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A Programming 20-30nm Rectangular DNA Origami for Loading Doxorubicin to Penetrate Ovarian Cancer Cells Xin Li, Xun Wang, Member, IEEE Hong Li, Xiaolong Shi, Pan Zheng, Senior Member, IEEE
Abstract—In DNA nanotechnology, the aim in folding DNA origami is to find a good piece of rectangular DNA origami with desired sizes, which could be larger or smaller for different application purposes. In recent three years, the technique of folding smaller ones is paid heavily attentions. In this work, we design a programming rectangular DNA origami in size 20*30nm with M13p18, which is smallest and cheapest to the best acknowledge of the authors. Since it is not hard to prepare with 30 staple strands and short annealing time, the cost of folding our designed rectangular DNA origami is less than 100 dollars. Although the large origami give more space, the smaller ones are cheaper and has the potential applications in penetrating cancer cells. It is obtained by cell penetrating experiments that our designed rectangular DNA origami can penetrate ovarian cancer cells efficiently even loading doxorubicin, but the thermodynamic stability needs further improved. Our designed programming 20-30nm triangular DNA origami shows potential applications in precision control of nanoscale particles and anti-tumor drug delivery in vivo. Index Terms—Bio-inspired computing, Membrane computing, Spiking neural P system, Learning, Letter classification
I. I NTRODUCTION Structural DNA nanotechnology has been developed as an important branch of nanotechnology, which is completely different from conventional nanotechnology that uses physical or chemical methods to construct nanoscale materials [1]. DNA nanotechnology takes DNA as basis material to construct desired shape and pattern in nanoscale by bottom-up selfassembly of synthesis DNA strands with designed sequence. Seeman put forward the viewpoint of using DNA as a building This data collection work is supported by grants from Tai Shan Scholar Foundation (No. tsqn201812029), National Natural Science Foundation of China (61873280, 61672033 and 61672248), Key Research and Development Program of Shandong Province (No. 2017GGX10147) and Natural Science Foundation of Shandong Province (No. ZR2017MF004, ZR2019MF012). The analysis and simulation is supported by Fundamental Research Funds for the Central Universities (No. 18CX02152A, 19CX05003A-6). The theoretical investigation is supported by Project TIN2016-81079-R (MINECO AEI/FEDER, Spain-EU), and the InGEMICS-CM Project (SB2017/BMD3691, FSE/FEDER, Comunidad de Madrid-EU), Research Project TIN201681079-R (AEI/FEDER, Spain-EU) and Grant 2016-T2/TIC-2024 from Talento-Comunidad de Madrid. Asterisk indicates corresponding author. Xin Li is with Department of Gynecology 2, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei, China. X. Wang∗ is with the College of Computer and Communication Engineering, China University of Petroleum, Qingdao 266580, Shandong, China. Xiaolong Shi is with School of Computer Science and Cyber Engineering, Guangzhou University, Guangzhou 510006, Guangdong, China. (e-mail: [email protected]). Pan Zheng is with Department of Accounting and Information Systems, University of Canterbury, Christchurch 8041, New Zealand.
material rather than a linear polymer [2]. In 2006, Rothemund presented DNA origami which could fold DNA to arbitrary 2D shapes with simple ‘one-pot’ anneal [3]. The concept of DNA origami is to fold a long single strand (namely scaffold strand) DNA to form a designed shape with the binding of multiple smaller strands (namely staple strand) in various places. Folding DNA depends on crossover formed by scaffold strand and staple strand in the adjacent helix, and the crossover is actually Holliday junctions. DNA origami possesses many outstanding merits such as simplicity of design and production, programmability and fully addressable [4]. Basing on wellestablished DNA nanotechnology many potential applications about DNA origami have been studied including enzyme immobilization [5], [6], drug carry capsules [7], [8], [9], and self-assembly of nanoscale materials etc. Especially DNA origami as a flat 2D structure, which is programmable and fully addressable, has a significant application as molecules platform to place and arrange nanoparticle with the precision down to a single-digit nanometer scale [10]-[23]. In DNA nanotechnology, one challenge in folding DNA origami is to find a good piece of ‘paper’, a long single strand DNA, with desired size. The scaffold of DNA origami generally uses nature long circled DNA strand: M13p18, 7249nucleotide DNA, which could be larger or smaller for different application purposes [24]-[29]. In 2014, LaBeans group addressed this challenge by create the largest DNA origami with the production of the largest to-date biologically derived single-stranded scaffold using a /M13 hybrid virus to produce a 51,466-nucleotide DNA [30]. To avoid the expensive cost of synthesis staple strand up to 51,466-nucleotide, they came up with inexpensive staple strands synthesis method via an ink jet printing process on a chip embossed with functionalized micro pillars made from cyclic olefin copolymer [30]. In 2015, LaBeans group produced a mini scaffold DNA origami with only with 2404-base long scaffold which is about one third length of M13 [31]. The main difference between our method and Labeans method is that we use long circled DNA strand M13p18 as template to assemble DNA rectangular origami. In this work, we create the smallest (to the best acknowledge) rectangular DNA origami with M13p18, which is cheaper and easier to prepare. This origami only use 30 staple strands to fold 960-base ssDNA scaffold in comparison with 2404-base in LaBeans work [31]. Consequently our design cost less than 100 dollars. The folded structure demonstrate here is a 2D rectangle about 20 × 30 nm in size. Smaller DNA origami has less space to load drugs but can access cancer
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cells easily. DNA origamis of small sizes has functioning of endocytosis, while for big molecules transition, it may need protein channel to enter the cell. It is obtained by cell penetrating experiments that our designed rectangular DNA origami can penetrate ovarian cancer cells efficiently even loading doxorubicin, but the thermodynamic stability needs further improved. Our designed programming 20-30nm triangular DNA origami shows potential applications in precision control of nanoscale particles and anti-tumor drug delivery in vivo. II. DESIGN A. DNA origami object design The process of designing a DNA origami is summarized to five steps [3]. One of most important step is to merge adjacent fractions of staples designed in previous steps. The length of each staple strand should not exceed 60 bases considering the difficulty of artificially synthesis. In addition, it’s necessary to change the position of the crossover by plus or minus a single nucleotide of staple strand between the neighbouring crossover in the direction of helix domains to avoid the deformation caused by design criterion of 32 base-pair per three turn for B-DNA which characterized 10.4 base-pair per turn. In principle, the process of self-assembly of DNA origami is thermodynamic equilibrium, with mixture of scaffold strand and staple strand in solution. Target of the design and simulation process is to reach the lowest energy point coincided with desired design to promote the possibility of high yield of designed DNA origami, and reduce the possibility of mispairing and contaminants. Our designed programming DNA origami of rectangle shape is shown in Figure 1.
