•Supramolecular polymers with blocks of different DNA sequences along their lengths•Unique thermosetting mechanism in which small pieces are fused end-to-end at 90°C•Fiber-fiber interactions controlled by the stereochemical sequence of the DNA polymer•Protein-like folding of DNA-polymer building blocks critical to fiber morphology Supramolecular polymers perform a range of important functions in nature—from cellular movement to muscle contraction—enabled by their complex nanoscale structure. Realizing this complexity in synthetic systems requires a controlled hierarchical self-assembly pathway. Branched DNA polymers assemble into supramolecular fibers in a unique, heat-driven, multistep process. By controlling this process, smaller fiber fragments can be “thermoset” together in a piece-by-piece fashion to make segmented supramolecular polymers that display different DNA sequences in discrete blocks along their lengths. Further control is afforded by varying the stereosequences of the DNA polymers, resulting in different fiber-fiber interactions. Due to their water-based chemistry and biocompatibility, these fibers may be used in softer, greener technologies that require a nanoscale spatial organization of different components, such as in biomaterials or energy-harvesting structures. DNA nanostructures are highly addressable and compatible with biological systems but often require hundreds of unique strands for their assembly. On the other hand, nature can assemble complex structures from identical building blocks by compartmentalizing the process: assembling molecules into sub-components and bringing these together across multiple length scales. Inspired by this process, we report DNA-polymer conjugates that assemble through a unique heat-driven hierarchical mechanism to form fibers displaying blocks of different DNA sequences along their axis of polymerization. These one-dimensional “thermoset” DNA fibers retain the compartmentalization programmed in the pre-assembled segments. Length control over fiber segments is also achieved through gel purification of pre-assembled cylindrical micelles. Importantly, we show that the stereochemical sequence of the hydrophobic core can be amplified into distinctive morphological traits in the DNA fibers. Molecular dynamics simulations are used to model the structure and elucidate how stereochemical sequence manifests itself in different fiber-fiber interactions. DNA nanostructures are highly addressable and compatible with biological systems but often require hundreds of unique strands for their assembly. On the other hand, nature can assemble complex structures from identical building blocks by compartmentalizing the process: assembling molecules into sub-components and bringing these together across multiple length scales. Inspired by this process, we report DNA-polymer conjugates that assemble through a unique heat-driven hierarchical mechanism to form fibers displaying blocks of different DNA sequences along their axis of polymerization. These one-dimensional “thermoset” DNA fibers retain the compartmentalization programmed in the pre-assembled segments. Length control over fiber segments is also achieved through gel purification of pre-assembled cylindrical micelles. Importantly, we show that the stereochemical sequence of the hydrophobic core can be amplified into distinctive morphological traits in the DNA fibers. Molecular dynamics simulations are used to model the structure and elucidate how stereochemical sequence manifests itself in different fiber-fiber interactions. Supramolecular polymers are exciting examples of hierarchical nanomaterials built from repetitive molecular units.1De Greef T.F. Smulders M.M. Wolffs M. Schenning A.P. Sijbesma R.P. Meijer E.W. Supramolecular polymerization.Chem. 