DNA Origami as Seeds for Promoting Protein CrystallizationBo Zhang†,‡, Andy R. Mei‡,§, Mark Antonin Isbell‡,§, Dianming Wang‡, Yiwei Wang⊥, Suk F. Tan‡, Xsu L. Teo‡, Lijin Xu*,†, Zhongqiang Yang*,‡, Jerry Y. Y. Heng*,§

†Department of Chemistry, Renmin University of China, Beijing 100872, PR China.‡Key Laboratory of Organic Optoelectronics & Molecular Engineering of the Ministry of Education, Department of Chemistry, Tsinghua University, Beijing 100084, PR China.§Surfaces and Particle Engineering Laboratory (SPEL), Department of Chemical Engineering, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom.⊥State Key Laboratory of Biomembrane, Center for Structural Biology School of Life Sciences and School of Medicine, Tsinghua University, Beijing 100084, PR China.

Corresponding authors: Lijin Xu (20050062@ruc.edu.cn); Zhongqiang Yang (zyang@tsinghua.edu.cn); Jerry Y. Y. Heng (jerry.heng@imperial.ac.uk)

Supporting Information Placeholder

ABSTRACT: This study reports the first experimental evidence of DNA origami as a seed resulting in the increase in probability of protein crystallization. Using DNA origami constructed from long single-stranded M13 DNA scaffolds folded with short single-stranded DNA staples, it was found that the addition of the DNA origami in concentrations of 2-6 nM to mixtures of a well-characterized protein (Catalase) solution (1.0-7.0 mg/mL) resulted in a higher proportion of mixtures with successful crystallization, up to 11x greater. The improvement to crystallization is evident particularly for mixtures with low concentrations of Catalase (< 5 mg/mL). DNA origami in different conformations: a flat rectangular sheet and a tubular hollow cylinder, were examined. Both conformations improved crystallization as compared to control experiments without M13 DNA or non-folded M13 DNA, but exhibited little difference in the extent of protein crystallization improvement. This work confirms predictions of the potential use of DNA origami to promote protein crystallization, with potential application to systems with limited protein available or difficult to crystallize.KEYWORDS: DNA Nanotechnology, DNA origami, DNA nanostructure, protein crystallization, DNA-protein interaction.

IntroductionDNA origami is one of the most promising

materials in biotechnology with a plethora of wide-ranging applications, largely used as the primary building block for self-assembled structures.1 Since the time when DNA origami techniques were first put forward by Rothemund et al. in 2006,2 DNA origami has been applied in many fields including nanomaterials and nanomedicine.3-7 To create DNA origami, hundreds of short single strands of DNA (staples) are used to direct the folding of a long single strand of circular DNA (scaffold, ca. ~8000 bases), allowing for the building of highly customizable 2D and 3D

nanostructures.8-12 The highly versatile nature of the size and molecular arrangement of DNA origami allow for its design into an enormous variety of geometric shapes.2,13-19 The ability to make precise individual molecule adjustments in the DNA origami structure along with its great biological compatibility makes DNA origami a powerful tool for the precise organization and manipulation of molecules at the nanometer length scale.4,20-24 The huge potential for DNA origami in the control of micromolecular assembly or macromolecular organization,25 along with the many underlying complexities, presents research significant opportunities.26,27 The powerful

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capabilities of DNA origami have already begun to be proven, such as its use as templates for the patterning of targeted proteins,28-32 organic or inorganic molecules33-36 and their synthesis.37,38 In addition to these applications, Professor Seeman proposed 30 years prior that protein crystallization could be attained for an array of molecules arranged in a highly ordered structure of DNA building blocks. Moreover, regardless of the fact that attempts of templating protein crystallization with DNA frameworks have thus far been unsuccessful, Seeman and his co-workers have crystallized a variety of self-assembling DNA crystals by designing periodic nucleic acid structures in one, two and three dimensions.39-43

Based on these researches, we proposed to apply DNA scaffold into protein crystallization.