Fig. 1.
origami is approximately 29 × 32.64 nm with the inter-helical gap size of 1nm resulting from 1.5-turn crossover spacing rule. As for design software tools, caDNAno was employed to aid visual design of 2D DNA origami [32]. It can rapidly and accurately determine staple sequences according to known scaffold sequence with mouse click. In order to analysing the design result of caDNAno, the design data need be submitted for analysis with CanDo (http://cando.dna-origami.org/) [33]. Users obtain the simulation result of formed shape and heat maps as show in Figure 2.
Fig. 2. Determine staple sequences according to known scaffold sequence with mouse click
Combined utilization of caDNAno for design and CanDo for analysis provides rapid and efficient feedback to guide whole design of DNA origami. It is especially useful for shape analysis and thermodynamic simulation. As shown in Figure 3, the shape has serious distort from the side view, which indicates that the global twist is not balanceable.
Our designed programming DNA origami of rectangle shape Fig. 3.
It totally used a one eighth fragment of 7249 bases M13mp18 DNA, which is about 900 bases. The number of staple strand is 30, with different lengths from 24nt to 48nt, which is fully addressable. Every staple can be used as a programmable site, which allows these 30 staple segments using as hybridization anchors for site-directed attachments. In detail, the central seam is adopted on purpose that there is serious possibility to prevent rectangle origami from deformation into an hourglass shape [3]. In addition, the four corners extend with 10 ’T’s to avoid sticking and stacking. This is much simpler than omitting staples along vertical edges. The distance of crossover along alternating sides of a helix is 1.5 turns in the design. Estimated the size of the rectangle
Users obtain the simulation result of formed shape and heat maps
The deeper reasons present as follows. The caDNAno assumed that the helical twist of a DNA molecule is 10.67 base pairs per turn, which is a conventional 2D DNA origami design method. But double-stranded B-DNA behaves as 10.4 base pairs per helical turn [34]. As a result, this distinction makes DNA helices under-twists compared with the native BDNA. Consequently, it is the imbalance of inner stress at the whole plane that caused DNA origami rolling up. Combined utilization of caDNAno for design and CanDo for analysis provides rapid and efficient feedback to guide whole design of DNA origami. It is especially useful for shape analysis and thermodynamic simulation. As shown in Figure
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This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TNB.2019.2943923, IEEE Transactions on NanoBioscience T. SONG et al.: A PROGRAMMING 20-30NM RECTANGULAR DNA ORIGAMI FOR LOADING DOXORUBICIN TO PENETRATE OVARIAN CANCER CELLS
3, the shape has serious distort from the side view, which indicates that the global twist is not balanceable. The deeper reasons present as follows. The caDNAno assumed that the helical twist of a DNA molecule is 10.67 base pairs per turn, which is a conventional 2D DNA origami design method. But double-stranded B-DNA behaves as 10.4 base pairs per helical turn [34]. As a result, this distinction makes DNA helices under-twists compared with the native B-DNA. Consequently, it is the imbalance of inner stress at the whole plane that caused DNA origami rolling up. Tensegrity is an idea to mechanically design of DNA nanostructures that utilizes a network of tensed elements balanced by internal compression struts to form a self-equilibrated mechanical structure that demands tensile pre-stress for its mechanical stability [35]. DNA origami can contain singlestranded domains with some segments not binding to the scaffold. It is a common method as used in the accommodation of this design. In essence, it is also on the purpose of eliminating stress increased by under-twists of crossover spacing rule, 10.67 base pairs per turn. An alternative is to directly adopt the crossover spacing rule of 10.4 base pairs per turn. In our design, rectangular DNA origami is adjusted by inserting or skipping some nucleotide of the staple strand at its beginning and ending to change its shape. The final result given by CanDo is shown in Figure 4. Though the top view appeared some crack, the origami became relatively flat from the perspective of front view and left view. A flat DNA origami is beneficial to adhere to the mica surface and then it may impact quantification of yield with suitable well-formed definition.
1× TAE buffer (10 mM M g2+, 20ml Tris with 7.6 − 8.0, 2mM EDTA). Then one pot annealing from 94◦ Cto 4◦ C in refrigerate. Purify objects with gel electrophoresis Samples were purified by 1% Agarose gel in 1× TAE buffer at 80V for 3 hours in 4◦ C refrigerate. The desired bands were physically excised, and then the excised chip was crunched and filtered through Centrifuge 5415D and stored in 4◦C refrigerate ready for AFM. The gel image result was shown in Figure 5.
Fig. 5.
Fig. 4.
The final result given by CanDo
III. EXPERIMENT AND RESULTS Prepare scaffold DNA and synthesized staples Scaffold DNA M13mp18 and Single-stranded staple DNA can be purchased from vendors, Sangon Biotech, with HAP purified. Run molecular self-assembly reactions Samples (between 30 and 100 µL) were prepared by mixing the scaffold strand (between 5 and 0.5 nM ) with the staple (in a 10× molar excess as compared to scaffold concentration) in
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The gel image results
AFM imaging 5µL annealed sample was pipetted to the surface of freshly cleaved mica. Imaging was performed with BRUKER MultiMode-8 Nano Scope using silicon nitride probes and scan analysis mode, keep the set point of contact force less than 0.02V , scan rate under 1HZ. Analysis on biocompatibility We test the performance of our designed DNA rectangular origami in penetrating ovarian cancer cells with loading doxorubicin. The complex is obtained by incubating our DNA origami with doxorubicin in 12h, 24h and 48h, respectively. And all the samples are tested in 2nM doxorubicin solution. The biocompatibility is tested by CCK-8 (Cell Counting Kit8). It allows sensitive colorimetric assays for the determination of cell viability in cell proliferation and cytotoxicity assays. Performance of penetrating ovarian cancer cells Doxorubicin is a chemotherapy medication used to treat cancer, which is known to be fluorescent. Doxorubicin fluorescence is quenched by binding to DNA. It is visible in fluorescence microscope by orangey-red in [36]. DAPI is a
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fluorescent stain that binds strongly to A-T rich regions in DNA, which is used extensively in fluorescence microscopy. As shown in Figures ?? and ??, the shape of desired DNA origami was a acceptable rectangle.
