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Hierarchical Assembly of DNA Filaments with Designer Elastic Properties Barbara Sacca, Wolfgang Pfeifer, Pascal Lill, and Christos Gatsogiannis ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b06012 • Publication Date (Web): 15 Nov 2017 Downloaded from http://pubs.acs.org on November 16, 2017

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Hierarchical Assembly of DNA Filaments with Designer Elastic Properties Wolfgang Pfeifer,1 Pascal Lill,2 Christos Gatsogiannis2 and Barbara Saccà1*

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Centre for Medical Biotechnology (ZMB) and Centre for Nano Integration Duisburg-Essen

(CENIDE), University of Duisburg-Essen Universitätstr. 2, 45117 Essen (Germany) *E-mail: [email protected].

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Department of Structural Biochemistry

Max-Planck-Institute of Molecular Physiology Otto-Hahn-Straße 11, 44227 Dortmund (Germany)

KEYWORDS: DNA nanotechnology, scaffolded DNA origami, self-assembly, filaments, persistence length.

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ABSTRACT: The elastic features of protein filaments are encoded in their component units and in the way they are connected, thus defining a biunivocal relationship between the monomer and the result of its self-assembly. Using DNA origami approaches, we constructed a reconfigurable module, composed of two quasi-independent domains and four possible interfaces, capable of facial and lateral growing through specific recognition patterns. Whereas the flexibility of the intra-domains region can be regulated by switchable DNA motifs, the inter-domain interfaces feature mutually and self-complementary shapes, whose pairwise association leads to filaments of programmable periodicity and variable persistence length. Thus, we show here that the assembly pathway leading to oligomeric chains can be finely tuned and fully controlled, enabling to emulate the formation of protein-like filaments using a single construction principle. Our approach results into artificial materials with a large variety of ultrastructures and bending strengths comparable, or even superior, to their natural counterparts.


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Natural fibers, such as actin filaments, microtubules and bacterial flagella, are striking examples of what can be done with a fairly limited selection of building blocks and a hierarchical assembly rule.1 Although being composed by the periodic repetition of identical units, often displaying relatively poor intrinsic properties, their spatial organization at multiple length scales results in supramolecular fibers with mechanical properties surpassing those of the single components by orders of magnitude.2 Nature’s modular and hierarchical design approaches are therefore effective pathways to highly ordered molecular architectures with superior traits and have been source of inspiration for the synthesis of artificial materials with applications in drug delivery, cellular scaffolding and tissue regeneration.3,4 Despite the notable progresses, still one of the major challenges in the fabrication of biomimetic systems is the achievement of full control of nanometer-sized features in structures built at the micro- and even macroscopic scale. A promising approach to overcome this drawback relies in the use of DNA.5-13 Using this molecule as the building block of supramolecular objects and employing hierarchical self-assembly rules, the atomic precision of base pair recognition can be extended to larger scales till the micrometer regime,14-16 eventually enabling the challenging realization of macroscopic three-dimensional crystals.17,18 In this way, a variety of filamentous-like structures have been realized, such as nanotubes with diameters ranging from 7 to 20 nm,19-21 nanoribbons,22 six-helix bundles23 and compliant DNA elements,24,25 characterized by a contour length of several micrometers and a bending stiffness about 100-fold higher than that of the double helix. Notably, the architecture and mechanical properties of such DNA polymers are uniquely dictated by the structural features of the constituent monomers and their interaction modes, thus recapitulating a typical aspect of natural


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protein filaments, that is, the biunivocal relationship between the starting unit and the result of its monodimensional growth. So far, structural studies on linear homo-oligomeric proteins allowed identifying two distinct and mutually exclusive mechanisms of non-covalent assembly.26,27 The first, so-called commutative mechanism, involves the head-to-tail interaction between heterologous surfaces, i.e. distinct patches from two individual subunits. Sequential association of monomers according to this assembly strategy typically gives rise to open filamentous-like structures, such as actin or microtubules, with important scaffolding roles. Contrarily, the non-commutative mechanism is often associated to isologous binding between identical patches of distinct subunits. This binding mode saturates the assembly and results into finite structures, usually described by a point-group symmetry. Self-association of such intermediates into one-dimensional arrays often occurs via shape-induced stacking and is permitted only when each subunit comprises multiple binding sites. Typical structures of this type are chaperones and other barrel-like proteins, among which the 13.5 MDa mega-hemocyanin (a sort of giant protein origami) is probably one of the most prominent examples.28 The emerging scenario thus embraces two separate assembly pathways, which evolved from geometrically distinct units to end up into filamentous architectures with specialized cellular functions. The next challenge in the design of biomimetic filaments is therefore to develop a more general manufacturing technique, which allows the production of functionally different filaments starting from a single building component. The idea is to design a DNA module which holds the potential to undertake distinct assembly routes, but whose final fate can be rationally programmed by selecting the forces acting between desired pairs of interfaces. Such a modular approach would be not only more convenient from a design perspective, but most importantly


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would enable to directly correlate a tiny variation in the subunits interactions with a macroscopic change in the physical properties of the resulting polymer, as for example its bending stiffness, expressed in terms of persistence length. In this sense, such a method would permit to achieve a high control of materials properties: from the chemical to the physical level and from the nanoto the macroscopic scale. To reach this goal, we realized a DNA-based building unit capable of facial and lateral associations through a combination of non-covalent interactions. Our approach relies on the exploitation of specific, directional and tunable forces, such as hydrogen bonding and base stacking often in a combined fashion, to induce the hetero- or isologous binding of, respectively, distinct or identical interfaces from the same subunit. In this way, we show that different building principles of natural protein filaments can be merged into a single construction program, using a segmental DNA unit and guiding its self-assembly through step-wise association of isolated intermediates. Thus, by manipulating the assembly process at selected stages, a simple “mix-and-match” procedure can be applied to fabricate a number of hierarchically structured materials with controlled composition and bending stiffness comparable, or even superior, to their natural counterparts.