The investigation of new methods to enhance and control protein crystallization is particularly valuable,44,45 especially for studies involving long nucleation times or the crystallization of target proteins that are difficult to obtain.46-53 Seeding method is an easy way to crystallize protein, especially heterogeneous nucleants. The main

researches of nucleation agents have studied some natural surfaces such as horse tail hairs, minerals, fibers, lipid layers;54-57 fabricated surfaces like silicons and polymeric films, showing specific pores and wrinkles;58,59 epitaxic nucleants which require a correlation between the lattice of the heterogeneous nucleating agent and the nascent protein crystal;60 However, it is difficult to make these materials consistent in nanoscale from batch to batch, such as physical and chemical property, which is unfavorable for repeating experiment and explaining the crystallization mechanism. Herein, we propose for the first time that DNA origami can be utilized as a seed to promote protein crystallization. With programmable property and precise recognition, we can accurately control size and morphology of DNA origami and ensure perfectly consistent performance of this material. In this study Catalase was used as a model protein, along with DNA origami constructed from M13 and staple strands.

Scheme 1. Experimental setup of the hanging-drop vapor diffusion chamber, containing 5 droplets of different mixture types, with a reservoir of precipitant solution at the bottom.

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Figure 1. (a) Crystal percentage, the percentage of droplets with observable crystals in them, after 11 days with 6 nM DNA origami (triangle), unfolded M13rs with unfolded M13 plus random staple strands (pentagon) and Blank without any additives (five-pointed star) for Catalase （ Cat ） at 1.0 mg/mL at 4 °C, (b) crystal percentage with 6 nM DNA origami (triangle) and Blank without additives (five-pointed star) for Catalase (Cat) at 1.5 mg/mL (red line) and 7.0 mg/mL (pink line) at 4 °C, and (c) crystal percentage with 6 nM DNA origami (triangle) and Blank without additives (five-pointed star) for Catalase (Cat) at 3.0 mg/mL (blue line) and 5.0 mg/mL (orange line) at 4 °C

As illustrated in Scheme 1, we chose DNA origami with two conformations: ‘Rectangle’ which consists of rectangular-shaped DNA origami, ‘Tube’ which consists of tubular-shaped DNA origami (Dimeter = 11 nm) folded from Rectangle origami with the aid of staple strands at the sides. These origami structures have the same total surface area but different geometries. As control, we chose ‘M13rs’ which consists of the M13 framework and random non-binding staple strands, ‘M13’ which consists of the M13 framework, and ‘Blank’ which had no additives other than the precipitant solution. The crystallization was carried out using the hanging-drop vapor diffusion technique. This work would investigate how the addition of DNA origami in

protein crystallization buffer would enhance the probability of crystallization and to demonstrate unequivocally that the structure of DNA origami as seeds is essential in promoting protein crystallization. The attempt to crystallize protein with DNA origami not only opens a new application of DNA origami, but also provides a new platform for protein crystallization for isolation and purification. In the long term, with the advantage of precise control over DNA origami, this approach may reveal the fundamental protein nucleation process and generate a step change in development of biopharmaceutical products.

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RESULTS AND DISCUSSIONThe influence of DNA origami on protein crystallization was first examined. As depicted in Scheme 1, the hanging-drop vapor diffusion technique was employed, with the arrangement of DNA origami in two conformations; M13rs and M13 with a further control experiment (Blank, without DNA present). Each mixture type was repeated 144 times. The degree of crystallization was evaluated with the quantity denoted as crystal percentage, i.e., the percentage of droplets with observable crystals present within them over a period of time, usually when crystal growth reaches a plateau. Figures 1a, 1b and 1c show the increase in the number of droplets with crystals over a period of 11 days. Figure 1a demonstrates that 6 nM DNA origami (average value of Tube and Rectangle) had a clear effect on the crystallization probability of Catalase, where the presence of DNA origami resulted in a significant increase in crystal percentage, from 1% to 17%. The presence of M13rs provided a small improvement in crystallization but to a much smaller extent, suggesting that the ordered structure of the DNA origami is one of the main contributions to this effect. We believe that the nonspecific intermolecular interactions between