Fig. 6. The shape of desired DNA origami by AFM from −10nm to 10nm scope
Fig. 7. scope
The shape of desired DNA origami by AFM from −5nm to 5nm
It had a raised part in the middle of one side. It reasoned that it was the remainder of the long scaffold strand M13mp18 that is unfolded and circular under the natural state. What’s more, the beginning and end of scaffold strand in our design, which is a segment of the whole M13mp18 DNA strand, locates in the middle of the bottom side of the small origami shown in Figure 8, and the AFM results was coincident to the design. However, not sheared circular M13mp18 DNA strand have a fair chance of undermining the stability of DNA origami. In our experiment, the origami will dissolve within several days while kept in room temperature.
By the way, the theoretical size of the DNA origami we designed here is 20 ∗ 32 nm, which is the smallest DNA origami reported so far, also proved by practical size measured in Figure 9, which is 20 × 25nm by section analysis. Comparing to the size of Rothemund’s DNA origami which is more than 100nm and LaBean’s mini-scaffold DNA origami is around 50nm in size, our minimal origami is the smallest known DNA origami, which is cheaper and easy to prepare compare to previous origamis. The lager area scan in Figure 8 also demonstrated our design of origami is highly yield, and basically the same size distribution. The zoomed in and zoomed out AFM image showed origamis have the same shape and size. Moreover, this smallest DNA origami could be a useful for anti-tumor drug delivery [9], [37], since drug delivery carrier in the size of 100nm were mostly cleared from plasma by the biological particulate filter known as the reticuloendothelial system [38]. As large origami has the barrier of creating long single strand. Challenge of small origami is to make it possible due to its instability. We found that the key to address this problem was to keep temperature lower (4◦ C) during purification and AFM, or the smallest DNA origami would fall apart. Generally speakingassembling of single-layer objects can be completed in a few hours with nearly 100% yield [38], [39]. As for relatively longer annealing time of minimal DNA origami that is about 16 hours and the relatively lower yield, it could be reasoned that the seam was not spanned by staples. As previous works, to strengthen a seam an additional pattern of breaks and merges may be imposed to yield staples that cross the seam when pairs of adjacent staples are merged cross nicks to yield fewer, longer, staples. So it’s also reason of its unstable in some extent. Of course, instability inevitably causes low yield. But the yield is moderate from Figure 10. There is the serious possibility that stability of the minimal DNA origami has a dramatically improvement by making staple strand traverse the central seam. It is shown in Figure 10 the biocompatibility tested by incubating our rectangular DNA origamis loading doxorubicin with A2780 ovarian cancer cells solution in 24h, 48h and 72h, where the concentrations ranges are 0.1nM, 0.2nM, 0.4nM, 0.8nM, 1.6nM and 3.2nM . It is shown in Figure 10 that our designed rectangular DNA origami with doxorubicin have well biocompatibility with A2780 ovarian cancer cells, achieving cell viability above 75% by incubating into A2780 ovarian cancer cells solution in 24h, 48h and 72h. In Figure 10, fluorescence microscope images show the uptake of 0.5µM our designed rectangular DNA origami loading doxorubicin, and 0.5µM doxorubicin by A2780 ovarian cancer cells. This indicates our designed triangular DNA origami uptake doxorubicin performs better than doxorubicin penetrating into A2780 ovarian cancer cells, i.e., combined with DNA triangular origamis can help doxorubicin penetrating into cells. IV. CONCLUSIONS For the DNA origami technique almost constructing arbitrary shape and pattern, enormous potential applications are
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This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TNB.2019.2943923, IEEE Transactions on NanoBioscience T. SONG et al.: A PROGRAMMING 20-30NM RECTANGULAR DNA ORIGAMI FOR LOADING DOXORUBICIN TO PENETRATE OVARIAN CANCER CELLS
Fig. 8.
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The practical size of our deigned DNA origami which is measured by AFM
Fig. 9. The biocompatibility tested by incubating our rectangular DNA origamis loading doxorubicin with A2780 ovarian cancer cells solution in 24h, 48h and 72h
Fig. 10. The fluorescence microscope images of the uptake of 0.5µM our designed rectangular DNA origami loading doxorubicin and 0.5µM doxorubicin by A2780 ovarian cancer cells.