RESULTS AND DISCUSSION

One DNA unit with four interfaces and multiple binding modes. Our DNA building block is composed of two subunits, called A and B, joined at opposite sides by two unpaired scaffold segments about 100 bases long (Fig. 1a). Each subunit of the module consists of a 24-helix bundle, with a diameter of ca. 18 nm and a length, respectively, of 64 nm (A) and 41 nm (B). The module displays four interfaces, named as A-, A+, B- and B+ (indicated by a two-colors


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arrow in Fig. 1a): whereas the A+ and B- interfaces are involved in intra-unit interactions, the Aand B+ interfaces mediate inter-unit interactions. The positive or negative sign defines here the “polarity” of the interface and provides a convenient way to classify the interaction between two subunits according to their relative orientation. Thus, for example, interfaces of opposite polarity associate when the corresponding subunits are oriented in parallel (Fig. 1b-e), whereas antiparallel subunits interact through faces of the same polarity (Fig. 1f and g). Besides the polarity, each interface is characterized by a unique patch of helices whose blunt ends belong to different planes perpendicular to the helical axes and positioned at n base-pair distance from a virtual reference layer (indicated as 0). Thus, for example, interface A+ (Fig. 1b, left panel) displays four domains of helices, with blunt ends located at 10 bp, 7 bp and 3 bp distance from the reference plane and is therefore defined by the “shape-sequence” (10.7.3.0) (Fig. 1c, left panel and Supplementary Fig. 1). Similarly, interface B- features four patches of blunt ends denoted as (0.3.7.10) (Fig. 1b and c, right panels and Supplementary Fig. 2). The complementarity of the two shape-sequences (10.7.3.0/0.3.7.10) ensures full matching of A+ and B- interfaces upon a rotation of 180° along the direction perpendicular to the helical axes and results in a 10 bp helical bridge between the two reference planes (Supplementary Fig. 3). The outer interfaces, A- and B+, instead display three patches of blunt ends with (0.3.7) and (7.4.0) shape-sequences (Fig. 1d, left and right panels, respectively and Supplementary Figs. 4 and 5). Also in this case, their full complementarity guarantees tight binding through formation of additional 24 helical segments, each 7 bp long, between the two patches at position 0 (Fig. 1e and Supplementary Fig. 6). Notably, A- as well as B+, are also capable of self-recognition and binding due to the partial self-complementarity of their shapes (Fig. 1f and g, respectively). Matching of two A- interfaces occurs by fixing the orientation of the former and rotating the


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latter of -60° around the axis parallel to the helices, followed by a 180° rotation in the direction perpendicular to them (Fig. 1f, left panel). In this way, the two patches of helices at position 0 and 7 can merge into a continuous double helical region, whereas the two sets of blunt ends at position 3 do not touch, leaving 1bp gap in between (Fig. 1f, right panel and Supplementary Fig. 7). Performing a specular geometric operation on two B+ interfaces leads also to their partial self-complementarity (Fig. 1g, left panel). However, in this case, only the patches at position 4 match, leaving two 1 bp-long gaps between the sets of helices at position 0 and 7 (Fig. 1g, right panel and Supplementary Fig. 8). Summarizing, our design of the building block enabled four distinct unit-to-unit interactions, namely, A+/B-, B+/A-, A-/A- and B+/B+, each one defined by unique polarity and shape-complementarity rules. In particular, mutual shape-complementarity between patches of opposite polarity (i.e. A+/B- or B+/A-) resulted in the head-to-tail interaction between AB subunits, leading respectively to AB monomers or (AB)n oligomers. Both species emulated the commutative association mechanism of protein assembly (indicated by parallel blue and orange arrows in Fig. 1c and e). On the other hand, the non-commutative assembly mechanism was exemplified by the isologous binding between patches of the same polarity (indicated by antiparallel arrows of the same color in Fig. 1f and g) and resulted in the tail-to-tail (A-/A-) or head-to-head (B+/B+) orientation of connected subunits, yielding respectively BA2B or AB2A homodimeric species. Clearly, in the next assembly step another pair of interfaces can be selected for binding and isolated intermediates from different pathways can be linked together, thus notably increasing the number of structural combinations accessible from one single precursor. Applying this strategy, we created more than fifteen homo-oligomeric filaments, all displaying exactly the


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same chemical content but differing in the pattern of subunits, as well as in the type and number of interactions between them. We created two classes of polymer chains, a totally flexible form and a less flexible, semi-rigid form (indicated as “flex” and rig”, respectively). These shared the same subunits sequence but featured either a lack or a maximal number of A+/B- interactions in the central region of the DNA module, thus allowing us to explore the relationship between chemical bonds and mechanical rigidity in large-scale assemblies. The bending stiffness of the DNA chains, expressed in terms of persistence length, was measured by observing the trajectories of the filaments upon sample deposition on a surface (all constructs realized in this work are reported in Supplementary Figs. 9-11 and their relative structural parameters are listed in Supplementary Tables 1-5).

From monomers to homo-dimers. The fundamental unit, precursor of all our constructs, is represented by the flexible AB monomer (ABflex, Fig. 2a, left side). Its structural features were described by the angle (γ) between the two segments, A and B, and by the end-to-end distance (R) between their edges (ensemble electrophoretic analysis of monomers and dimers is given in Supplementary Fig. 12, whereas analysis of all our flexible constructs is provided in Supplementary Fig. 13. Additional atomic force microscopy (AFM) and transmission electron microscopy (TEM) images for each construct are given in Supplementary Figs. 14-55). AFM imaging of the flexible AB monomer revealed a broad angle distribution centered at 182° (Fig. 2b and c), indicating that the two subunits are relatively free to move and that the entropic force generated by the single-stranded DNA segments connecting them is basically negligible (Supplementary Fig. 56 and Supplementary Note 1). Such a rotational freedom was completely suppressed upon addition of hybridization strands that linked together the A+ and B- interfaces