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This allows the synthesis of robust fibers displaying unique DNA sequences in compartments along their lengths, endowing the supramolecular polymers with specifically addressable separate segments. With this mechanism, the same building blocks can be used to assemble different hierarchical structures with tunable block sizes, simply by controlling temperature and ion concentration. Stable fibers with very low dispersity in length are also generated using simple electrophoretic separation. Furthermore, we show for the first time that the morphology of the fibers and fiber-fiber association can be finely tuned by controlling the stereochemical sequence of the core. Using molecular dynamics simulations, an atomistic model of the DNA fibers is proposed that helps illuminate the microscopic mechanism of fiber-fiber interaction as dictated by stereochemical sequence. Thermosetting technology—in which high temperatures are used to covalently crosslink polymer chains into solid resins—dates back to the beginning of the traditional polymer industry.41Goodman S.H. Hanna D. Handbook of Thermoset Plastics. William Andrew Publishing, 2014Google Scholar Instead of cross-linking, the distinctive mechanism demonstrated here is an ion-programmed, piece-by-piece heat-driven fusion: a one-dimensional thermosetting supramolecular polymerization of pre-formed segments. Ion concentration and temperature are used together to guide the polymerization through a hierarchical assembly pathway. This mechanism introduces the term “ion-programmed thermoset” into the modern field of supramolecular polymerization and propels the potential for soft materials to be controllably manufactured on multiple length scales. bC1212-DNAa was synthesized using phosphoramidite-based solid-phase synthesis, affording a monodisperse block co-oligomer with a hydrophilic block of 19 DNA nucleotides (DNAa) and a hydrophobic block composed of twelve bC12 units (Figure 1A). bC1212-DNAa is an oligo (phosphodiester); the monomers—including those in the hydrophobic bC1212 block—are punctuated by negative charges. This characteristic confers two noteworthy properties to bC1212-DNAa: it is highly soluble in aqueous solutions, and the presence of cations dictates the self-assembly. A structural isomer of bC1212-DNAa in which the C12 monomers are completely linear has previously been shown to form spherical micelles,36Edwardson T.G. Carneiro K.M. Serpell C.J. Sleiman H.F. An efficient and modular route to sequence-defined polymers appended to DNA.Angew. Chem. Int. Ed. Engl. 2014; 53: 4567-4571Crossref PubMed Scopus (99) Google Scholar or fibers when combined with additional hydrophobic monomers.38Bousmail D. Chidchob P. Sleiman H.F. Cyanine-mediated DNA nanofiber growth with controlled dimensionality.J. Am. Chem. Soc. 2018; 140: 9518-9530Crossref PubMed Scopus (39) Google Scholar Block-copolymer theory predicts that changes in the relative hydrophobic to hydrophilic volume ratio determine the self-assembly morphology. However, this prediction is complicated by the presence of negative charges within the hydrophobic block.42Israelachvili J. Intermolecular and Surface Forces.Third Edition. Academic Press, 2011: 1-674Crossref Google Scholar Divalent cation concentration was observed to dramatically affect the self-assembly of bC1212-DNAa. At 1.25 mM Mg2+ bC1212-DNAa formed spheres upon annealing (slow cooling from 95°C; see experimental procedures), and as Mg2+ concentration increased, we observed a gradual change from spheres to fibers (Figures 1B and S1). At 12.5 mM Mg2+, fibers with lengths over 1 μm were observed by atomic force microscopy (AFM) in air (Figures 1B, S2, and S3), in liquid (Figures 1C and S4), and by transmission electron microscopy (TEM) (Figures 1D and S5). Mg2+ dependent fiber assembly was confirmed by non-denaturing agarose gel electrophoresis (Figures S6 and S7) and dynamic light scattering (DLS) (Figures S8–S12), and fibers were also observed by AFM in the presence of Ca2+ (Figure S13). The increase in Mg2+ concentration likely improves the shielding of negative charges within the micelle corona, resulting in its shrinkage and hence a decrease in interfacial curvature. Ultimately, this can lead to a preference of cylinders over spheres.42Israelachvili J. Intermolecular and Surface Forces.Third Edition. Academic Press, 2011: 1-674Crossref Google Scholar Complexation of Mg2+ by addition of EDTA resulted in the disassembly of the fibers. However, further addition of Mg2+ followed by reannealing brought about their reassembly (Figure S14). Small-angle X-ray scattering (SAXS) was used to examine the fine structural details of the assemblies at different Mg2+ concentrations. At 12.5 mM Mg2+—a concentration at which long fibers are observed—we observed a sharp scattering peak at high q values indicating ordering or crystallinity with a characteristic distance of 3.27 nm (Figure 1E). This