more likely to induce revolutionary breakthrough that makes biotechnology as important as current information technology
considering rapid development of biotechnology for years. Here we have described a method to produce a minimal DNA
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scaffold with a size 20 × 30nm as rectangle. The advantages of our smallest design of DNA origami are its lower cost and its potential application in anti-tumour drug delivery in Vivo which could avoid being cleared from plasma by the liver. Furthermore, larger DNA origami is limited by lengths of single stranded scaffolds, it is a natural defect of DNA origami technology, but the minimal DNA scaffold have no this secret worry and it very suitable for teaching and low cost experiment. In a word, as for minimal DNA origamiits low cost advantage could contribute to rapid design and testing of various DNA origami structures and will surely expand the application of DNA origami and structural DNA nanotechnology. It is obtained by cell penetrating experiments that our designed rectangular DNA origami can penetrate ovarian cancer cells efficiently even loading doxorubicin, but the thermodynamic stability needs further improved. Our designed programming 20 − 30nm triangular DNA origami shows potential applications in precision control of nanoscale particles and anti-tumor drug delivery in vivo. Some other bio-inspired computing models, see e.g. [40]-[45] will provide potential platform for drug delivering. R EFERENCES [1] D.B. Souvik Modi, Friedrich C Simmel,and Yamuna Krishnan, The Journal of Physical Chemistry Letters, 2010, 1, 1994-2005. [2] J. T. Seeman N C, Biol, 1982, 99, 237-247. [3] P. W. Rothemund, Nature, 2006, 440, 297-302. [4] M. Endo, Y. Yang and H. Sugiyama, Biomater. Sci., 2013, 1, 347-360. [5] C. Timm and C. M. Niemeyer, Angewandte Chemie, 2015, 54, 67456750. [6] K. Numajiri, T. Yamazaki, M. Kimura, A. Kuzuya and M. Komiyama, Journal of the American Chemical Society, 2010, 132, 9937-9939. [7] J. S. Yonggang Ke, Minghui Liu,Kasper Jahn,Yan Liu and Hao Yan, Nano letters, 2009, 9, 2445-2447. [8] New Scientist, 2012, 215, 17. [9] Q. Jiang, C. Song, J. Nangreave, X. Liu, L. Lin, D. Qiu, Z.-G. Wang, G. Zou, X. Liang, H. Yan and B. Ding, Journal of the American Chemical Society, 2012, 134, 13396-13403. [10] G. P. B. Acuna, M.; Stein, I. H.; Steinhauer, C.; Kuzyk, A.;Holzmeister, P.; Schreiber, R.; Moroz, A.; Stefani, F. D.; Liedl, T.;Simmel, F. C.; Tinnefeld, P, ACS Nano, 2012, 6, 3189-3195. [11] H. O. Bui, C.; Kidwell, C.; Tan, Y.; Graugnard, E.; Kuang,W.; Lee, J.; Knowlton, W. B.; Yurke, B.; Hughes, W. L, Nano letters, 2010, 10, 3367-3372. [12] H. C. Gu, J.; Xiao, S.-J.; Seeman, N. C., Nature, 2010, 465, 202-205. [13] S. R. Helmig, A.; Arian, D.; Kovbasyuk, L.; Arnbjerg, J.;Ogilby, P. R.; Kjems, J.; Mokhir, A.; Besenbacher, F.; Gothelf, K. V, ACS Nano, 2010, 4, 7475-7480. [14] A. L. Kuzyk, K. T.; To?rma, P, Nanotechnology, 2009, 20, 235305. [15] C. J. Lin, R.; Leifer, A. M.; Li, C.; Levner, D.; Church, G.M.; Shih, W. M.; Yin, P. Nat, Chem, 2012, 4, 832-839. [16] H. T. H. Maune, S.P. Barish, R. D.; Bockrath, M.; Iii, W. A. G.;Rothemund, P. W. K.; Winfree, E. Nat, Nanotechnology, 2010, 5, 61-66. [17] M. G. Pilo-Pais, S.; Samano, E.; LaBean, T. H.;Finkelstein, G, Nano letters, 2011, 11, 3489-3492. [18] J. C. Sharma, R.; Andersen, C. S.; Gothelf, K. V.; Yan, H.;Liu, Y. J. m. Chem, Soc, 2008, 130, 7820?7821. [19] I. H. S. l. Stein, V.; Bo?hm, P.; Tinnefeld, P.; Liedl, T, ChemPhysChem, 2011, 12, 689-695. [20] N. L. Stephanopoulos, M.; Tong, G. J.; Li, Z.; Liu, Y.; Yan, H.;Francis, M. B, Nano letters, 2010, 10, 2714-2720. [21] L. K. S. M. B. DJ., Physical Review Letters, 2003, 9122, 7402-7402. [22] D. Baoquan, D. Zhengtao, Y. Hao, S. Cabrini, R. N. Zuckermann and J. Bokor, Journal of the American Chemical Society, 2010, 132, 32483249. [23] C. J. D. Li H, Labean T H, Mater Today, 2009, 12, 24-32.
[24] T. Song, X. Zeng, P. Zheng, M. Jiang, R.P. Alfonso, A Parallel Workflow Pattern Modelling Using Spiking Neural P Systems With Colored SpikesIEEE Transactions on NanobioscienceDOI 10.1109/TNB.2018.2873221 [25] Shi X, Wang Z, Deng C, Song T, Pan L, Chen Z, A Novel Bio-Sensor Based on DNA Strand Displacement. PLoS ONE, 2014, 9(10): e108856. [26] Shi X, Wu X., Song T, Li X. Construction of DNA Nanotubes with Controllable Diameters and Patterns using Hierarchical DNA sub-tiles, Nanoscale, 2016,8, 14785-14792 [27] Shi X, Chen C, Li X, Song T., Chen Z. Zhang Z., Wang Y, Sizecontrollable DNA nanoribbons assembled from three types of reusable brick single-strand DNA tiles, Soft Matter, 2015,11, 8484-8492 [28] Li X, Hong L, Song T., A. Rodriguez-Paton, Highly Biocompatible Drug-Delivery Systems Based on DNA Nanotechnology, J. Biomed. Nanotechnol. 