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in the intra-unit region (Fig. 2a, right side). This resulted in one of the four possible initial species: i.e. the stiffer form of the monomer (ABrig), with an end-to-end distance of ca. 97 nm, slightly below the expected theoretical value of 105 nm (Fig. 2d and Supplementary Table 1). Although being more rigid than the ABflex precursor, this monomer still displays an intrinsic flexibility, as predicted by analysis of the thermal fluctuations of the structure using the free online CanDo software (https://cando-dna-origami.org)29 (Supplementary Fig. 57). Insertion of hairpin loops between the two subunits reduced their free movement and provided a tool to trigger and monitor the structural reconfiguration of the monomer from the flexible to the semirigid analog (Supplementary Fig. 58a). To this end, biotin-modified strands complementary to the hairpin loops were used as fuels and the sample was imaged by AFM upon streptavidin addition, confirming the structural change of the seam region (Supplementary Fig. 58b-e). Both the flexible and the semi-rigid monomer were additionally characterized by TEM. When compared to the AFM data, the flexible form showed a narrower angle distribution centered at about the same value (Fig. 2e and f), which could be partially due to preferred orientations adopted by the particles on the carbon layer of the EM grid. However, the stiffer analog displayed a more homogeneous length, in optimal match with the theoretical expected value (Fig. 2g and Supplementary Table 1), thus suggesting that in general, TEM settings and sample preparation better preserve the structural properties and integrity of the specimen. Addition of strands to create blunt ends at one of the two outer interfaces (either A- or B+) led to formation of homodimers (respectively, BA2B and AB2A; Fig. 3). Both structures are the consequence of isologous matching between partially complementary shapes and represent the intermediate species attainable through assembly saturation at one of the two inter-unit interfaces (Fig. 3a and g, respectively). Correct and almost quantitative formation of the dimers


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was visualized by electrophoretic analysis (Supplementary Fig. 12) as well as by AFM and TEM, with the latter technique showing again the best agreement between the observed structural data and the theoretical values from design. The flexible dimers revealed the expected structural features: BA2Bflex showed two outer segments of length 39±4 nm and a central segment of length 128±7 nm (Fig. 3b), in agreement with the theoretical values of 41 nm and 128 nm, respectively associated to the outer B subunits and two central A subunits linked together (Supplementary Table 2). The AB2Aflex dimer displayed instead two outer segments of length 64±5 nm and a central segment of length 81±6 nm (Fig. 3h), well corresponding to the expected values of 64 nm (A) and 82 nm (B2) (Supplementary Table 3). In both dimer constructs, the relative orientation of the freely jointed segments appeared to be broadly distributed around 180°, suggesting their almost negligible angle correlation (Supplementary Fig. 59 and Supplementary Tables 2 and 3). As for the monomers, the stiffer dimer analogs were obtained by targeting the A+/B- interface with hybridization strands; this produced in both cases linear structures about 200 nm long (Fig. 3d-e and j-k). These dimers were additionally characterized by AFM upon topographical marking of the reconfigurable A+/B- seam region (Fig. 3f and l, respectively, see also Supplementary Tables 2 and 3).

Multimers with different ultrastructure and bending stiffness. The fourth and last species accessible by self-assembly of the initial monomer at one pair of interfaces is the oligomeric (AB)n chain. This resulted from the front and back association of the linear AB module at the B+/A- interface and featured a periodic repetition of identical segments about 107 nm in length oriented in a parallel fashion (Fig. 4a). Despite the shape complementarity of B+ and Ainterfaces would allow them to be linked by stacking interactions, using hybridization forces to


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connect consecutive blocks together ensured full control over the directionality of binding. This guaranteed growth of a head-to-tail construct, preventing formation of competing isologous species. In its flexible form, that is, in absence of intra-unit interactions, the (AB)n,flex oligomer showed a tendency to cyclize into regular polygonal structures of identical edges but different sizes, as observed by AFM and TEM (Fig. 4b and c; see also Supplementary Figs. 23 and 45). We attribute this phenomenon to the relatively low concentration of the starting monomer (ca. 1 nM), which therefore enhances the probability of intra-oligomer binding between the last and the first unit of the chain. Statistical analysis of the contour lengths revealed a high prevalence of small structures (n = 3) at the AFM and a more even distribution of sizes (3 < n < 9) for structures observed at TEM (Fig. 4d, grey and red bars, respectively). Addition of hybridization staples at the A+/B- interface produced stiffer (AB)n oligomers (Fig. 4e and f; Supplementary Figs. 24 and 46), with a persistence length (Lp) of 11.9 ± 0.5 µm and 8.8 ± 0.8 µm, for chains observed at AFM and TEM, respectively (Fig. 4g). Interestingly, the maximal contour length observed for rigid chains of comparable growing time was also dependent on the technique used, with TEM samples reaching about 5 µm length and mica-adsorbed structures only 2 µm length (all structural parameters of the flexible and semi-rigid oligomeric chains are reported in Supplementary Tables 4 and 5, respectively). Altogether, these results again suggest that surfacerelated properties may strongly affect polymer trajectories, probably through kinetic trap of the chains in out-of-equilibrium conditions. In support to this hypothesis, we performed a Kurtosis test30,31 on a set of few tens of (AB)n,rig molecules, imaged by AFM and TEM, and in both cases obtained large deviations from the theoretical value of 3 expected for chains fully equilibrated on the surface (Supplementary Fig. 60; a theoretical treatment of polymer trajectories according to the worm-like chain model is reported in Supplementary Note 2). In particular, the mica support


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and/or the AFM imaging conditions appeared to have a more harmful impact on the structural properties of the chain, supposedly as a consequence of strong surface interactions that may hinder conformational equilibrium, induce bonds breakage or cause differential adsorption rates of the various species in solution (Supplementary Fig. 61). For this reason, comparison between the persistence lengths of different constructs was done using the trajectories of the DNA chains observed by TEM measurements. Until now we considered the simplest form of filament, originated after one-step assembly at one interface (B+/A-) and therefore characterized by one single segment (BA) periodically repeated along the chain. Starting from the ABflex precursor and applying step-wise assembly at distinct pairs of interfaces enabled to increase the structural complexity of the oligomer, in terms of pattern, periodicity and relative orientation of the constituent subunits. For example, upon a first stacking assembly at the B+/B+ interface, the resulting AB2A flexible dimers were linked at the A-/A- interface, thus yielding oligomers of the type (A2B2)n,flex (Fig. 5a). The same oligomer was obtained starting from BA2B and saturating the assembly at the B+ interface. In both cases, the ultrastructure of the oligomer featured a periodic repetition of two segments of different length (A2 and B2): the former derived from two divergent AB units and the latter from two convergent AB units (blue and orange arrows in Fig. 5a). This geometric arrangement well illustrates the isologous assembly principle based on building elements with two distinct binding sites. In another procedure, the same homodimers, i.e. AB2A and BA2B, were instead linked by hydrogen bonds at the B+/A- interface, thus leading to a larger module of four segments, namely B2, AB, A2 and BA, whose periodic repetition yielded the corresponding (AB2ABA2B)n,flex oligomer (Fig. 5b).