distance could correspond to a spacing between phosphodiester backbones within a highly ordered or crystalline fiber core.39Golder M.R. Jiang Y. Teichen P.E. Nguyen H.V. Wang W. Milos N. Freedman S.A. Willard A.P. Johnson J.A. Stereochemical sequence dictates unimolecular diblock copolymer assembly.J. Am. Chem. Soc. 2018; 140: 1596-1599Crossref PubMed Scopus (43) Google Scholar SAXS was also performed on the spheres formed by bC1212-DNAa at 1.25 mM Mg2+ and no sharp peaks were seen (Figures S15 and S16). This suggests that there is an important structural transformation from a disordered globular sphere to an ordered cylinder that occurs as Mg2+ concentration is increased and is likely facilitated in part by the decrease in interfacial curvature. We observed that fiber length also increased as temperature increased (Figures 2A and S17). bC1212-DNAa monomers in the presence of 12.5 mM of Mg2+ were slowly heated by sequentially holding for 10 min each at 30°C, 50°C, 70°C, and 90°C. Not only was self-assembly present at all temperatures up to and including 90°C, but upon cooling from 90°C back to room temperature, the elongated morphology was preserved (Figure S18). At lower temperatures, mixtures of spheres and intermediate fibers were present, which were converted into longer fibers as temperature increased, the short segments “thermoset” into long supramolecular polymers. The dependence of fiber length on both Mg2+ concentration and temperature allowed the possibility to deliberately control the polymerization mechanism by sequentially manipulating both parameters. We performed an initial test by assembling spheres from bC1212-DNAa at low Mg2+, which were subsequently converted into long fibers by first increasing Mg2+ concentration and then heating (Figure S19). Without heating, the resultant fibers were much smaller (Figure S20). Based on these observations, we propose a possible mechanism for the fiber elongation process. First, assembly of bC1212-DNAa at low Mg2+ concentration gives spheres as the preferred morphology due to inter-oligomer repulsion. Increasing Mg2+ concentration then decreases repulsion and makes the transformation from a spherical to cylindrical morphology thermodynamically favorable. At this higher Mg2+ concentration, the spheres and short fibers are now kinetically trapped and require heat to overcome an energy barrier and transform into long fibers: potentially a conformational rearrangement of the oligomer backbones and alkyl chains into a highly ordered or crystalline structure as indicated by SAXS.43Ryu J.H. Lee M. Transformation of isotropic fluid to nematic gel triggered by dynamic bridging of supramolecular nanocylinders.J. Am. Chem. Soc. 2005; 127: 14170-14171Crossref PubMed Scopus (69) Google Scholar As this rearrangement occurs, it drives the elongation of smaller cylinders into long fibers. Due to the stability of the fibers at 90°C and their ordered internal structure, we hypothesized that intermediate morphologies in the polymerization mechanism are stable and that the specific mechanism of elongation occurs through a progressive piece-by-piece fusion of shorter cylindrical segments. The polymerization of shorter segments into longer fibers raises the interesting possibility that we can create discrete regions within the fibers with different chemical compositions. However, this would require that the diffusion of individual bC1212-DNAa oligomers between and throughout segments and fibers be very slow. To investigate this potential, two fluorescently labeled oligomers bC1212-Cy3-DNAa and bC1212-Cy5-DNAa (Figure 2B) were separately pre-assembled in 3.125 mM of Mg2+ to form trapped short fibers of an average length of 80 nm (Figure 2C). These separate solutions of Cy3 and Cy5 short fibers were mixed with each other, the Mg2+ concentration increased to 12.5 mM, and then heated at 90°C for 30 min to polymerize the segments into long fibers (Figure 2C). An increase in size was observed by both AFM (Figures 2C and S21) and DLS (Figure S22), confirming the polymerization. A much lower Forster resonance energy transfer (FRET) efficiency (E) was observed for the fibers with separated bC1212-Cy3-DNAa and bC1212-Cy5-DNAa segments (E = 0.04 ± 0.07) compared with a positive control (E = 0.71 ± 0.09), in which the short segments themselves were mixtures of both oligomers assembled together in the first place (Figures 2D and S23–S26). Although very low, the non-zero FRET background (E = 0.04) observed for the segmented experiment may be due to a small degree of mixing between segments, in addition to FRET occurring at the segment interfaces. We also performed total internal reflection fluorescence microscopy (TIRF-M) on the segmented Cy3/Cy5 fibers and observed clear separation (Figures 2F, S27, and S28).44Gidi Y. Bayram S. Ablenas C.J. Blum A.S. Cosa G. Efficient one-step PEG-Silane passivation of glass surfaces for single-molecule fluorescence studies.ACS Appl. Mater. Interfaces. 