2017, 13, 747C757 [29] Li X, Song T, Chen Z, Shi X, A Universal Fast Colorimetric Method for DNA Signal Detection with DNA Strand Displacement and Gold Nanoparticles, Journal of Nanomaterials, 2015, Article ID 407184 [30] T. Song, S. Pang, S. Hao, R.P. Alfonso, P. Zheng, A Parallel Image Skeletonizing Method Using Spiking Neural P Systems with Weights, Neural Processing Letters, DOI: 10.1007/s11063-018-9947-9 [31] S. Brown, J. Majikes, A. Martinez, T. M. Giron, H. Fennell, E. C. Samano and T. H. LaBean, Nanoscale, 2015, 7, 16621-16624. [32] G. Amoako, M. Zhou, R. Ye, L. Zhuang, X. Yang and Z. Shen, Chinese Science Bulletin, 2013, 58, 3019-3022. [33] C. E. Castro, F. Kilchherr, D. N. Kim, E. L. Shiao, T. Wauer, P. Wortmann, M. Bathe and H. Dietz, Nature methods, 2011, 8, 221-229. [34] R. D. Rebeca S. Mathew, Pehr A. B.Harbury science, 2008, 322, 446449. [35] T. Song, L. Pan, T. Wu, P., Zheng, Wong, M. L. Dennis and R.P., Alfonso, Spiking Neural P Systems with Learning Functions, IEEE Trans Nanobioscience, 2019, DOI: 10.1109/TNB.2019.2896981 [36] S. S. Simmel, P. C. Nickels and T. Liedl, Accounts of chemical research, 2014, 47, 1691-1699. [37] T. Song, P. Zheng, M.L. Dennis, X. Wang, Design of Logic Gates Using Spiking Neural P Systems with Homogeneous Neurons and Astrocyteslike Control, Information Sciences, 372, 2016, 380C391 [38] Akerman ME, Chan WCW, Laakkonen P, Bhatia SN, Ruoslahti E, Proceedings of the National Academy of Sciences of the United States of America, 2002, 99, 12617C12621. [39] X. Shi, W. Lu, Z. Wang, L. Pan, G. Cui, J. Xu and T. H. LaBean, Nanotechnology, 2014, 25, 075602. [40] Chen Z, Song T, Huang Y., Shi X., Solving Vertex Cover Problem Using DNA Tile Assembly Model, Journal of Applied Mathematics, 2013, Article ID 407816 [41] Wang X, Gong F, Pan Z, On the Computational Power of Spiking Neural P Systems With Self-Organization, Scientific Reports, 2016, Article No. srep27624 [42] Song T, Liu X, Zhao Y, Zhang X, Spiking Neural P Systems with White Hole Neurons, IEEE Trans on Nanobioscience, 2016, 15(7) 666-673. [43] T. Song, R.P. Alfonso, P. Zheng, X. Zeng, Spiking Neural P Systems With Colored Spikes, IEEE Transactions on Cognitive and Developmental Systems, 2018. DOI 10.1109/TCDS.2017.2785332 [44] D Benes, A Rodriguez-Paton, P Sosik, Directed evolution of biocircuits using conjugative plasmids and CRISPR-Cas9: design and in silico experiments, Natural Computing, 2017 16 (3), 497-505 [45] A Rodriguez-Paton, IS de Murieta, P Sosik, DNA strand displacement system running logic programs, Biosystems, 2015, 115, 5-12
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A Programming 20-30nm Rectangular DNA Origami for Loading Doxorubicin to Penetrate Ovarian Cancer Cells Xin Li, Xun Wang, Member, IEEE Hong Li, Xiaolong Shi, Pan Zheng, Senior Member, IEEE
Abstract—In DNA nanotechnology, the aim in folding DNA origami is to find a good piece of rectangular DNA origami with desired sizes, which could be larger or smaller for different application purposes. In recent three years, the technique of folding smaller ones is paid heavily attentions. In this work, we design a programming rectangular DNA origami in size 20*30nm with M13p18, which is smallest and cheapest to the best acknowledge of the authors. Since it is not hard to prepare with 30 staple strands and short annealing time, the cost of folding our designed rectangular DNA origami is less than 100 dollars. Although the large origami give more space, the smaller ones are cheaper and has the potential applications in penetrating cancer cells. It is obtained by cell penetrating experiments that our designed rectangular DNA origami can penetrate ovarian cancer cells efficiently even loading doxorubicin, but the thermodynamic stability needs further improved. Our designed programming 20-30nm triangular DNA origami shows potential applications in precision control of nanoscale particles and anti-tumor drug delivery in vivo. Index Terms—Bio-inspired computing, Membrane computing, Spiking neural P system, Learning, Letter classification
I. I NTRODUCTION Structural DNA nanotechnology has been developed as an important branch of nanotechnology, which is completely different from conventional nanotechnology that uses physical or chemical methods to construct nanoscale materials [1]. DNA nanotechnology takes DNA as basis material to construct desired shape and pattern in nanoscale by bottom-up selfassembly of synthesis DNA strands with designed sequence. Seeman put forward the viewpoint of using DNA as a building This data collection work is supported by grants from Tai Shan Scholar Foundation (No. tsqn201812029), National Natural Science Foundation of China (61873280, 61672033 and 61672248), Key Research and Development Program of Shandong Province (No. 2017GGX10147) and Natural Science Foundation of Shandong Province (No. ZR2017MF004, ZR2019MF012). The analysis and simulation is supported by Fundamental Research Funds for the Central Universities (No. 18CX02152A, 19CX05003A-6). The theoretical investigation is supported by Project TIN2016-81079-R (MINECO AEI/FEDER, Spain-EU), and the InGEMICS-CM Project (SB2017/BMD3691, FSE/FEDER, Comunidad de Madrid-EU), Research Project TIN201681079-R (AEI/FEDER, Spain-EU) and Grant 2016-T2/TIC-2024 from Talento-Comunidad de Madrid. Asterisk indicates corresponding author. Xin Li is with Department of Gynecology 2, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei, China. X. Wang∗ is with the College of Computer and Communication Engineering, China University of Petroleum, Qingdao 266580, Shandong, China. Xiaolong Shi is with School of Computer Science and Cyber Engineering, Guangzhou University, Guangzhou 510006, Guangdong, China. (e-mail: [email protected]). Pan Zheng is with Department of Accounting and Information Systems, University of Canterbury, Christchurch 8041, New Zealand.