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Observation of dimer-derived flexible chains by TEM revealed again formation of closed polygonal structures of different sizes (Fig. 5c and d). As expected, (A2B2)n,flex chains showed a periodic alternation of two segments about 120 nm and 80 nm long, consistent with A2 and B2 traits (Fig. 5c and Supplementary Figs. 26 and 47). Similarly, (AB2ABA2B)n,flex chains were composed by the periodic repetition of four segments (about 80 nm, 100 nm, 120 nm and 100 nm long), corresponding to B2, AB, A2 and BA traits (Fig. 5d and Supplementary Figs. 29 and 50). Both A2B2 and AB2ABA2B modules originated by step-wise assembly of the AB precursor and had a length correspondingly equal to twice and four-fold 100 nm; such module´s length was then periodically repeated along the growing chain. This structural aspect, defined as the periodicity of the oligomer, clearly emerged by statistical analysis of the contour lengths of the flexible chains that showed the appearance of distribution peaks centered at LC = (210±16)n nm, for the (A2B2)n,flex (Fig. 5e, grey peaks) and LC = (415±7)n nm, for the (AB2ABA2B)n,flex (Fig. 5e, red peaks), where n is the number of repeated modules. These periodical lengths are indeed, respectively, twice and four times the size of one AB unit. Interestingly, we observed a decrease in the yield of cyclization when going from the (AB)n,flex to the (A2B2)n,flex till the more complex (AB2ABA2B)n,flex oligomer (respectively 100%, 78% and 55%; see also Supplementary Table 4). This was accompanied by a larger presence of unreacted precursors and open chains, particularly evident in the samples imaged by AFM (cfr. Supplementary Figs. 29 and 50). To gain more insights into the nature of our flexible oligomeric structures in solution, we performed an electrophoretic mobility assay (Supplementary Fig. 13). We observed that, with the exception of (AB2ABA2B)n,flex, the chains were almost free of monomeric or dimeric precursors, indicating that chain elongation was quite efficient and that the presence of precursors and partially broken structures in the samples imaged by AFM and TEM was probably due to a post-assembly


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process, most likely the result of strong disrupting interactions with the surface or structural damages caused by sample preparation or sample scanning. Altogether, these data point out that the oligomerization yield and the extent of cyclization are affected by (i) the number of assembly steps, (ii) the nature and strength of the unit-to-unit interaction and (iii) the method used for sample observation, with surface deposition being probably more damaging than gel electrophoresis. Finally, saturating the assembly at the remaining A+/B- intra-units interface drastically reduced the flexibility of the chains, resulting in rods of several micrometers length and much higher stiffness. TEM analysis of the polymer trajectories (Fig. 5f and g) allowed to calculate values of persistence length of few tens of micrometers (Lp = 28.6 ± 0.8 µm and 19.1 ± 1.7 µm, for (A2B2)n,rig and (AB2ABA2B)n,rig, respectively; Fig. 5h and Supplementary Table 5).

Combining facial and lateral interactions. In our approach, facial growth of the 24-helix bundle essentially resulted into linear extension of the initial module along one direction. In this sense, only two of the four accessible interfaces were used for oligomerization (A- and B+), whereas the other pair (A+ and B-) mediated the switching between the flexible and the semirigid form. We therefore modified our approach to create a module capable of exploiting all faces for oligomerization, enabling to increase the structural complexity of the growing chains and generalize the method to any building unit made of N freely jointed segments. To permit facial growth of all four interfaces, the B subunit was flipped on top of the A subunit and linked to it through lateral associations, such that A and B pointed into opposite directions (Fig. 6a and Supplementary Figs. 51 and 62). Antiparallel flipped dimers were isolated upon connection of two A+/B- or two B+/A- interfaces, leading, respectively, to divergent or convergent flipped constructs (Fig. 6b and c, respectively; Supplementary Figs. 52 and 53). Whereas A+/B-


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connections (previously intra-units interfaces) worked equally well using hybridization or stacking forces, B+/A- (previously inter-units) interfaces could be linked only through hydrogen bonds to prevent formation of alternative stacked structures (Supplementary Fig. 11). Oligomerization of the flipped dimers resulted in formation of superbundles of 48 helices, formed by two antiparallel 24 helix-bundles one on top of the other (Fig. 6d to f). In particular, two types of superbundles were created, having identical ultrastructure but a different set of binding interactions: in the former, (AB)n,flip (h), all interfaces were connected through hydrogen bonds, in the latter, (AB)n,flip (s), alternating hybridization and stacking forces were instead employed (Supplementary Figs. 54 and 55), leading to the highest value of persistence length observed for our DNA-made filaments (about 40 µm; Fig. 6g, last orange bar, and Supplementary Table 5). Thus, combining lateral and facial interactions, all four interfaces of the initial unit could be made accessible for chain growing, resulting in much stiffer polymers with a persistence length almost 1000-fold higher than the double stranded DNA (50 nm) and more than twice the value of natural actin filaments (ca. 15 µm).

