DNA nanostructures (a) freshly prepared, (b) freeze-thawed or (c) lyophilized. Scale bars: 50 nm. (d) Quantification of the yields of correct structures with different treatments. Yields of the TDNs were quantified from the band densities on the gel images. Yields of origami structures were counted in the AFM images. “U”, freshly prepared; “F”, freeze-thawed; “L”, lyophilized. The error bars represent the standard deviations from three tests. We prepared several typical DNA nanostructures, including TDNs with 17 or 20 base pairs on each edge (TDN17 and TDN20), triangular origami, and rectangular origami, which are easy to prepare with high yields and have been widely used in recent studies.23-24 To evaluate the feasibility of lyophilization, we firstly examined the effects of freeze-thaw process on these DNA nanostructures, which is inevitable in lyophilization. We froze these structures in solution (containing 50 µM Mg2+) at -80 °C for 12 hours, thawed them at room temperature, and then characterized them with gel electrophoresis and AFM (Figure 2 and Figure S1). Results (Figure


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2b) showed that the bands of freeze-thawed DNA nanostructures presented migrations identical to that of freshly prepared ones (Figure 2a), indicating that their structural integrity was maintained after the freeze-thaw procedure. The AFM images of them also verified the integrity and dispersity of these structures after the freeze-thaw process. Next, we lyophilized these nanostructures under the same solution condition, and then reconstituted them by adding deionized water to their initial volume (as shown in Figure 1). The electrophoresis of which (Figure 2c) also presented bands with identical mobilities compared to which of the freshly prepared structures. In the AFM images, we did not observe obvious structural changes on the nanostructures reconstituted from lyophilization. Moreover, the proportions of intact structures (Figure 2d) quantified from the gels (for TDNs) or AFM images (for origamis) were very close to which of freshly prepared ones. These results demonstrated that the unmodified DNA nanostructures can withstand the lyophilization process under proper conditions without the help of stabilizing agents. Therefore, lyophilization can be widely applied in preservation of various DNA nanostructures in a facile way.


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Figure 3. Gel electrophoresis and AFM imaging of TDN20 structures (a) freshly prepared, (b) freeze-thawed or (c) lyophilized, from solutions of different Mg2+ concentrations. M: 20 bp DNA ladder; Scale bars: 50 nm. Yields of correct TDN structures were quantified from the band densities in the gel images. (d) HPLC spectra of TDN structures from different Mg2+ concentrations, freshly prepared (left) or reconstituted from lyophilization (right). The positions


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representing dispersed TDNs were marked with dashed boxes. (e) AFM images of reconstituted origami structures from different Mg2+ concentrations, and (f) the counts of intact structures. The error bars represent the standard deviations from three tests. Ionic concentration (especially Mg2+ concentration) is known critical for the structural stability of DNA nanostructures.2 Given that the salt concentration will rise hundreds of times (along with the solution volume drops from 1 mL to less than 10 µL) during the lyophilization process,25 we investigated the structural stability of lyophilized DNA nanostructures in solutions with different Mg2+ concentrations. We synthesized TDN20 with Mg2+ varied from 5 µM to 5 mM (5 µM, 50 µM, 0.5 mM, and 5 mM) respectively. The electrophoresis results of the products (Figure 3) showed that except for the 5µM Mg2+ concentration, all other conditions (50 µM, 5 mM, and 0.5 mM) enabled high-yield (around 80% quantified from the band densities on the gel) selfassembly of TDN20 structures. We then lyophilized these TDN solutions with different Mg2+ concentrations, and the electrophoresis results (Figure 3c) revealed that with higher Mg2+ concentrations (5 mM and 0.5 mM) in their initial solution, the bands representing the correct TDN structures almost disappeared. Most structures were remained in the gel wells, which seemed to be severe aggregation of DNA structures. The AFM images indeed revealed that these products (from 5 mM or 0.5 mM Mg2+) were mostly disordered aggregates. In addition, HPLC (SEC4000 column) spectra of them (Figure 3d) represents peaks appeared much earlier (~4 min for which from 5mM Mg2+ and ~8 min for which from 0.5 mM Mg2+) than which of dispersed TDNs (~10 min), indicating their increased charges resulted from larger sizes. This phenomenon might be attributed to the extremely high salt concentration caused by the drying process which facilitated mismatches between nucleic bases and unspecific adsorptions due to the salt-induced charge screening of the repulsive interparticle interactions.26 In contrary, the lowest Mg2+