the DNA origami and the protein molecules cause the protein molecules to adsorb onto the DNA nanostructure, resulting in an increase in the local protein concentration61-63 as well as potentially stabilizing the protein conformation. And also, DNA origami and Catalase both have overall negative charge47,64, remaining Mg2+ ions in DNA origami assemble process may act as salt bridge to link DNA origami and Catalase molecules, which possibly also facilitates protein molecules to interact with DNA origami. This ordered nanostructure may provide a significantly easier pathway to the super-saturation conditions required for crystal nucleation, compared to the disordered case for both M13rs and Blank.

Figure 1b shows a comparison of the crystal percentage for two different protein concentrations (1.5 and 7.0 mg/mL) demonstrating that the effect of 6 nM DNA origami is reduced at higher protein concentrations. This is further demonstrated in Figure 1c where the other concentrations of Catalase (3.0 and 5.0 mg/mL) are displayed. From Figure 1, it is concluded that the extent to which 6 nM DNA origami promoted protein crystallization was proportionally greater at lower concentrations and smaller at higher concentrations of Catalase.

Figure 2. Crystal percentage with Blank without additives (five-pointed star), 6 nM M13rs with unfolded M13 plus random staple strands (pentagon) and unfolded M13 (rhombus) for Catalase (Cat) at 1.0 mg/mL (black line), 1.5 mg/mL (red line), 3.0 mg/mL (blue line), 5.0 mg/mL (orange line) and 7.0 mg/mL (pink line) at 4 °C.

At a Catalase concentration of 1.0 mg/mL, the crystal percentage increased by more than 11 times from a success rate of 1.4% for the Blank mixture to 16.3% for the DNA origami. For the 1.5 mg/mL Catalase concentration however, the crystal percentage, proportionally, increased by

significantly less ~5 times from 4.2% for the Blank mixture to 24.7% for the DNA origami.

At higher protein concentrations, there is a greater probability of protein molecules aggregating together and achieving the necessary critical size for nucleation. Therefore,

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crystallization generally occurs more readily with increasing super-saturation conditions because of the larger number of protein molecules overcoming the energy barrier. The influence of DNA origami therefore became much smaller since crystals could readily form, independently of the DNA origami structure. At lower concentrations however, it seems the DNA origami provides an ordered framework on which protein molecules can form clusters more easily. This aspect of DNA origami’s influence on crystallization at low protein concentrations indicates the excellent potential of DNA origami as a crystallization booster for systems with scarcer amounts of the protein. In addition to improving systems where the protein is difficult to crystallize, DNA origami could significantly reduce the waste of valuable protein that may be difficult to acquire or synthesize and so maximize the efficient use of protein resources.

Figure 2 shows how both 6 nM M13 and M13rs did very little to improve crystallization versus Blank consistently at all five protein concentrations ranging from 1.0 to 7.0 mg/mL. So while DNA origami is seen to significantly promoted protein crystallization, it is clear that its constituent components in M13rs on their own appear to have nowhere near the same effect. In the ordered structure of DNA origami, all of the

DNA strands are held tightly together within the M13 framework. We suspect that this results in a greater collection of elevated DNA concentration sites, allowing for stronger intermolecular interactions between the DNA origami and the protein molecules. For the M13rs, which contains same amount of DNA as DNA origami, but with random sequence, therefore, the M13 and the staple strands are randomly dispersed in solution without ordered structure, which is a likely cause for the distinction seen in Figure 1a between DNA origami and M13rs. While the large M13 molecule plus random strands might still have provided an aggregation site for the protein molecules hence the small improvement in protein crystallization, its effect was not comparable to that of the highly structured DNA origami.