CONCLUSIONS In this work, we applied step-wise assembly to a segmented DNA unit and prepared more than fifteen DNA chains with distinct ultrastructures and persistence lengths. Interestingly, an increase in the length of the module (i.e. the periodic block of the chain), from 1 to 2 to 4 AB subunits, did not result in a parallel increase of bending stiffness and assumed a maximal value for the (AB2A)n,rig construct (Fig. 6g). This suggests that the type of forces used to link the units together and their geometric arrangement within the module, rather than their multiplicity, are the main determinants of the bending stiffness of the growing chain. In particular, stacking


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interactions appeared to play a key role, with the density of stacked bonds per module going with the increase in the persistence length of the corresponding chain (Lp (AB2A)n,rig > (BA2B)n,rig ≈ (AB2ABA2B)n,rig > (AB)n,rig; Supplementary Table 5). An anomalous case was the (BA2B)n,rig oligomer, obtained by isologous stacking of BA2B dimers at the B+ interfaces. Despite the efficient formation of relatively long chains with a contour length similar to the value obtained for (AB2A)n,rig (ca. 9 µm, Fig. 6g, cfr. 2nd and 3rd blue bars), the Lp of (BA2B)n,rig was significantly lower than that of the (AB2A)n,rig analog (20.3 ± 1.3 µm vs. 28.6 ± 0.8 µm; Fig. 6g, cfr. 2nd and 3rd orange bars). This was quite surprising, as the sequence of subunits and interaction modes are identical in the two constructs. A possible explanation could rely in the different assembly pathways employed for the construction of the two oligomers, assuming that the last step, i.e. dimers oligomerization, works less efficiently than the first one, i.e. monomer dimerization. Last-step elongation of the BA2B occurs at the B+/B+ interfaces, whose reduced shape-complementarity compared to A-/A- (Fig. 1f and g) would generate a lower joining force and therefore a less rigid oligomer. The important role of stacking forces was also visible by the difference in persistence length obtained for the two 48 superbundles (37.9 ± 1.6 µm and 40.0 ± 1.9 µm, for the (h) and (s) form, respectively; Fig. 6g, last two orange bars). Here of course the increased cross-section is the main responsible for the extreme stiffness of the structures, with the all-hybridized construct being slightly less rigid than the stacked analog. Finally, the orientation of the units within the chain seemed also to be critical, as evidenced by comparing for example the Lp values of (AB2A)n,rig and (AB)rand,rig (28.6 ± 0.8 µm and 19.1 ± 0.6 µm, respectively). Although both oligomers are built up by polymerization of the same precursor and have therefore an identical chemical content, the former displays a regular antiparallel arrangement of AB segments linked by alternating stacking and hydrogen bonds; on the contrary,


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the latter is the result of unsystematic associations of AB units, hybridized at the A+/B- intraregion and stacked one another in a random fashion at one of three possible interfaces: A-/A-, B+/B+ or B+/A-. Altogether, the results obtained show that appropriate design strategies and hierarchical assembly procedures can be used to surpass current limits of synthetic filaments, providing access to a large variety of DNA chains with tailored ultrastructure and improved persistence length. Differently from natural oligomeric filaments, whose assembly fate is encoded in the geometry and interactions of the component units, our synthetic strategy enables to reach distinct outcomes starting from a sole precursor. By controlling chain growth at different stages, the entire pathway can be reshaped in a rationale manner and guided to a desired end. This offers a main advantage: namely the possibility to program unit-to-unit interactions and, most importantly, to understand how tiny changes in the molecular forces between the monomers may affect the global macroscopic behavior of the assembled chain. Finally, our approach can be generalized to linear DNA modules formed by N freely jointed segments: using lateral associations, such modules will provide 2N interfaces, whose controlled pairwise association through selected binding forces would lead to formation of distinct intermediate species, down to a desired assembly pathway. In this way, the cross-section of the bundle can be increased at will, leading to interlinked filaments with enhanced stiffness and predefined orientation of the component chains, which can be in turn employed to construct more sophisticated biomimetic materials.

METHODS


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Design and assembly of the DNA filaments. DNA origami structures were designed with caDNAno (www.cadnano.org v2.2.0) and assembled using a 1:10 molar ratio between the M13mp18 ssDNA scaffold (10 nM) and each of the staple strands, in 1X TEMg buffer (5 mM Tris, 1 mM EDTA, 16 mM MgCl2, pH 8.0). Thermal annealing was performed on a Thermocycler Mastercycler nexus gradient (Eppendorf) by the following protocol: 65°C for 5 min,

65-55

°C

(-1°C/5 min), 55-30°C (-1°C/15 min), 30-20°C (-1°C/min), hold at 20°C. Monomers and dimers were purified via agarose gel extraction of the desired band and recovered using Freeze ‘N Squeeze (Biorad) spin columns: at this end, reaction tubes were incubated at -20 °C for 5 minutes and subsequently centrifuged for 4 minutes at 13,000 rcf at room temperature. Upon adjustment of the Mg2+ concentration, multimers were assembled by mixing intermediate species in equimolar amounts (when necessary) and adding the required staple strands in 10-fold excess for 20 h at 40 °C (if not stated differently). The concentration of monomer and dimers used for assembly of the oligomeric chains was, respectively, 1 nM and 0.5 nM (assuming a roughly 10% yield after gel extraction). Biotin-modified multimers were purified by PEG-purification, prior to incubation with streptavidin.32 In short, the sample was adjusted to 20 mM MgCl2 and then mixed with the same volume amount of PEG-buffer containing 5 mM Tris, 1 mM EDTA, 505 mM NaCl and 15% PEG 8,000 (w/v) followed by centrifugation for 25 min at 16,000 rcf. Subsequently, the supernatant was removed, the pellet was dissolved in TEMg buffer and left overnight for equilibration. The same sample was used for AFM and TEM analysis; in addition, sample preparation (i.e. surface adsorption onto mica or carbon grids) was performed on equally “aged” constructs and repeated several times for at least three independent assembly reactions. This led to hundreds of structures for each construct, enabling to check for reproducibility of the


