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Molecular strain can be introduced to influence the outcome of chemical reactions. Once a thermodynamic product is formed, however, reversing the course of a strain-promoted reaction is challenging. Here, a reversible, strain-promoted polymerization in cyclic DNA is reported. The use of nonhybridizing, single-stranded spacers as short as a single nucleotide in length can promote DNA cyclization. Molecular strain is generated by duplexing the spacers, leading to ring opening and subsequent polymerization. Then, removal of the strain-generating duplexers triggers depolymerization and cyclic dimer recovery via enthalpy-driven cyclization and entropy-mediated ring contraction. This reversibility is retained even when a protein is conjugated to the DNA strands, and the architecture of the protein assemblies can be modulated between bivalent and polyvalent states. This work underscores the utility of using DNA not only as a programmable ligand for assembly but also as a route to access restorable bonds, thus providing a molecular basis for DNA-based materials with shape-memory, self-healing, and stimuli-responsive properties.

The structural and functional diversity of materials in nature depends on the controlled assembly of discrete building blocks into complex architectures via specific, multistep, hierarchical assembly pathways. Achieving similar complexity in synthetic materials through hierarchical assembly is challenging due to difficulties with defining multiple recognition areas on synthetic building blocks and controlling the sequence through which those recognition sites direct assembly. Here, we show that we can exploit the chemical anisotropy of proteins and the programmability of DNA ligands to deliberately control the hierarchical assembly of protein-DNA materials. Through DNA sequence design, we introduce orthogonal DNA interactions with disparate interaction strengths ("strong" and "weak") onto specific geometric regions of a model protein, stable protein 1 (Sp1). We show that the spatial encoding of DNA ligands leads to highly directional assembly via strong interactions and that, by design, the first stage of assembly increases the multivalency of weak DNA-DNA interactions that give rise to an emergent second stage of assembly. Furthermore, we demonstrate that judicious DNA design not only directs assembly along a given pathway but can also direct distinct structural outcomes from a single pathway. This combination of protein surface and DNA sequence design allows us to encode the structural and chemical information necessary into building blocks to program their multistep hierarchical assembly. Our findings represent a strategy for controlling the hierarchical assembly of proteins to realize a diverse set of protein-DNA materials by design., Competing Interests: The authors declare no competing interest.

A novel method for controlling the oligomerization of metastable DNA hairpins using the hybridization chain reaction (HCR) is reported. Control was achieved through the introduction of a base-pair mismatch in the duplex of the hairpins. The mismatch modification allows one to kinetically differentiate initiation versus propagation events, leading to DNA oligomers up to 10 monomers long and improving dispersities from 2.5 to 1.3-1.6. Importantly, even after two consecutive chain extensions, dispersity remained unaffected, showing that well-defined block co-oligomers can be achieved. As a proof-of-concept, this technique was then applied to hairpin monomers functionalized with a mutant green fluorescent protein to prepare protein oligomers. Taken together, this work introduces an effective method for controlling living macromolecular HCR oligomerization in a manner analogous to the controlled polymerization of small molecules.

Proteins are a class of nanoscale building block with remarkable chemical complexity and sophistication: their diverse functions, shapes, and symmetry as well as atomically monodisperse structures far surpass the range of conventional nanoparticles that can be accessed synthetically. The chemical topologies of proteins that drive their assembly into materials are central to their functions in nature. However, despite the importance of protein materials in biology, efforts to harness these building blocks synthetically to engineer new materials have been impeded by the chemical complexity of protein surfaces, making it difficult to reliably design protein building blocks that can be robustly transformed into targeted materials. Here we describe our work aimed at exploiting a simple but important concept: if one could exchange complex protein-protein interactions with well-defined and programmable DNA-DNA interactions, one could control the assembly of proteins into structurally well-defined oligomeric and polymeric materials and three-dimensional crystals. As a class of nanoscale building block, proteins with surface DNA modifications have a vast design space that exceeds what is practically and conceptually possible with their inorganic counterparts: the sequences of the DNA and protein and the chemical nature and position of DNA attachment all play roles in dictating the assembly behavior of protein-DNA conjugates. We summarize how each of these design parameters can influence structural outcome, beginning with proteins with a single surface DNA modification, where energy barriers between protein monomers can be tuned through the sequence and secondary structure of the oligonucleotide. We then explore challenges and progress in designing directional interactions and valency on protein surfaces. The directional binding properties of protein-DNA conjugates are ultimately imposed by the amino acid sequence of the protein, which defines the spatial distribution of DNA modification sites on the protein. Through careful design and mutagenesis, bivalent building blocks that bind directionally to form one-dimensional assemblies can be realized. Finally, we discuss the assembly of proteins densely modified with DNA into crystalline superlattices. At first glance, these protein building blocks display crystallization behavior remarkably similar to that of their DNA-functionalized inorganic nanoparticle counterparts, which allows design principles elucidated for DNA-guided nanoparticle crystallization to be used as predictive tools in determining structural outcomes in protein systems. Proteins additionally offer design handles that nanoparticles do not: unlike nanoparticles, the number and spatial distribution of DNA can be controlled through the protein sequence and DNA modification chemistry. Changing the spatial distributions of DNA can drive otherwise identical proteins down distinct crystallization pathways and yield building blocks with exotic distributions of DNA that crystallize into structures that are not yet attainable using isotropically functionalized particles. We highlight challenges in accessing other classes of architectures and establishing general design rules for DNA-mediated protein assembly. Harnessing surface DNA modifications to build protein materials creates many opportunities to realize new architectures and answer fundamental questions about DNA-modified nanostructures in both materials and biological contexts. Proteins with surface DNA modifications are a powerful class of nanomaterial building blocks for which the DNA and protein sequences and the nature of their conjugation dictate the material structure.