Additionally, the conformation of DNA origami, in the form of rectangular sheets or tubular hollow cylinders, was investigated. Figure 3 shows that 6 nM DNA origami in the form of Rectangles or Tubes, unexpectedly, did not improve crystallization rates further. The structural differences between the two are expected to have a noticeable impact on the crystallization rates, given that the rectangular conformation has twice the exposed surface area versus the tubular configuration, and therefore would allow for double the potential sites for intermolecular

Figure 3. Crystal percentage over a period of 11 days with 6 nM Rectangle conformation DNA origami (square) and Tube conformation DNA origami (circle) for Catalase (Cat) at 1.0 mg/mL (black line), 1.5 mg/mL (red line), 3.0 mg/mL (blue line), 5.0 mg/mL (orange line) and 7.0 mg/mL (pink line) at 4 °C.

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Figure 4. Crystal percentage for 5 mg/mL Catalase (Cat) over a period of 11 days with the Tube conformation DNA origami at different concentrations of 0 nM (black line), 2 nM (red line), 4 nM (blue line) and 6 nM (pink line) at 4°C.

interactions with the protein molecules. On the other hand, the tubular hollow cylinders may provide a confinement effect.47,65-67 As we know, only in pores with suitable size, can protein molecules accumulate efficiently.65,67 Here, it may be likely that the effects of point interactions are as effective as confinement, since trapping protein molecules in the pseudo-pores of the DNA origami would result in an increase in local protein concentration. And also, we designed different pore sizes of tubular DNA origami (diameter = 8, 11, 22 nm) at the same concentration to promote Catalase crystallization respectively. As a result (Figure S2), all the three pore sizes of Tube can remarkably improve Catalase crystallization success rate compared with Blank condition, but has little difference to promote protein crystallization success rate among these DNA origami with various diameters. Figure 4 displays the crystal percentage with increasing Tube conformation DNA origami concentration from 0 to 6 nM. From these results, Tube concentration was increased from 2 to 4 nM, but the crystallization success rate respectively did not double in the same way. This indicates a non-linear relationship between the exposed surface area of the DNA origami and the rate of crystallization, suggesting that other factors are behind the Rectangle and Tube conformations having similar crystal growth rates despite their shape difference. One possible explanation is that beyond a certain concentration, there may exist a critical seed-loading amount, above which additional DNA origami seeding has no effect on the amount of crystallization rate. Within this system, the data in Figure 4 suggests that this potential limit has been reached at a DNA origami concentration of 4 nM, with very little further improvement in crystallization at 6 nM.

The many factors that affect DNA origami in promoting protein crystallization, including the

intermolecular forces, the structure of the DNA, potential artificial porosity and seeding effects, are all still relatively unexplored in terms of their specific mechanics with crystallization and are areas for future study. Future efforts in applying our methodology would focus on more complicated structures of DNA origami with size tuned at 1 nm resolution, adapting it to fragile and difficult to crystallize proteins, in order to establish a general scheme for protein crystallization and separation.

CONCLUSIONWe report a new approach using DNA origami as seeds to improve the crystallization probability of complex molecules such as proteins. The probability for protein crystallization was enhanced with the presence of DNA origami, especially at low protein concentrations. The extent of this improvement was, unexpectedly, similar for both rectangular and tubular conformations of DNA origami, suggesting that several mechanisms may be involved in protein crystallization promoted by DNA origami. Additionally, it was seen that there exists a critical loading of the DNA origami, above which there was no improvement to the probability of crystallization. The increase in crystallization was not observed in cases where the DNA was not ordered. We are trying to realize Seeman’s idea that protein can be crystallize within DNA scaffold. This may be successful with DNA origami clusters with numerous pores designed by Y. Ke’s group,69,70 research which is on-going in our group. The ability to precisely manipulate DNA origami structure and chemistry will allow for the potential to finely control macromolecular assembly such as nucleation and crystallization to a much greater extent.

