Controlled Hierarchical Assembly of Spider Silk-DNA Chimeras into

Jun 13, 2014 - A combination of 5′-conjugated silk moieties via complementary nucleic acids enhanced fibril association, whereas mixing complementar...
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Controlled Hierarchical Assembly of Spider Silk-DNA Chimeras into Ribbons and Raft-Like Morphologies Martin Humenik,*,† Markus Drechsler,‡ and Thomas Scheibel†,§ †

Biomaterials, Faculty of Engineering Science, ‡Bayreuth Institute of Macromolecular Research (BIMF) - Soft Matter Electron Microscopy, and §Bayreuth Center for Colloids and Interfaces (BZKG), Research Center Bio-Macromolecules (BIOmac), Bayreuth Center for Molecular Biosciences (BZMB), and Bayreuth Center for Material Science (BayMAT), University of Bayreuth, D-95440 Bayreuth, Germany S Supporting Information *

ABSTRACT: Spider silk−DNA conjugates comprising the recombinant spider silk protein eADF4(C16) and short oligonucleotides were arranged in a linear antiparallel and parallel as well as in a branched manner via designed complementarity of the DNA moieties. After cross-β fibril self-assembly, temperature-induced annealing of the DNA moieties triggered fibril association into ribbons, composed of aligned nanofibrils, and rafts composed of ribbons ordered into sharply bordered, squared fibrous microstructures. The formation of the superstructures was clearly dependent on the individual silk−DNA conjugate. A combination of 5′conjugated silk moieties via complementary nucleic acids enhanced fibril association, whereas mixing complementary 5′- and 3′-silk conjugates inhibited the formation of higher-order structures. KEYWORDS: Assembly, conjugates, DNA, fibril, spider silk

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nucleic acids to either alter their self-assembly properties29 or to expand their functionality.30 We previously demonstrated the conjugation of the recombinant spider silk protein eADF4(C16), a derivative of one major ampullate spidroin from the European garden spider Araneus diadematus, namely, A. diadematus fibroin 4 (ADF4),31 and short oligonucleotides using “click” chemistry (Figure 1A, Table S1). Morphology and protein secondary structure of self-assembled fibrils were identical to those prepared from the unmodified silk protein. The morphologies and structures were even preserved when the silk moieties were arranged in a linear antiparallel (55DNA) and parallel (53DNA) manner or in a branched three way junction (TWJ) (Figure 1B). Moreover, DNA moieties remained accessible on the fibril surfaces enabling attachment of gold nanoparticles or fluorescent labels via appropriately modified complementary DNA strands.30 To further explore the fibrils made of spider silk−DNA conjugates, self-assembly of the conjugates possessing different topologies (55DNA, 53DNA, and TWJ, Figure 1B) was studied at elevated temperatures. Strikingly, different temperature regimes yielded lateral fibril alignment into ribbons and raftlike morphologies depending on the designed nucleic acid complementarities.

n the past two decades, the ability of DNA to hybridize in a defined and programmable manner has allowed the development of self-assembled nanomaterials. Nucleic acid design has resulted in a plethora of 2D and 3D nano-objects.1−3 The specificity of DNA recognition properties and the reversibility of its association have enabled bioanalytical applications especially in conjugation with enzymes or particles.4,5 Using DNA sequences to functionalize traditional materials such as block copolymers,6,7 lipids,8 and metallic nanoparticles9 has made it possible to build superstructures in a “bottom-up” fashion inspired by nature. Structural proteins are another class of molecules utilized for creating a variety of new self-assembly materials.10,11 Favorable characteristics of self-assembled cross-β nanofibrils such as good mechanical properties,12−15 suitability for chemical modifications, and thermodynamic and environmental stability16,17 have rendered them an attractive scaffold for “bottom up” material assembly,14,18 such as preparation of nanowires upon metal deposition,19,20 light harvesting materials upon modification with fluorescent dyes,21 electron transport upon enzyme attachment,22 and self-assembling biocatalysts.23,24 Recently, we and other groups could show that recombinant spider silk variants enable the formation of nanofibrils25 and self-assembled superstructures such as particles26 and capsules.27,28 In contrast to other polymeric6,7 and metallic13 nanomaterials, structural proteins have been only rarely combined with © XXXX American Chemical Society

Received: April 16, 2014 Revised: June 12, 2014

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Figure 2. Assembly kinetics of eADF4(C16)−DNA conjugates. Cartoons represent the topologies of assembled units. Kinetics of fibril assembly of unmodified eADF4(C16) were compared to that of the basic conjugates and hybridized constructs using identical protein concentrations. Assembly was initiated upon addition of 100 mM potassium phosphate (KPi), and fibril formation was followed using turbidity measurements at 340 nm. Turbidity signals were normalized and fitted according to the Finke−Watzky two step model (see Supporting Methods).