A strategy that utilizes DNA for controlling the association pathway of proteins is described. This strategy uses sequence-specific DNA interactions to program energy barriers for polymerization, allowing for either step-growth or chain-growth pathways to be accessed. Two sets of mutant green fluorescent protein (mGFP)-DNA monomers with single DNA modifications have been synthesized and characterized. Depending on the deliberately controlled sequence and conformation of the appended DNA, these monomers can be polymerized through either a step-growth or chain-growth pathway. Cryo-electron microscopy with Volta phase plate technology enables the visualization of the distribution of the oligomer and polymer products, and even the small mGFP-DNA monomers. Whereas cyclic and linear polymer distributions were observed for the step-growth DNA design, in the case of the chain-growth system linear chains exclusively were observed, and a dependence of the chain length on the concentration of the initiator strand was noted. Importantly, the chain-growth system possesses a living character whereby chains can be extended with the addition of fresh monomer. This work represents an important and early example of mechanistic control over protein assembly, thereby establishing a robust methodology for synthesizing oligomeric and polymeric protein-based materials with exceptional control over architecture.

Asymmetric functionality and directional interactions, which are characteristic of noncentrosymmetric particles, such as Janus particles, present an opportunity to encode particles with properties, but also a great synthetic challenge. Here, we exploit the chemical anisotropy of proteins, and the versatile chemistry of DNA to synthesize a protein-based Janus nanoparticle comprised of two proteins encoded with sequence-specific nucleic acid domains, tethered together by an interprotein "DNA bond". We use these novel nanoparticles to realize a new class of three-dimensional superlattice, only possible when two sides of the particle are modified with orthogonal oligonucleotide sequences. The low symmetry, intrinsic to Janus particles, enables the realization of unprecedented multicomponent nanoparticle superlattices with unique, hexagonal layered architectures. In addition, the interprotein "DNA bond" can be modulated to selectively expand the lattice in a single direction. The results presented herein not only emphasize the power of rationally designing nanoscale building blocks to create highly engineered colloidal crystals, but also establish a precedent for applications of multidomain DNA-encoded nanoparticles, especially in the field of colloidal crystallization.

Molecular strain can be introduced to influence the outcome of chemical reactions. Once a thermodynamic product is formed, however, reversing the course of a strain-promoted reaction is challenging. Here, a reversible, strain-promoted polymerization in cyclic DNA is reported. The use of nonhybridizing, single-stranded spacers as short as a single nucleotide in length can promote DNA cyclization. Molecular strain is generated by duplexing the spacers, leading to ring opening and subsequent polymerization. Then, removal of the strain-generating duplexers triggers depolymerization and cyclic dimer recovery via enthalpy-driven cyclization and entropy-mediated ring contraction. This reversibility is retained even when a protein is conjugated to the DNA strands, and the architecture of the protein assemblies can be modulated between bivalent and polyvalent states. This work underscores the utility of using DNA not only as a programmable ligand for assembly but also as a route to access restorable bonds, thus providing a molecular basis for DNA-based materials with shape-memory, self-healing, and stimuli-responsive properties. [ABSTRACT FROM AUTHOR]