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concentration (5 µM) tested in our study resulted in multiple bands in the gel with higher mobilities compared with which of intact TDNs (Figure 3c). The AFM image of which showed many linear structures rather than particle-like structures, and the HPLC spectra also presented multiple peaks around the expected time point (Figure 3d). These results suggest that under the 5 µM Mg2+ condition, the efficiency of TDN self-assembly was too low (~20% yield of intact TDNs quantified from the band density on the gel shown in Figure 3), and many component strands were left unassembled or partially assembled. Therefore, the freeze-thaw or lyophilization treatment on them could not lead to better yield of TDN structures. As compared, the products came from the 50 µM Mg2+ solution obtained the best yield of dispersed TDN structures after reconstituted from lyophilization (Figure 3c), which was almost identical to the original yield of TDN synthesis. The AFM images and the HPLC analysis27 (Figure 3d) also verified the high structural homogeneity of them resulted from this condition. Therefore, we suggest that 50 µM Mg2+ is an appropriate ionic strength to prepare and lyophilize TDNs, which can ensure their structural integrity and meanwhile prevent them from irreversible aggregation after lyophilization. For triangular and rectangular DNA origami structures, we fabricated them in the preparation buffer containing 12.5 mM Mg2+ and then exchanged the buffer into that with different Mg2+ concentrations (50 µM, 0.5 mM and 5 mM) via ultrafiltration. We studied their structural integrity in these solution environments with AFM. As results (Figure 3e), we did not find obvious structural deformations, destructions or aggregations under all these conditions. Meantime, the counts of intact origami structures did not obviously change with these Mg2+ concentrations (Figure 3f, counted from Figure S2). Therefore, we can conclude that DNA


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origami structures can be well lyophilized and reconstituted from solutions within a range of Mg2+ concentrations (50 µM to 5 mM) lower than which in their preparation solution. To evaluate the stability of lyophilized DNA nanostructures for a longer period of time, we treated the lyophilized triangular DNA origami structures (5 mM Mg2+) with an accelerated aging test (by raising the storage temperature to 60 ˚C).28 After a 10-day test (equivalent to about 100 days at room temperature),28 AFM imaging results (Figure 4a) showed that a large part of structures stored in liquid solution had been disassembled or degraded. In contrast, almost all the structures kept in dry powder form remained uniformly intact after reconstitution (Figure 4b, counted from Figure S3). Thus, these results revealed that although without the protection from chemical modifications or stabilizers, the lyophilized DNA nanostructures are significantly more stable in storage under the non-frozen condition compared with which kept in liquid solution.


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Figure 4. Accelerated aging test of DNA origami structures. (a) AFM images of triangular DNA origamis stored in liquid solution (upper row) or in lyophilized dry powder form (lower row), which were incubated at 60 ˚C for up to 10 days. Scale bars: 50 nm. (b) Proportions of intact structures after accelerated aging tests, counted from AFM images. The error bars represent the standard deviations from three tests.


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Taken together, we conclude that the lyophilization method can be used for storage of DNA nanostructures at ambient temperature.29 The improved stability can be attributed to the minimized degradative reaction (e.g. hydrolysis and oxidation) activity in the dehydrated environment.28 The salt concentration of which before drying is an important factor to the quality of reconstituted structures. We have not observed high-salt induced denaturation of DNA nanostructures, which is however a common problem for lyophilization of proteins.30 Although for TDNs, the Mg2+ concentration in their original preparation solution may induce aggregations during lyophilization, this problem can be solved by simply minimize the salt concentration in their preparation. Exploiting this facile method for keeping DNA nanostructures has obvious advantages. It facilitates the transportation and distribution of DNA nanostructures without cold preservation. For laboratory use, one batch of properly lyophilized DNA nanostructures will be able to satisfy multiple rounds of experiments for a long period of time, which can largely reduce the batch-to-batch variations and improve the reproducibility. More significantly, in practical applications, lyophilization-based preservation makes a meaningful step towards improved commercial availability of DNA nanostructures and their point-of-care applications in diagnosis or therapies. Nevertheless, the effects of lyophilization on the specific functioning and/or biomedical applications of these nanostructures, deserves further investigation. In conclusion, we investigated the structural integrity of DNA nanostructures treated by lyophilization. We showed that under proper Mg2+ concentrations, the structures could be well reconstituted from freeze-dried powder. Results from the ten-day accelerated aging test further indicated that the freeze-dried DNA nanostructures are significantly more stable than which stored in liquid solution. Overall, we concluded that the lyophilization strategy with optimized ionic strength is suitable for storage of DNA nanostructures. Based on which, we envision that


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this reliable storage method should benefit the practical biomedical applications of DNA nanotechnology.

ASSOCIATED CONTENT Supporting Information. Materials and methods; large view AFM images of TDNs and DNA origamis reconstituted from lyophilization (Figure S1), DNA origamis from lyophilization with different Mg2+ concentrations (Figure S2), DNA origamis treated with accelerated aging tests (Figure S3); DNA sequences used for preparing TDNs (Table S1) and DNA origamis (Table S2 and Table S3). This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]; [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21329501, U1532119, 21227804). REFERENCES


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Preservation of DNA nanostructure carriers: Effects of freeze-thawing

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