structural transformation of eADF4(C16) from an intrinsically unfolded toward a β-sheet rich structure,35 the conjugation of the protein with highly charged nucleic acid chains significantly slowed down both nucleation (Table S2, compare lag times) and fibril growth (k2, Table S3) due to repulsive forces as well as overall higher molecular weights of the conjugates (accompanied by lower diffusion coefficients). 53DNA, however, differed in the close proximity of the silk moieties (Figure 1B), resulting in faster nucleation and fibril growth (Tables S2 and S3) in comparison to 55DNA, although possessing an identical total charge and molecular weight. Therefore, positioning of silk moieties on the same site of a DNA double helix facilitates protein dimerization, which is an initial step for oligomerization/nucleation. This finding is confirmed by assembly of 53DNA in the presence of 300 mM NaCl, since higher ionic strength shields charged nucleic acids and allows faster nucleation of all constructs with the exception of 53DNA (Figure S3). Assembly of Spider Silk Conjugates Upon Steep Temperature Gradients Terminating at Ambient Temperature. Initially, unmodified eADF4(C16), basic conjugates 1a−c and 2a−c (Figure 1A, Table S1), or hybridized constructs 55DNA, 53DNA, and TWJ (Figure 1B) were incubated with a linear gradient from 95 to 25 °C within 70 min. Similar to fibrils assembled at ambient temperature30 (Figure 3A−C), unmodified eADF4(C16) and basic conjugates 1a−c and 2a−c formed individual fibrils (Figure S4A−C). In contrast, linear 55DNA and branched TWJ assembled into ribbons/bundles of fibrils aligned in a micrometer regime (Figure 3E−G, I and J, Figures S5 and S6). To exclude interface,38 surface, and deposition effects along fibril association, assemblies were visualized using cryogenic transmission electron microscopy (cryo-TEM) (Figure 3A and E) in comparison to visualization of dried samples on hydrophobic grids (TEM) (Figure 3B and F) and on hydrophilic mica (atomic force microscopy (AFM)) (Figure 3C and G). In contrast to eADF4(C16) fibrils (Figure S4B), aligned TWJ and 55DNA fibrils were clearly separated. Interestingly, temperature-guided assembly of 53DNA constructs resulted in formation of individual fibrils (Figure 3K and

Figure 1. Schematic representation of spider silk−DNA conjugates and hybridized constructs. (A) Spider silk-oligonucleotide “click” conjugates were prepared from azide modified eADF4(C16) and different 5′- or 3′-alkyne modified oligonucleotides (Table S1); (B) “Click” conjugates were hybridized according to designed complementarities of the oligonucleotide moieties to prepare 55DNA, 53DNA, and three way junction (TWJ) constructs to arrange silk moieties in an antiparallel, parallel, and branched manner, respectively.

Secondary Structure of Fibrils Assembled at Ambient and Elevated Temperatures. Fibrils assembled from basic conjugates 1a−c and 2a−c (Figure 1A, Table S1) or hybridized constructs 53DNA, 55DNA, and TWJ (Figure 1B) showed similar secondary structure content as indicated by Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy independent of the assembly temperature. The position of amide I (1624 cm−1) and amide II (1534 cm−1) bands in FT-IR spectra (Figure S1) are typical for β-sheet rich silk structures,32−34 as well as minima at 218 nm in CD spectra.30,35 Kinetics of Fibril Assembly. Recombinant spider silk eADF4(C16) has been shown to self-assemble into fibrils through a nucleation-dependent mechanism.35 Here, fibrilization kinetics of eADF4(C16) moieties conjugated with different DNA strands (Table S1) as well as those of hybridized 53DNA, 55DNA, and TWJ (Figure 1) were determined using turbidity measurements35 at ambient temperature (Figure 2, S2, Table S2 and S3 and Supporting Methods). Assembly of all constructs followed sigmoidal growth curves with lag, growth, and saturation phases (Figure 2). The initial lag phase corresponds to the formation of thermodynamically disfavored oligomers.36,37 Once a critical nucleus size is reached, fibril growth starts by monomer addition onto nucleus/fibril ends until monomer depletion (final plateau). Since nucleation and growth of the fibrils is based on the B

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Figure 3. Assembly of conjugate fibrils upon a steep temperature gradient. Cartoons on the left panel represent the topology of assembled units and temperature conditions. (A−D) Single TWJ fibrils assembled at ambient temperature; (E−H) TWJ ribbons and (I and J) 55DNA ribbons of associated fibrils upon a linear temperature gradient from 95 to 25 °C for 70 min. (K and L) No fibril association was observed for 53DNA using the temperature gradient. Assemblies were visualized by cryo-TEM (A, E, I−L), TEM (B and F), and AFM (C and G). Cross sectional areas in D and H revealed similar fibril heights of ∼3 nm for single as well as for aligned fibrils. Scale bars represent 100 nm in A−C, E−G, and J; 200 nm in L; and 0.5 μm in I and K. The color bar represents heights from −5 nm (dark) to 5 nm (white) for AFM scans.

