Highly flexible PEG-LifeAct constructs act as tunable biomimetic actin crosslinkers.

In vitro studies of actin filament networks crosslinked with dynamic actin binding proteins provide critical insights into cytoskeletal mechanics as well as inspiration for new adaptive materials design. However, discontinuous variance in the physiochemical properties of actin binding proteins impedes holistic relationships between crosslinker molecular parameters, network structure, and mechanics. Bio-synthetic constructs composed of synthetic polymer backbones and actin binding motifs would enable crosslinkers with engineered physiochemical properties to directly target the desired structure-property relationships. As a proof of concept, bio-synthetic crosslinkers composed of highly flexible polyethylene glycol (PEG) polymers functionalized with the actin binding peptide LifeAct, are explored as actin crosslinkers. Using bulk rheology and fluorescence microscopy, these constructs are shown to modulate actin filament network structure and mechanics in a contour length dependent manner, while maintaining the stress-stiffening behavior inherent to actin filament networks. These results encourage the design of more diverse and complex peptide-polymer crosslinkers to interrogate and control semi-flexible polymer networks.

Symbiotic assembly of peptide nano-mosaics at solid interfaces.

The spontaneous co-organization of distinct biomolecules at interfaces enables many of Nature's hierarchical organizations involving both hard and soft materials. Engineering efforts to mimic such hybrid complexes rely on our ability to rationally structure biomolecules at inorganic interfaces. Control over the nanoscale structure of patterned biomolecules remains challenging due to difficulties in controlling the multifarious interactions involved. This work discusses binary peptide assembly as a means to fabricate biomolecular nano-mosaics at graphite surfaces with predictable structures. Distinct peptide-substrate interactions lead to divergent crystallographic growth directions, molecular scale immiscibility, and a symbiotic assembly phenomenon. We present a symbiotic assembly model that accurately predicts the binary assembly structure relying solely on the constituent peptide nucleation kinetics and molar fractions. The ability to tune such biomolecular nano-mosaic structures facilitates the bottom up fabrication of high-density, multifunctional interfaces for nanotechnology.

Thermal Selection of Aqueous Molecular Conformations for Tailored Energetics of Peptide Assemblies at Solid Interfaces.

Key to the development of functional bioinorganic soft interfaces is the predictive control over the micron-scale assembly structure and energetics of biomolecules at solid interfaces. While assembly of labile biomolecules, such as short peptides, at interfaces is a great deal affected by the shape of the molecule, biomolecular conformations are prompted by external solution conditions, involving temperature, pH, and salt concentration. In this light, one can expect that the environmental conformational selection of aqueous biomolecules could potentially allow for fine-tuning of the equilibrium assembly structure at interfaces, as well as, the binding strength and molecular mobility within these assemblies. Here, we demonstrate the energetic and structural tailoring of two-dimensional surface assemblies of graphite-binding dodecapeptides, through the thermal selection of aqueous peptide conformations. Our findings based on a scanning probe energetic analysis, supplemented by molecular dynamics modeling, show that peptide-graphite and peptide-peptide intermolecular interactions strongly depend on the thermally selected molecular conformation and that the extent of the conformational change is directly related to the observed assembled structure. Enabled by these results was the design of a peptide with predictable binding and assembled structure, thus, suggesting environmental preconditioning of peptides as a means for controlling self-assembling active bioinorganic interfaces for bioelectronic implementations such as biomolecular fuel cells and biosensors.

Conformationally directed assembly of peptides on 2D surfaces mediated by thermal stimuli.

Dynamic and environmentally directed assembly of molecules in biological systems is essential for the fabrication of micronscale, hierarchical, functional structures. Here, we demonstrate the directed assembly of genetically selected graphite binding peptides on 2D solid surfaces upon thermal stimuli. Structural and kinetic analyses as well as molecular dynamics simulations yield the self-assembly process as thermally controllable upon tuning the solvated peptide conformational states. The ability to tailor the structure of two-dimensional soft bio/nano interfaces via external stimuli shows the potential for the bottom-up fabrication of complex materials with nanotechnological importance, such as biosensors, bioelectronics, and biomolecular fuel cells.

Terminating DNA Tile Assembly with Nanostructured Caps.

Precise control over the nucleation, growth, and termination of self-assembly processes is a fundamental tool for controlling product yield and assembly dynamics. Mechanisms for altering these processes programmatically could allow the use of simple components to self-assemble complex final products or to design processes allowing for dynamic assembly or reconfiguration. Here we use DNA tile self-assembly to develop general design principles for building complexes that can bind to a growing biomolecular assembly and terminate its growth by systematically characterizing how different DNA origami nanostructures interact with the growing ends of DNA tile nanotubes. We find that nanostructures that present binding interfaces for all of the binding sites on a growing facet can bind selectively to growing ends and stop growth when these interfaces are presented on either a rigid or floppy scaffold. In contrast, nucleation of nanotubes requires the presentation of binding sites in an arrangement that matches the shape of the structure's facet. As a result, it is possible to build nanostructures that can terminate the growth of existing nanotubes but cannot nucleate a new structure. The resulting design principles for constructing structures that direct nucleation and termination of the growth of one-dimensional nanostructures can also serve as a starting point for programmatically directing two- and three-dimensional crystallization processes using nanostructure design.

Self-Assembly of Hierarchical DNA Nanotube Architectures with Well-Defined Geometries.

An essential motif for the assembly of biological materials such as actin at the scale of hundreds of nanometers and beyond is a network of one-dimensional fibers with well-defined geometry. Here, we demonstrate the programmed organization of DNA filaments into micron-scale architectures where component filaments are oriented at preprogrammed angles. We assemble L-, T-, and Y-shaped DNA origami junctions that nucleate two or three micron length DNA nanotubes at high yields. The angles between the nanotubes mirror the angles between the templates on the junctions, demonstrating that nanoscale structures can control precisely how micron-scale architectures form. The ability to precisely program filament orientation could allow the assembly of complex filament architectures in two and three dimensions, including circuit structures, bundles, and extended materials.