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MATERIALS AND METHODSMaterials

Catalase as lyophilized powders (C40) and 2-methyl-1, 3-propanediol (MPD) were purchased from Sigma-Aldrich. M13 was purchased from New England Biolabs. All staple strands purified by HPLC were purchased from Zixi Bio (Beijing, China). HEPES was purchased from Xinjing Biological Science Technologies Co. (Beijing, China). PEG-4000 was purchased from Alfa Aesar. CH3COOH was purchased from Guangfu (Tianjing, China). Mg(OAc)2, EDTA and Tris were purchased from Tianjin Fuchen (Tianjing, China). For all reagents, analytical grade was purchased.Protein samples

Catalase was dissolved in 25 mM HEPES (pH 7.0). Filtration of protein samples and all the solution for crystallization through 0.22 µm mesh size filters was the standard procedure used before setting up trials for crystallization. The concentrations of Catalase were 1.0, 1.5, 3.0, 5.0 and 7.0 mg/mL.DNA origami preparation

DNA origami was prepared using an established procedure detailed in literature.68 The DNA origami was prepared in an assembly buffer containing: 12.5 mM Mg(OAc)2, 20 mM CH3COOH, 40 mM Tris, 2 mM EDTA, pH 8.0. It was annealed at 95 C for 5 mins, then exposed to a temperature decrease at a rate of 1 C/min until 4 C. The size of the rectangular DNA origami (Rectangle) used was 35 nm × 100 nm. The diameter of the corresponding rolled-up tubular DNA origami (Tube) was 11 nm × 100 nm with 2 nm in wall thickness. Atomic force microscopy (AFM) was employed to characterize the morphology of the rectangular and tubular structure (Supporting Information, Figure 1).

As the protein crystallization required a precipitant solution of 25 mM HEPES (pH 7.0), 5% w/v PEG-4000 and 5% v/v MPD, the assembly buffer of the DNA origami needs to be removed and replaced with said precipitant solution, using buffer exchange.68 After removal of the assembly buffer, DNA origami was dissolved in 25 mM HEPES (pH 7.0), 5% w/v PEG-4000 and 5% v/v MPD. The concentration of DNA origami after buffer exchange was 6 nM as determined with a UV-Vis Spectrophotometer. The morphology of DNA origami was characterized by AFM. It was verified that DNA origami can stay intact for 15 days at least (Supporting Information, Table S1). Therefore, we are certain that DNA origami remains undamaged over the crystallization period.

Three control conditions were made to compare to the DNA origami mixtures: M13rs - M13 with random strands incapable of hybridizing or assembly into DNA origami collectively dissolved in precipitant solution at 6 nM concentration, M13 - M13 dissolved in precipitant solution at 6 nM concentration, Blank – precipitant

solution with no additional chemicals.Protein crystallization experiment

Protein crystallization was performed with custom-made plates using the hanging-drop vapor-diffusion method. Crystallization drops were made by mixing 1.5 μL of protein solution and an equal volume of one of the five precipitant solution mixtures designated as: Rectangle, Tube, M13rs, M13, and Blank. The drops were equilibrated against 333 μL of the precipitant solution in the reservoir well as shown in Scheme 1. The precipitant solution for Catalase was comprised of 25 mM HEPES (pH 7.0), 5% w/v PEG-4000 and 5% v/v MPD. After the crystallization plates were sealed, the plates were set in an incubator and incubated at 4 C for two weeks with daily examination. The effects of the DNA on the nucleation of protein crystals were studied by counting the number of drops with crystals. Each drop type was repeated 144 times for one data set of all five mixtures at all five concentrations. The crystals in the crystallization solution were observed using an optical microscope (SZM-T4).

Supporting Information.AFM pictures of DNA origami, Figures of protein crystallization success rate with 8, 11, 22 nm diameter, crystal pictures of all the conditions and base sequence of DNA origami.

ACKNOWLEDGMENTSThis work was supported by the National

Natural Science Foundation of China (21474059, 21372258). ZY and JYYH acknowledge the Royal Academy of Engineering – Research Exchange China and India (RECI) programme (Reference: 1314RECI047). JYYH also acknowledges the EPSRC (EP/N015916/1) for funding. We acknowledge the Tsinghua University Branch of China National Center for Protein Sciences Beijing for technical support.

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