Figure 4. Hierarchical assembly of conjugate fibrils upon incubation at elevated temperature. Cartoons on the left panel represent the topologies of assembled units. Raft-like morphologies assembled from TWJ (A and B) and 55DNA (C and D). Disordered fibrous entanglements assembled from 53DNA (E and F). CLSM images of TWJ (G) and 55DNA (H) assemblies were processed in an ImageJ 3D viewer to render three-dimensional visualizations from Z-confocal sections (30 stacks, 0.25 μm per section). Objects were labeled with 5′-fluorescein oligonucleotides (green) or 5′rhodamine oligonucleotides (red), which were complementary to corresponding oligonucleotide moieties of the hybridized constructs (Table S1). Scale bars represent 50 μm in A−F and 100 μm in G and H.

L and Figure S7). In comparison to assembly at ambient temperatures proceeding within several hours (Figure 2), elevated temperature resulted in complete assembly during the time of the gradient (70 min), due to increased diffusion coefficients enhancing the nucleation and growth phases. Fibril association observed for the assembly of the unmodified

protein at elevated temperatures (Figure S4A and B) probably resulted from enhanced hydrophobic interaction of the fibril surfaces rather than from structural changes of the silk protein. As seen in the FT-IR spectra (Figure S1C) the typical β-sheet maximum of amide I bands of the assemblies remained unchanged at ambient and elevated temperatures. In basic silk− C

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Figure 5. Model of spider silk conjugate assembly into higher-order structures. As an example, TWJ (A) and 53DNA (B) are presented at ambient temperature. At 95 °C they completely dissociate as shown in C and D, respectively. The concomitant fast nucleation process (E) inhibits the proper TWJ rehybridization (as shown in A) during the temperature drop. Possible recombination of DNA strands, attached through 5′-ends to the protein, occurs via lateral alignment of fibrils (G). In case of 53DNA, cooling also results in formation of mixed nuclei (F) and fibrils (H). However, due to proper 5′/3′-orientation of the DNA strands on the fibril surface, the majority of complementary strands undergo intra-fibril hybridization. This capping prevents fibril−DNA−fibril interactions. Prolonged incubation of associated TWJ fibrils (G) close to the DNA melting temperature (∼50 °C) enables optimal DNA hybridization and fibrils annealing, resulting in the formation of raft-like morphologies (I). Capped DNA strands on the surface of 53DNA fibrils (H) inhibit formation of regular structures resulting in fibrous entanglements (J). Cryo-TEM images (G and H), scale bars 100 nm; fluorescence microscopy images (I and J), scale bars 50 μm.

and a thickness of 7−10 μm, as shown by Z-confocal sections of CSLM images (Figure 4G and H). 53DNA (Figure 4E and F) and the basic eADF4(C16)−DNA conjugates (1a−c and 2a−c, Table S1, Figure S8C and D) showed fibril entanglements. Shear forces readily disintegrated the rafts into small pieces (Figure S8A and B), indicating low mechanical stability. Model of Spider Silk-DNA Conjugate Assembly into Higher-Order Structures. Hybridized TWJ, 55DNA, and 53DNA (Figure 5A and B) completely dissociate at 95 °C (Figure 5C and D). Recombination of the complementary nucleic acid strands occurs during a linear temperature decrease. However, based on the established nucleation mechanism of eADF4(C16) fibril assembly,35 nucleus formation of the eADF4(C16) moieties (Figure 5E and F) and successive fibril elongation (Figure 5G and H) compete with proper DNA association, resulting in formation of fibrils possessing different DNA strands on their surface (Figure 5G and H). In the case of “mixed” fibrils assembled from 55DNA or TWJ (Figure 5G), all nucleic acids moieties are attached onto the fibril surface through their 5′-ends. Therefore, formation of thermodynamically stable DNA double helices yields ribbons due to interfibril hybridization. Keeping the system close to the Tm (∼50°) of the complementary DNA strands for a prolonged time leads to dynamic association/ dissociation of the DNA moieties between fibrils, resulting in the maximized number of DNA interactions and formation of a thermodynamically stable configuration,39,40 yielding higherorder raft like structures (Figure 5I). In contrast, 53DNA mixed fibrils (Figure 5H) possess alternating 3′- and 5′-complementary DNA strands which could readily recombine on the fibril surface prevented the formation of higher order structures (Figure 5J). Conclusions. β-sheet rich fibrils represent attractive scaffolds for the formation of spatially oriented arrays41−43 due to a propensity to form superstructures15,38,43−46 based on inherent properties of their amino acid building blocks.13,47