material rather than a linear polymer [2]. In 2006, Rothemund presented DNA origami which could fold DNA to arbitrary 2D shapes with simple ‘one-pot’ anneal [3]. The concept of DNA origami is to fold a long single strand (namely scaffold strand) DNA to form a designed shape with the binding of multiple smaller strands (namely staple strand) in various places. Folding DNA depends on crossover formed by scaffold strand and staple strand in the adjacent helix, and the crossover is actually Holliday junctions. DNA origami possesses many outstanding merits such as simplicity of design and production, programmability and fully addressable [4]. Basing on wellestablished DNA nanotechnology many potential applications about DNA origami have been studied including enzyme immobilization [5], [6], drug carry capsules [7], [8], [9], and self-assembly of nanoscale materials etc. Especially DNA origami as a flat 2D structure, which is programmable and fully addressable, has a significant application as molecules platform to place and arrange nanoparticle with the precision down to a single-digit nanometer scale [10]-[23]. In DNA nanotechnology, one challenge in folding DNA origami is to find a good piece of ‘paper’, a long single strand DNA, with desired size. The scaffold of DNA origami generally uses nature long circled DNA strand: M13p18, 7249nucleotide DNA, which could be larger or smaller for different application purposes [24]-[29]. In 2014, LaBeans group addressed this challenge by create the largest DNA origami with the production of the largest to-date biologically derived single-stranded scaffold using a /M13 hybrid virus to produce a 51,466-nucleotide DNA [30]. To avoid the expensive cost of synthesis staple strand up to 51,466-nucleotide, they came up with inexpensive staple strands synthesis method via an ink jet printing process on a chip embossed with functionalized micro pillars made from cyclic olefin copolymer [30]. In 2015, LaBeans group produced a mini scaffold DNA origami with only with 2404-base long scaffold which is about one third length of M13 [31]. The main difference between our method and Labeans method is that we use long circled DNA strand M13p18 as template to assemble DNA rectangular origami. In this work, we create the smallest (to the best acknowledge) rectangular DNA origami with M13p18, which is cheaper and easier to prepare. This origami only use 30 staple strands to fold 960-base ssDNA scaffold in comparison with 2404-base in LaBeans work [31]. Consequently our design cost less than 100 dollars. The folded structure demonstrate here is a 2D rectangle about 20 × 30 nm in size. Smaller DNA origami has less space to load drugs but can access cancer
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cells easily. DNA origamis of small sizes has functioning of endocytosis, while for big molecules transition, it may need protein channel to enter the cell. It is obtained by cell penetrating experiments that our designed rectangular DNA origami can penetrate ovarian cancer cells efficiently even loading doxorubicin, but the thermodynamic stability needs further improved. Our designed programming 20-30nm triangular DNA origami shows potential applications in precision control of nanoscale particles and anti-tumor drug delivery in vivo. II. DESIGN A. DNA origami object design The process of designing a DNA origami is summarized to five steps [3]. One of most important step is to merge adjacent fractions of staples designed in previous steps. The length of each staple strand should not exceed 60 bases considering the difficulty of artificially synthesis. In addition, it’s necessary to change the position of the crossover by plus or minus a single nucleotide of staple strand between the neighbouring crossover in the direction of helix domains to avoid the deformation caused by design criterion of 32 base-pair per three turn for B-DNA which characterized 10.4 base-pair per turn. In principle, the process of self-assembly of DNA origami is thermodynamic equilibrium, with mixture of scaffold strand and staple strand in solution. Target of the design and simulation process is to reach the lowest energy point coincided with desired design to promote the possibility of high yield of designed DNA origami, and reduce the possibility of mispairing and contaminants. Our designed programming DNA origami of rectangle shape is shown in Figure 1.
Fig. 1.
origami is approximately 29 × 32.64 nm with the inter-helical gap size of 1nm resulting from 1.5-turn crossover spacing rule. As for design software tools, caDNAno was employed to aid visual design of 2D DNA origami [32]. It can rapidly and accurately determine staple sequences according to known scaffold sequence with mouse click. In order to analysing the design result of caDNAno, the design data need be submitted for analysis with CanDo (http://cando.dna-origami.org/) [33]. Users obtain the simulation result of formed shape and heat maps as show in Figure 2.
Fig. 2. Determine staple sequences according to known scaffold sequence with mouse click
Combined utilization of caDNAno for design and CanDo for analysis provides rapid and efficient feedback to guide whole design of DNA origami. It is especially useful for shape analysis and thermodynamic simulation. As shown in Figure 3, the shape has serious distort from the side view, which indicates that the global twist is not balanceable.
Our designed programming DNA origami of rectangle shape Fig. 3.
It totally used a one eighth fragment of 7249 bases M13mp18 DNA, which is about 900 bases. The number of staple strand is 30, with different lengths from 24nt to 48nt, which is fully addressable. Every staple can be used as a programmable site, which allows these 30 staple segments using as hybridization anchors for site-directed attachments. In detail, the central seam is adopted on purpose that there is serious possibility to prevent rectangle origami from deformation into an hourglass shape [3]. In addition, the four corners extend with 10 ’T’s to avoid sticking and stacking. This is much simpler than omitting staples along vertical edges. The distance of crossover along alternating sides of a helix is 1.5 turns in the design. Estimated the size of the rectangle
Users obtain the simulation result of formed shape and heat maps
The deeper reasons present as follows. The caDNAno assumed that the helical twist of a DNA molecule is 10.67 base pairs per turn, which is a conventional 2D DNA origami design method. But double-stranded B-DNA behaves as 10.4 base pairs per helical turn [34]. As a result, this distinction makes DNA helices under-twists compared with the native BDNA. Consequently, it is the imbalance of inner stress at the whole plane that caused DNA origami rolling up. Combined utilization of caDNAno for design and CanDo for analysis provides rapid and efficient feedback to guide whole design of DNA origami. It is especially useful for shape analysis and thermodynamic simulation. As shown in Figure
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This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TNB.2019.2943923, IEEE Transactions on NanoBioscience T. SONG et al.: A PROGRAMMING 20-30NM RECTANGULAR DNA ORIGAMI FOR LOADING DOXORUBICIN TO PENETRATE OVARIAN CANCER CELLS
3, the shape has serious distort from the side view, which indicates that the global twist is not balanceable. The deeper reasons present as follows. The caDNAno assumed that the helical twist of a DNA molecule is 10.67 base pairs per turn, which is a conventional 2D DNA origami design method. But double-stranded B-DNA behaves as 10.4 base pairs per helical turn [34]. As a result, this distinction makes DNA helices under-twists compared with the native B-DNA. Consequently, it is the imbalance of inner stress at the whole plane that caused DNA origami rolling up. Tensegrity is an idea to mechanically design of DNA nanostructures that utilizes a network of tensed elements balanced by internal compression struts to form a self-equilibrated mechanical structure that demands tensile pre-stress for its mechanical stability [35]. DNA origami can contain singlestranded domains with some segments not binding to the scaffold. It is a common method as used in the accommodation of this design. In essence, it is also on the purpose of eliminating stress increased by under-twists of crossover spacing rule, 10.67 base pairs per turn. An alternative is to directly adopt the crossover spacing rule of 10.4 base pairs per turn. In our design, rectangular DNA origami is adjusted by inserting or skipping some nucleotide of the staple strand at its beginning and ending to change its shape. The final result given by CanDo is shown in Figure 4. Though the top view appeared some crack, the origami became relatively flat from the perspective of front view and left view. A flat DNA origami is beneficial to adhere to the mica surface and then it may impact quantification of yield with suitable well-formed definition.