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results and ensuring reliable statistical analysis of the data. All DNA sequences are provided in the Supplementary Materials. Electrophoretic mobility assays. Agarose gel electrophoresis was performed with 0.4% in 1X TBEMg (45 mM Tris base, 45 mM boric acid, 1 mM EDTA, 11 mM MgCl2, pH 8.0) or 1% agarose in 1X TBEMg (40 mM Tris base, 20 mM boric acid, 2 mM EDTA, 12.5 mM Mg acetate, pH 8.0) for 3h at 80V. Gel chambers were submerged into an ice-water bath and used at 4 °C. Gels were stained with ethidium bromide and imaged with a Typhoon FLA 9000 laser scanner at a resolution of 100 µm/pixel. 1 kbp DNA-ladder was used as reference for electrophoresis analysis (New England Biolabs and Roth, respectively, for 0.4% and 1% agarose gels). Atomic force microscopy. The sample was deposited on freshly cleaved mica surface (Plano GmbH) and adsorbed for 3 min at room temperature. After washing with ddH2O, the sample was dried under gentle argon flow and scanned in ScanAsyst Mode using a MultiModeTM microscope (Bruker) equipped with a Nanoscope V controller. 0.4 N/m force constant cantilevers with sharpened pyramidal tips (ScanAsyst-Air tips, Bruker) were used for scanning. After engagement the peak force setpoint was typically 0.02 volt and the scan rates about 1 Hz. Topographical characterization of the reconfigurable region (A+/B- interface) was performed by mixing an aliquot of purified biotin-containing sample 1:1 (v/v) with 2 µM streptavidin. After 10 minutes of incubation, 5 µl of the sample were applied onto freshly cleaved mica and processed as described above. All images were analyzed using the Nanoscope Analysis v1.5 software. Negative stain transmission electron microscopy and data analysis. DNA origami structures were prepared for TEM as previously described.33,34 4 µl droplets were applied on freshly glow discharged copper grids, covered with a 6-nm continuous carbon film. The sample was applied


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for 60 seconds and blotted from the side with a filter paper (Whatman no. 4). This procedure was repeated several times in order to increase the concentration of DNA origami particles on the TEM grid. Afterwards, the grids were washed with a 10 µl droplet of ddH2O and stained with two droplets of 1% uranyl acetate. All micrographs were taken with a FEI Tecnai Spirit G2 equipped with a LaB6 cathode at an operation voltage of 120 kV. Digital micrographs were recorded with a 4k x 4k CMOS Camera F416 (TVIPS) at a magnification between 6500x – 30000x. Digital micrographs of the ABflex, ABrig, ABflip, 2ABflip(inter) and 2ABflip(intra) were recorded at a nominal magnification of 30.000x with a pixel size of 0.358 nm. We analyzed a total of 12000, 13000, 19700, 2500 and 1000 single particles, extracted from 184, 302, 404, 314 and 156 digital micrographs, respectively. Single particles were selected manually using e2boxer35 and subsequently sorted using the reference free ISAC alignment and clustering approach as implemented in the SPHIRE software package.36 The final class-averages include between 60 and 100, 70-100, 80-200, 20-50 and 20-50 members, respectively for ABflex, ABrig, ABflip, 2ABflip(inter) and 2ABflip(intra). Data analysis and theoretical model. The polymer traces observed by AFM and TEM were measured using a custom-made software suite coded in MATLAB (Supplementary Materials) and analyzed applying the worm-like chain model for structures equilibrated in 3D and trapped on the surface (Supplementary Note 2).30,31

ASSOCIATED CONTENT

Supporting Information. The following files are available free of charge: Experimental and theoretical details, Figures S1- S68, Tables S1-S5, DNA sequences and supplementary references (PDF).


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Author Contributions W.P. performed research, B.S. conceived the project and wrote the manuscript, P.L. performed the TEM measurements, P.L. and C.G. analyzed and discussed TEM data; all authors discussed the results, prepared figures and commented on the manuscript. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft (grant from the CRC initiative 1093 to B.S. and grant GA 2519/1-2 to C.G.) and by the Max Planck Society (to C.G.). C.G. thanks Stefan Raunser for continuous support. We also thank S. Saccà for helpful discussions and development of the MATLAB suite code for image analysis.

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(7) Rothemund, P. W. Folding DNA to Create Nanoscale Shapes and Patterns. Nature 2006, 440, 297-302. (8) He, Y.; Ye, T.; Su, M.; Zhang, C.; Ribbe, A. E.; Jiang, W.; Mao, C. Hierarchical SelfAssembly of DNA into Symmetric Supramolecular Polyhedra. Nature 2008, 452, 198-201. (9) Douglas, S. M.; Dietz, H.; Liedl, T.; Hogberg, B.; Graf, F.; Shih, W. M. Self-Assembly of DNA into Nanoscale Three-Dimensional Shapes. Nature 2009, 459, 414-418. (10) Ke, Y.; Ong, L. L.; Shih, W. M.; Yin, P. Three-Dimensional Structures Self-Assembled from DNA Bricks. Science 2012, 338, 1177-1183. (11) Wei, B.; Dai, M.; Yin, P. Complex Shapes Self-Assembled from Single-Stranded DNA Tiles. Nature 2012, 485, 623-626. (12) Zhang, F.; Jiang, S.; Wu, S.; Li, Y.; Mao, C.; Liu, Y.; Yan, H. Complex Wireframe DNA Origami Nanostructures with Multi-Arm Junction Vertices. Nat. Nanotechnol. 2015, 10, 779784. (13) Benson, E.; Mohammed, A.; Gardell, J.; Masich, S.; Czeizler, E.; Orponen, P.; Hogberg, B. DNA Rendering of Polyhedral Meshes at the Nanoscale. Nature 2015, 523, 441-444. (14) Gerling, T.; Wagenbauer, K. F.; Neuner, A. M.; Dietz, H. Dynamic DNA Devices and Assemblies Formed by Shape-Complementary, Non-Base Pairing 3D Components. Science 2015, 347, 1446-1452. (15) Woo, S.; Rothemund, P. W. Programmable Molecular Recognition Based on the Geometry of DNA Nanostructures. Nat. Chem. 2011, 3, 620-627. (16) Pfeifer, W.; Saccà, B. From Nano to Macro through Hierarchical Self-Assembly: The DNA Paradigm. Chembiochem 2016, 1063-1080. (17) Zheng, J.; Birktoft, J. J.; Chen, Y.; Wang, T.; Sha, R.; Constantinou, P. E.; Ginell, S. L.; Mao, C.; Seeman, N. C. From Molecular to Macroscopic Via the Rational Design of a SelfAssembled 3D DNA Crystal. Nature 2009, 461, 74-77. (18) Zhang, T.; Hartl, C.; Fischer, S.; Frank, K.; Nickels, P.; Heuer-Jungemann, A.; Nickel, B.; Liedl, T. 3D DNA Origami Crystals. arXiv.1706.06965v1 2017. (19) Liu, D.; Park, S. H.; Reif, J. H.; LaBean, T. H. DNA Nanotubes Self-Assembled from Triple-Crossover Tiles as Templates for Conductive Nanowires. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 717-722. (20) Rothemund, P. W.; Ekani-Nkodo, A.; Papadakis, N.; Kumar, A.; Fygenson, D. K.; Winfree, E. Design and Characterization of Programmable DNA Nanotubes. J. Am. Chem. Soc. 2004, 126, 16344-16352. (21) Schiffels, D.; Liedl, T.; Fygenson, D. K. Nanoscale Structure and Microscale Stiffness of DNA Nanotubes. ACS Nano 2013, 7, 6700-6710.