DNA conjugates (1a−c and 2a−c) distinct hydrophobic interactions were clearly suppressed due to the charged nucleic acids chains, which are accessible on the surface of eADF4(C16)−DNA fibrils.30 To confirm the importance of both DNA accessibility and elevated temperature for the observed fibril association, mature fibrils made of conjugates 2a, 2b, and 2c were equivalently mixed at ambient temperature resulting in entanglements (Figure S4E), whereas the same mixture produced fibril bundles upon incubation with the temperature gradient (Figure S4F). Next, TWJ was mixed with an excess of unmodified complementary nucleic acids strands yielding individual fibrils (Figures S4D) during thermally triggered assembly. Similarly to DNA modified metallic nanorods,39 here the excess of free DNA strands apparently blocked lateral association of complementary fibril counterparts. Assembly of Spider Silk Conjugates upon Flat Temperature Gradients Terminating at Elevated Temperature. Next, the hybridized constructs were incubated using flat linear temperature gradients from 95 to 50 °C over 4 h, followed by incubation at 50 °C for 24 h, which is slightly below the melting temperatures (Tm) of 55DNA and 53DNA (Tm ∼ 55 °C) as well as of strand pairs of TWJ, i.e., 2a/2b (∼58 °C) or 2a/2c (∼65 °C) and 2b/2c (∼56 °C). TWJ and 55DNA ribbons, similar in appearance to fibrils associated during the steep temperature gradient (Figure 3E−J), were also formed at the end of the flat gradient. However, prolonged incubation at 50 °C induced formation of morphologies of a higher-order. Fluorescently labeled complementary oligonucleotides (3−5, Table S1) were added after this procedure, and the resulting self-assembled structures were visualized using confocal laser scanning microscopy (CLSM) (Figure 4). Strikingly, well-defined squared microscopic objects resembling raft-like patterns of aligned and layered fibrils were detected in case of TWJ (Figure 4A and B) and 55DNA (Figure 4C and D). The rafts typically had a length and width of 50−100 μm D

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Furthermore, the DNA properties allowed the design and controlled organization of particulate nanomaterials.48−51 The assembly of cross-β fibrils made of the recombinant spider silk eADF4(C16) into higher-ordered patterns can be affected by functional groups such as nucleic acids. Distinct spider silk−DNA conjugates allowed the precise organization of silk moieties into linear antiparallel and parallel as well as into branched topologies. Coupling eADF4(C16) opposite to each other onto 5′-ends of hybridized constructs 55DNA and TWJ yielded ribbons and raft-like morphologies, whereas the proximity of silk monomers placed onto 3′- and 5′-ends of 53DNA inhibited formation of such configurations. Since both steep and flat gradients yielded fibrillar ribbons, their formation seems to be thermodynamically controlled. However, appropriate design of nucleic acid sequences and/or lowering the annealing temperatures to slow down the de- and rehybridization would also allow kinetic control according to rules established recently for DNA−particulate systems.50−53 The development of programmable silk assembly via “ondemand” design of conjugated DNA strands presents a step toward triggered and controlled formation of ordered nanomaterials for technological applications.52,53

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ASSOCIATED CONTENT

S Supporting Information *

Figures: FT-IR and CD spectra in S1, turbidity data in S2, lag times comparison in S3, AFM of fibril assembly in S4, large scale cryo-TEM images in S5−S7, fluorescence imaging of micro-objects in S8; Tables: DNA sequences and modifications in S1, kinetic parameters in S2 and S3; Methods: conjugate synthesis and assembly, oligonucleotide labeling, CD and FTIR spectroscopy, microscopy techniques, assembly kinetics, theoretical background. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: (M.H.) [email protected]. Author Contributions

M.H. designed research, performed synthesis and purification of conjugates, kinetic measurements, fibril assembly experiments, AFM and fluorescence microscopy imaging, and wrote the manuscript; M.D. performed cryo-TEM experiments and wrote the manuscript; T.S. designed research and wrote the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeindschaft (SFB 840 TP A8). We thank M. Hermann and A. Heidebrecht for CLSM, M. Heim for TEM imaging and E. Lintz for critical comments on the manuscript.

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