2018; 10: 39505-39511Crossref PubMed Scopus (16) Google Scholar Our results suggest spatial separation of Cy3 and Cy5 within a single fiber with slow monomer exchange between segments. The utility of addressable supramolecular polymers in nanotechnology is broad and exciting.3Aida T. Meijer E.W. Stupp S.I. Functional supramolecular polymers.Science. 2012; 335: 813-817Crossref PubMed Scopus (2459) Google Scholar The construction of fibers with discrete, addressable segments opens the door to the design of structures with even greater hierarchy and function, such as spatially arranged enzyme cascades, multi-component nanowires, or circuits for DNA-based computing, all from simple, repetitive monomers. To demonstrate this potential, we used the thermosetting polymerization mechanism to produce fibers displaying different DNA sequences in discrete segments along their lengths. We synthesized bC1212-DNAb, which is composed of twelve bC12 units appended to a 19mer of DNA (as in bC1212-DNAa), but with a different DNA sequence (see SI-II-a). Similar to our segmented fluorescent fibers, solutions of short segments of bC1212-DNAa and bC1212-DNAb were pre-formed separately from each other, then polymerized together into fibers (Figure 3A) by increasing Mg2+ concentration and temperature. The polymerized fibers of bC1212-DNAa and bC1212-DNAb were hybridized (in situ on mica) with 11 nm gold nanoparticles decorated with DNA sequences complementary only to DNAa, and AFM was used to visualize the fibers. Clearly distinguishable discrete segments with and without attached gold nanoparticles were observed along the fibers (Figures 3B and S29). Some gold nanoparticles are observed attached to the DNAb segments, suggesting some oligomer exchange; however, we also see a small degree of non-specific labeling in DNAb only negative controls (Figures S30 and S31). This method of “tagging” DNA fibers aids segment visibility and demonstrates the utility of these materials for nanoscale organization. Furthermore, the same two monomers bC1212-DNAa and bC1212-DNAb could be used to produce fibers with different segment sizes by changing the Mg2+ concentration used to pre-assemble the segments (Figures 3C and S32–S34). Length and dispersity (Đ) analysis of the different segments was performed before and after polymerization (Figures S35 and S36). For segments pre-assembled at 1.25 mM Mg2+, the segment length (LN) was 23.3 nm (Đ = 1.00) before polymerization and 41.9 nm (Đ = 1.34) after. The increase in length and dispersity of the segments supports a statistical mode of copolymer elongation in which bC1212-DNAa segments join to both bC1212-DNAa and bC1212-DNAb segments stochastically. Similar increases in length and dispersity were also seen for segments pre-assembled at 3.125 mM and 6.25 mM Mg2+ (Figure 3C). Despite the high dispersity, statistical significance in length distribution was still observed between polymerized segments pre-assembled at different Mg2+ concentrations. For example, LN following polymerization for segments pre-assembled at 3.125 and 6.25 mM were 204 nm (Đ = 1.69) and 382 nm (Đ = 1.82), respectively, (p < 0.001). Highly defined building blocks are required to make hierarchical materials in a controlled manner. The DNA corona of the bC1212-DNAa fibers and their potential segmentation facilitate their use as components in super-structured materials such as bundles, junctions, and networks through DNA-DNA interactions. However, to truly demonstrate control over hierarchical assemblies, control over the length of the fibers themselves is required. In other systems, living crystallization-driven self-assembly has been used to control the length of cylindrical micelles, facilitating their use in hierarchical structures.23Gilroy J.B. Gäd