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(22) Jungmann, R.; Scheible, M.; Kuzyk, A.; Pardatscher, G.; Castro, C. E.; Simmel, F. C. DNA Origami-Based Nanoribbons: Assembly, Length Distribution, and Twist. Nanotechnology 2011, 22, 275301. (23) Liedl, T.; Hogberg, B.; Tytell, J.; Ingber, D. E.; Shih, W. M. Self-Assembly of ThreeDimensional Prestressed Tensegrity Structures from DNA. Nat. Nanotechnol. 2010, 5, 520-524. (24) Castro, C. E.; Su, H. J.; Marras, A. E.; Zhou, L.; Johnson, J. Mechanical Design of DNA Nanostructures. Nanoscale 2015, 7, 5913-5921. (25) Zhou, L.; Marras, A. E.; Su, H. J.; Castro, C. E. DNA Origami Compliant Nanostructures with Tunable Mechanical Properties. ACS Nano 2014, 8, 27-34. (26) Angelucci, F.; Bellelli, A.; Ardini, M.; Ippoliti, R.; Saccoccia, F.; Morea, V. One Ring (or Two) to Hold Them All - on the Structure and Function of Protein Nanotubes. FEBS J. 2015, 282, 2827-2845. (27) Goodsell, D. S.; Olson, A. J. Structural Symmetry and Protein Function. Annu. Rev. Biophys. Biomol. Struct. 2000, 29, 105-153. (28) Gatsogiannis, C.; Hofnagel, O.; Markl, J.; Raunser, S. Structure of Mega-Hemocyanin Reveals Protein Origami in Snails. Structure 2015, 23, 93-103. (29) Kim, D. N.; Kilchherr, F.; Dietz, H.; Bathe, M. Quantitative Prediction of 3D Solution Shape and Flexibility of Nucleic Acid Nanostructures. Nucleic Acids Res. 2012, 40, 2862-2868. (30) Lamour, G.; Kirkegaard, J. B.; Li, H.; Knowles, T. P.; Gsponer, J. Easyworm: An OpenSource Software Tool to Determine the Mechanical Properties of Worm-Like Chains. Source Code Biol. Med. 2014, 9, 16. (31) Rivetti, C.; Guthold, M.; Bustamante, C. Scanning Force Microscopy of DNA Deposited onto Mica: Equilibration versus Kinetic Trapping Studied by Statistical Polymer Chain Analysis. J. Mol. Biol. 1996, 264, 919-932. (32) Stahl, E.; Martin, T. G.; Praetorius, F.; Dietz, H. Facile and Scalable Preparation of Pure and Dense DNA Origami Solutions. Angew. Chem. Int. Ed. Engl. 2014, 53, 12735-12740. (33) Gatsogiannis, C.; Lang, A. E.; Meusch, D.; Pfaumann, V.; Hofnagel, O.; Benz, R.; Aktories, K.; Raunser, S. A Syringe-Like Injection Mechanism in Photorhabdus Luminescens Toxins. Nature 2013, 495, 520-523. (34) Sprengel, A.; Lill, P.; Stegemann, P.; Bravo-Rodriguez, K.; Schoneweiss, E. C.; Merdanovic, M.; Gudnason, D.; Aznauryan, M.; Gamrad, L.; Barcikowski, S.; Sanchez-Garcia, E.; Birkedal, V.; Gatsogiannis, C.; Ehrmann, M.; Sacca, B. Tailored Protein Encapsulation into a DNA Host Using Geometrically Organized Supramolecular Interactions. Nat. Commun. 2017, 8, 14472. (35) Tang, G.; Peng, L.; Baldwin, P. R.; Mann, D. S.; Jiang, W.; Rees, I.; Ludtke, S. J. Eman2: An Extensible Image Processing Suite for Electron Microscopy. J. Struct. Biol. 2007, 157, 38-46. (36) Moriya, T.; Saur, M.; Stabrin, M.; Merino, F.; Voicu, H.; Huang, Z.; Penczek, P. A.; Raunser, S.; Gatsogiannis, C. High-Resolution Single Particle Analysis from Electron CryoMicroscopy Images Using Sphire. J. Vis. Exp. 2017, 123, 55448.


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Figure 1. Design of the modular AB unit. (a) The basic building block is a single DNA origami structure constituted by two 24-helix bundles, A (64 nm x 18 nm) and B (41 x 18 nm), covalently connected by two singlestranded regions (97 and 98 bases long) of the M13mp18 scaffold. This generates four interfaces, indicated as A-, A+, B- and B+ (blue and orange arrows), that can be independently addressed to guide the (b and c) intra-molecular binding within the central region of one unit (A+/B-) or the (d and e) inter-molecular binding between the edges of two distinct units (A-/B+). Each interface displays distinct layers of helical edges lying on planes perpendicular to the helical axes and spaced 3bp, 4bp, 7bp or 10 bp away from a reference plane 0 (edges belonging to the same layer are indicated by circles of the same color). For clarity, helix number 0 is indicated by a striped circle. Full shape matching between extruding and intruding edges of distinct interfaces allows for their tight binding, either via base stacking or base hybridization. (c) Schematic representation of the A+/B- interface showing the four layers of helical edges and their pairwise shape-complementarity (10.7.3.0/0.3.7.10). (e) Schematic representation of the B+/Ainterface showing the three layers of edges and their pairwise shape-complementarity (0.3.7/7.4.0). A- and B+ display a partial self-complementarity in shape upon a 60° rotation, which leads to their tail-to-tail (f) and head-tohead (g) self-association. Partially matching sequences are respectively (0.3.7/7.3.0) for A-/A-rot (1 bp gap) and (7.4.0/0.4.7) for B+/B+rot (twice 1 bp gap). Each of the four interactions is also schematically represented by colorcode arrows oriented either in a parallel or antiparallel fashion.