1× TAE buffer (10 mM M g2+, 20ml Tris with 7.6 − 8.0, 2mM EDTA). Then one pot annealing from 94◦ Cto 4◦ C in refrigerate. Purify objects with gel electrophoresis Samples were purified by 1% Agarose gel in 1× TAE buffer at 80V for 3 hours in 4◦ C refrigerate. The desired bands were physically excised, and then the excised chip was crunched and filtered through Centrifuge 5415D and stored in 4◦C refrigerate ready for AFM. The gel image result was shown in Figure 5.
Fig. 5.
Fig. 4.
The final result given by CanDo
III. EXPERIMENT AND RESULTS Prepare scaffold DNA and synthesized staples Scaffold DNA M13mp18 and Single-stranded staple DNA can be purchased from vendors, Sangon Biotech, with HAP purified. Run molecular self-assembly reactions Samples (between 30 and 100 µL) were prepared by mixing the scaffold strand (between 5 and 0.5 nM ) with the staple (in a 10× molar excess as compared to scaffold concentration) in
3
The gel image results
AFM imaging 5µL annealed sample was pipetted to the surface of freshly cleaved mica. Imaging was performed with BRUKER MultiMode-8 Nano Scope using silicon nitride probes and scan analysis mode, keep the set point of contact force less than 0.02V , scan rate under 1HZ. Analysis on biocompatibility We test the performance of our designed DNA rectangular origami in penetrating ovarian cancer cells with loading doxorubicin. The complex is obtained by incubating our DNA origami with doxorubicin in 12h, 24h and 48h, respectively. And all the samples are tested in 2nM doxorubicin solution. The biocompatibility is tested by CCK-8 (Cell Counting Kit8). It allows sensitive colorimetric assays for the determination of cell viability in cell proliferation and cytotoxicity assays. Performance of penetrating ovarian cancer cells Doxorubicin is a chemotherapy medication used to treat cancer, which is known to be fluorescent. Doxorubicin fluorescence is quenched by binding to DNA. It is visible in fluorescence microscope by orangey-red in [36]. DAPI is a
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fluorescent stain that binds strongly to A-T rich regions in DNA, which is used extensively in fluorescence microscopy. As shown in Figures ?? and ??, the shape of desired DNA origami was a acceptable rectangle.
Fig. 6. The shape of desired DNA origami by AFM from −10nm to 10nm scope
Fig. 7. scope
The shape of desired DNA origami by AFM from −5nm to 5nm
It had a raised part in the middle of one side. It reasoned that it was the remainder of the long scaffold strand M13mp18 that is unfolded and circular under the natural state. What’s more, the beginning and end of scaffold strand in our design, which is a segment of the whole M13mp18 DNA strand, locates in the middle of the bottom side of the small origami shown in Figure 8, and the AFM results was coincident to the design. However, not sheared circular M13mp18 DNA strand have a fair chance of undermining the stability of DNA origami. In our experiment, the origami will dissolve within several days while kept in room temperature.
By the way, the theoretical size of the DNA origami we designed here is 20 ∗ 32 nm, which is the smallest DNA origami reported so far, also proved by practical size measured in Figure 9, which is 20 × 25nm by section analysis. Comparing to the size of Rothemund’s DNA origami which is more than 100nm and LaBean’s mini-scaffold DNA origami is around 50nm in size, our minimal origami is the smallest known DNA origami, which is cheaper and easy to prepare compare to previous origamis. The lager area scan in Figure 8 also demonstrated our design of origami is highly yield, and basically the same size distribution. The zoomed in and zoomed out AFM image showed origamis have the same shape and size. Moreover, this smallest DNA origami could be a useful for anti-tumor drug delivery [9], [37], since drug delivery carrier in the size of 100nm were mostly cleared from plasma by the biological particulate filter known as the reticuloendothelial system [38]. As large origami has the barrier of creating long single strand. Challenge of small origami is to make it possible due to its instability. We found that the key to address this problem was to keep temperature lower (4◦ C) during purification and AFM, or the smallest DNA origami would fall apart. Generally speakingassembling of single-layer objects can be completed in a few hours with nearly 100% yield [38], [39]. As for relatively longer annealing time of minimal DNA origami that is about 16 hours and the relatively lower yield, it could be reasoned that the seam was not spanned by staples. As previous works, to strengthen a seam an additional pattern of breaks and merges may be imposed to yield staples that cross the seam when pairs of adjacent staples are merged cross nicks to yield fewer, longer, staples. So it’s also reason of its unstable in some extent. Of course, instability inevitably causes low yield. But the yield is moderate from Figure 10. There is the serious possibility that stability of the minimal DNA origami has a dramatically improvement by making staple strand traverse the central seam. It is shown in Figure 10 the biocompatibility tested by incubating our rectangular DNA origamis loading doxorubicin with A2780 ovarian cancer cells solution in 24h, 48h and 72h, where the concentrations ranges are 0.1nM, 0.2nM, 0.4nM, 0.8nM, 1.6nM and 3.2nM . It is shown in Figure 10 that our designed rectangular DNA origami with doxorubicin have well biocompatibility with A2780 ovarian cancer cells, achieving cell viability above 75% by incubating into A2780 ovarian cancer cells solution in 24h, 48h and 72h. In Figure 10, fluorescence microscope images show the uptake of 0.5µM our designed rectangular DNA origami loading doxorubicin, and 0.5µM doxorubicin by A2780 ovarian cancer cells. This indicates our designed triangular DNA origami uptake doxorubicin performs better than doxorubicin penetrating into A2780 ovarian cancer cells, i.e., combined with DNA triangular origamis can help doxorubicin penetrating into cells. IV. CONCLUSIONS For the DNA origami technique almost constructing arbitrary shape and pattern, enormous potential applications are
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This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TNB.2019.2943923, IEEE Transactions on NanoBioscience T. SONG et al.: A PROGRAMMING 20-30NM RECTANGULAR DNA ORIGAMI FOR LOADING DOXORUBICIN TO PENETRATE OVARIAN CANCER CELLS
Fig. 8.
5
The practical size of our deigned DNA origami which is measured by AFM
Fig. 9. The biocompatibility tested by incubating our rectangular DNA origamis loading doxorubicin with A2780 ovarian cancer cells solution in 24h, 48h and 72h
Fig. 10. The fluorescence microscope images of the uptake of 0.5µM our designed rectangular DNA origami loading doxorubicin and 0.5µM doxorubicin by A2780 ovarian cancer cells.
more likely to induce revolutionary breakthrough that makes biotechnology as important as current information technology
considering rapid development of biotechnology for years. Here we have described a method to produce a minimal DNA
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scaffold with a size 20 × 30nm as rectangle. The advantages of our smallest design of DNA origami are its lower cost and its potential application in anti-tumour drug delivery in Vivo which could avoid being cleared from plasma by the liver. Furthermore, larger DNA origami is limited by lengths of single stranded scaffolds, it is a natural defect of DNA origami technology, but the minimal DNA scaffold have no this secret worry and it very suitable for teaching and low cost experiment. In a word, as for minimal DNA origamiits low cost advantage could contribute to rapid design and testing of various DNA origami structures and will surely expand the application of DNA origami and structural DNA nanotechnology. It is obtained by cell penetrating experiments that our designed rectangular DNA origami can penetrate ovarian cancer cells efficiently even loading doxorubicin, but the thermodynamic stability needs further improved. Our designed programming 20 − 30nm triangular DNA origami shows potential applications in precision control of nanoscale particles and anti-tumor drug delivery in vivo. Some other bio-inspired computing models, see e.g. [40]-[45] will provide potential platform for drug delivering. R EFERENCES [1] D.B. Souvik Modi, Friedrich C Simmel,and Yamuna Krishnan, The Journal of Physical Chemistry Letters, 2010, 1, 1994-2005. [2] J. T. Seeman N C, Biol, 1982, 99, 237-247. [3] P. W. Rothemund, Nature, 2006, 440, 297-302. [4] M. Endo, Y. Yang and H. Sugiyama, Biomater. Sci., 2013, 1, 347-360. [5] C. Timm and C. M. Niemeyer, Angewandte Chemie, 2015, 54, 67456750. [6] K. Numajiri, T. Yamazaki, M. Kimura, A. Kuzuya and M. Komiyama, Journal of the American Chemical Society, 2010, 132, 9937-9939. [7] J. S. Yonggang Ke, Minghui Liu,Kasper Jahn,Yan Liu and Hao Yan, Nano letters, 2009, 9, 2445-2447. [8] New Scientist, 2012, 215, 17. [9] Q. Jiang, C. Song, J. Nangreave, X. Liu, L. Lin, D. Qiu, Z.-G. Wang, G. Zou, X. Liang, H. Yan and B. Ding, Journal of the American Chemical Society, 2012, 134, 13396-13403. [10] G. P. B. Acuna, M.; Stein, I. H.; Steinhauer, C.; Kuzyk, A.;Holzmeister, P.; Schreiber, R.; Moroz, A.; Stefani, F. D.; Liedl, T.;Simmel, F. C.; Tinnefeld, P, ACS Nano, 2012, 6, 3189-3195. [11] H. O. Bui, C.; Kidwell, C.; Tan, Y.; Graugnard, E.; Kuang,W.; Lee, J.; Knowlton, W. B.; Yurke, B.; Hughes, W. L, Nano letters, 2010, 10, 3367-3372. [12] H. C. Gu, J.; Xiao, S.-J.; Seeman, N. C., Nature, 2010, 465, 202-205. [13] S. R. Helmig, A.; Arian, D.; Kovbasyuk, L.; Arnbjerg, J.;Ogilby, P. R.; Kjems, J.; Mokhir, A.; Besenbacher, F.; Gothelf, K. V, ACS Nano, 2010, 4, 7475-7480. [14] A. L. Kuzyk, K. T.; To?rma, P, Nanotechnology, 2009, 20, 235305. [15] C. J. Lin, R.; Leifer, A. M.; Li, C.; Levner, D.; Church, G.M.; Shih, W. M.; Yin, P. Nat, Chem, 2012, 4, 832-839. [16] H. T. H. Maune, S.P. Barish, R. D.; Bockrath, M.; Iii, W. A. G.;Rothemund, P. W. K.; Winfree, E. Nat, Nanotechnology, 2010, 5, 61-66. [17] M. G. Pilo-Pais, S.; Samano, E.; LaBean, T. H.;Finkelstein, G, Nano letters, 2011, 11, 3489-3492. [18] J. C. Sharma, R.; Andersen, C. S.; Gothelf, K. V.; Yan, H.;Liu, Y. J. m. Chem, Soc, 2008, 130, 7820?7821. [19] I. H. S. l. Stein, V.; Bo?hm, P.; Tinnefeld, P.; Liedl, T, ChemPhysChem, 2011, 12, 689-695. [20] N. L. Stephanopoulos, M.; Tong, G. J.; Li, Z.; Liu, Y.; Yan, H.;Francis, M. B, Nano letters, 2010, 10, 2714-2720. [21] L. K. S. M. B. DJ., Physical Review Letters, 2003, 9122, 7402-7402. [22] D. Baoquan, D. Zhengtao, Y. Hao, S. Cabrini, R. N. Zuckermann and J. Bokor, Journal of the American Chemical Society, 2010, 132, 32483249. [23] C. J. D. Li H, Labean T H, Mater Today, 2009, 12, 24-32.
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