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Figure 2. Structural characterization of the AB monomer. Lack or addition of connecting staples at the A+/Bintra-block region results in formation of a flexible (flex) or a semi-rigid (rig) AB monomer (a, left and right side, respectively), leaving the two terminal interfaces (A- and B+) available for further hierarchical assembly. The orientation (or “polarity”) of the monomer´s subunits, A and B, is schematically represented by a color-coded arrow (from A- to A+, in blue, and from B- to B+, in orange). The angle (γ) between the two subunits and their end-to-end distance (R) was measured both by AFM (b-d) and TEM (e-g), for both the flexible (b, c and e, f) and the stiffer (d and g) form. The flexible form appears slightly bent under both imaging conditions (2° or 3° deviation from 180°, corresponding to the configuration in which A and B are aligned), although the narrower Gaussian distribution observed by TEM (ω = 74° ± 4°) indicates higher homogeneity of the sample and a weaker impact of the surface and experimental settings on the structural properties of the monomer. This is further confirmed by the total length of the ABrig form observed by TEM (ca. 101 nm) that, when compared to the AFM value (ca. 97 nm), better matches the theoretical expectation (105 nm). Scale bars are 100 nm. Additional EM characterizations and class averages of the constructs are reported in Supplementary Figs. 39 and 40.


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Figure 3. Isologous assembly of AB monomers into BA2B and AB2A dimers. The self shape-complementarity of the A- and B+ interface enables base stacking of two AB monomers into, respectively, BA2B (a-f) and AB2A (g-l) dimers. Further addition of hybridization staples at the A+/B- intra-region allows reconfiguring the flexible dimers into their semi-rigid analogues, resulting in both cases in anti-parallel orientation of the two units (represented by divergent and convergent arrows in a and g, respectively). Both BA2B and AB2A have been characterized by TEM and AFM, in both the flexible (respectively, b, c and h, i) and stiffer configuration (respectively, d, e and j, k), revealing the expected structural features. Topographical marking of the A+/B- interfaces with biotin-modified strands, followed by streptavidin addition, allowed to differentiate the two dimers under AFM inspection and confirmed correct formation of the structures (f and l, respectively for, BA2B and AB2A). Scale bars are 100 nm.


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Figure 4. Hierarchical assembly of AB monomers into (AB)n polymers. (a) Hybridization of the A-/B+ interfaces led to connection of several AB monomers at their terminal edges. Further addition of hybridization strands targeting the A+/B- intra-region resulted in the reconfiguration of the flexible (AB)n polymer into its semirigid counterpart and alignment of the building blocks along the same direction (indicated by parallel oriented arrows). AFM (b) and TEM (c) characterization of the flexible (AB)n polymer revealed the formation of ring-like structures of different sizes but identical edges, whose length was equal to that of a single monomer unit. Scale bars are 100 nm (b, c). Analysis of the size distributions (d) in the two imaging modes confirmed the strong impact of the surface and sample preparation in the structural features of the macromolecule. Analysis of the trajectories described by the stiffer polymers upon surface adsorption and imaging by AFM (e) and TEM (f) allowed calculating the persistence length in both experimental conditions (g). At this end, we applied the worm-like chain model for a polymer chain equilibrated in 3D and trapped on the surface (Supplementary Note 2). Scale bars are 100 nm (e) and 400 nm (f). Additional AFM and TEM images are provided in the Supplementary Information.


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Figure 5. Hierarchical assembly of AB monomers into (A2B2)n and (AB2ABA2B)n polymers. (a) Upon base stacking at A-/A- (or B+/B+) interfaces, identical dimers can be combined in a second step through base stacking of the opposite free edges, namely B+/B+ (or A-/A-), respectively. This leads to polymers constituted by the periodic repetition of A2 and B2 segments. (b) Hybridization of two distinct dimers at their A-/B+ interface yields to polymers made up by the periodic repetition of four segments, namely B2, AB, A2 and BA. In both cases, the stiffer forms are obtained by hybridization at the A+/B- intra-region, leading either to a simple antiparallel orientation of the building units, schematically represented by consecutive head-to-head and tail-to-tail interactions (a), or to a more complex ultrastructure, where head-to-head and tail-to-tail contacts are spaced by a head-to-tail bond (b). TEM characterization of the flexible polymers showed closed structures (c, d) whose size and shape well agree with the expected periodical pattern (e). Scale bars are 100 nm (c, d). Application of the WLC model to the trajectories of the polymers observed by TEM (f, g) led to calculation of the corresponding persistence lengths (h). Scale bars are 1 µm (f) and 100 nm (g).


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Figure 6. Hierarchical assembly of AB monomers into superbundles. (a) Flipped monomer obtained by linking the A and B subunits along the direction perpendicular to their helical axes and resulting in two antiparallel 24 helixbundles (indicated by blue and orange arrows pointing in opposite directions). TEM class averages for the flipped monomer in bottom, side and top view are also reported (respectively, in the left, middle and right panel; Supplementary Fig. 51). Scale bar is 25 nm. Linking the A+/B- (b) or the A-/B+ (c) interfaces of two flipped monomers gives rise to two distinct dimers, displaying antiparallel AB modules, oriented either in a tail-to-tail (b) or head-to-head (c) fashion. Class averages for the dimers in side (left panels) and top/bottom (right panels) view are also reported (see also Supplementary Figs. 52 and 53). Scale bar is 50 nm. (d) Oligomerization of flipped dimers leads to formation of super-bundles of 48 helices, constituted by two 24-helix bundles tightly connected one another and oriented along opposite directions. (e) Cross section of the super-bundle. (f) TEM characterization of the 48helix bundle and close-up view of the polymer showing the merging of two chains along their helical axes. Scale bar is 400 nm. (g) Persistence lengths for all constructs analyzed by TEM and calculated applying the WLC model indicate that the ultrastructure of the polymer strongly affects the contour length (LC, blue bars) and the elastic properties of the macromolecule, reaching values of Lp (orange bars) comparable or even superior to naturally occurring actin protein filaments (numerical values are reported in Supplementary Table 5).


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