Designer peptide-DNA cytoskeletons regulate the function of synthetic cells.

The bottom-up engineering of artificial cells requires a reconfigurable cytoskeleton that can organize at distinct locations and dynamically modulate its structural and mechanical properties. Here, inspired by the vast array of actin-binding proteins and their ability to reversibly crosslink or bundle filaments, we have designed a library of peptide-DNA crosslinkers varying in length, valency and geometry. Peptide filaments conjoint through DNA hybridization give rise to tactoid-shaped bundles with tunable aspect ratios and mechanics. When confined in cell-sized water-in-oil droplets, the DNA crosslinker design guides the localization of cytoskeletal structures at the cortex or within the lumen of the synthetic cells. The tunable spatial arrangement regulates the passive diffusion of payloads within the droplets and complementary DNA handles allow for the reversible recruitment and release of payloads on and off the cytoskeleton. Heat-induced reconfiguration of peptide-DNA architectures triggers shape deformations of droplets, regulated by DNA melting temperatures. Altogether, the modular design of peptide-DNA architectures is a powerful strategy towards the bottom-up assembly of synthetic cells.

Uncovering supramolecular chirality codes for the design of tunable biomaterials.

In neurodegenerative diseases, polymorphism and supramolecular assembly of ß-sheet amyloids are implicated in many different etiologies and may adopt either a left- or right-handed supramolecular chirality. Yet, the underlying principles of how sequence regulates supramolecular chirality remains unknown. Here, we characterize the sequence specificity of the central core of amyloid-ß 42 and design derivatives which enable chirality inversion at biologically relevant temperatures. We further find that C-terminal modifications can tune the energy barrier of a left-to-right chiral inversion. Leveraging this design principle, we demonstrate how temperature-triggered chiral inversion of peptides hosting therapeutic payloads modulates the dosed release of an anticancer drug. These results suggest a generalizable approach for fine-tuning supramolecular chirality that can be applied in developing treatments to regulate amyloid morphology in neurodegeneration as well as in other disease states.

A Programmable Toolkit to Dynamically Signal Cells Using Peptide Strand Displacement.

The native extracellular matrix communicates and interacts with cells by dynamically displaying signals to control their behavior. Mimicking this dynamic environment in vitro is essential in order to unravel how cell-matrix interactions guide cell fate. Here, we present a synthetic platform for the temporal display of cell-adhesive signals using coiled-coil peptides. By designing an integrin-engaging coiled-coil pair to have a toehold (unpaired domain), we were able to use a peptide strand displacement reaction to remove the cell cue from the surface. This allowed us to test how the user-defined display of RGDS ligands at variable duration and periodicity of ligand exposure influence cell spreading degree and kinetics. Transient display of αVß3-selective ligands instructed fibroblast cells to reversibly spread and contract in response to changes in ligand exposure over multiple cycles, exhibiting a universal kinetic response. Also, cells that were triggered to spread and contract repeatedly exhibited greater enrichment of integrins in focal adhesions versus cells cultured on persistent RGDS-displaying surfaces. This dynamic platform will allow us to uncover the molecular code by which cells sense and respond to changes in their environment and will provide insights into ways to program cellular behavior.

Encoding Reversible Hierarchical Structures with Supramolecular Peptide-DNA Materials.

Creating soft materials with the tunable hierarchical structures observed in nature remains an enormous challenge. Synthetic hierarchical systems have been reported, yet strategies to reversibly modulate their structure and function are scarce. We report on the programmable self-assembly of peptide-DNA brush copolymers into supramolecular architectures that can be tuned with changes in temperature, pH, or addition of a soluble trigger. A fiber to bundle transition occurs upon mixing peptides bearing complementary oligonucleotides. These hierarchical structures can be reversed using the programmable nature of DNA-DNA interactions. The ability to encode the final assemblies in the composition of both amino acid and DNA building blocks provides a strategy for constructing a unique class of reconfigurable supramolecular materials.

Meta-Alkoxy-Substituted Difluoroboron Dibenzoylmethane Complexes as Environment-Sensitive Materials.

The optical properties of meta-alkoxy-substituted difluoroboron dibenzoylmethane dyes were investigated in solution and in the solid state. Meta-alkoxy substitution induced strong intramolecular charge transfer (ICT) from the oxygen-donating substituent to the halide and boron acceptors in the excited state, as compared to the π-π* transition that is observed with para-alkoxy substitution. The optical properties of para- and meta-substituted alkoxy boron dyes were evaluated by calculations, in dilute solution, and in solid-state films. When embedded in amorphous matrixes (e.g., PLA, PMMA, PS, cholesterol), all dyes showed fluorescence (F) and phosphorescence (P) emission. In this report, we show that meta-substitution resulted in enhanced solvatochromism and an increased phosphorescence-to-fluorescence ratio in solid-state films compared to analogous para-substituted samples. With enhanced phosphorescence intensity via the heavy-atom effect, iodo-substituted dyes were further studied in PLA-PEG nanoparticles. Oxygen calibrations revealed stronger phosphorescence and a greater oxygen-sensing range for the meta- versus para-alkoxy-substituted dyes, features that are important for oxygen-sensing materials design.

Luminescent Difluoroboron ß-Diketonate PLA-PEG Nanoparticle.

Luminescent difluoroboron ß-diketonate poly(lactic acid) (BF2bdkPLA) materials serve as biological imaging agents. In this study, dye structures were modified to achieve emission colors that span the visible region with potential for multiplexing applications. Four dyes with varying π-conjugation (phenyl, naphthyl) and donor groups (-OMe, -NMe2) were coupled to PLLA-PEG block copolymers (∼11 kDa) by a postpolymerization Mitsunobu reaction. The resulting dye-polymer conjugates were fabricated as nanoparticles (∼55 nm diameter) to produce nanomaterials with a range of emission colors (420-640 nm). For increased stability, dye-PLLA-PEG conjugates were also blended with dye-free PDLA-PEG to form stereocomplex nanoparticles of smaller size (∼45 nm diameter). The decreased dye loading in the stereoblocks blue-shifted the emission, generating a broader range of fluorescence colors (410-620 nm). Tumor accumulation was confirmed in a murine model through biodistribution studies with a red emitting dimethyl amino-substituted dye-polymer analogue. The synthesis, optical properties, oxygen-sensing capabilities, and stability of these block copolymer nanoparticles are presented.

Multi-stimuli responsive luminescent azepane-substituted ß-diketones and difluoroboron complexes.

Difluoroboron ß-diketonate (BF2bdk) compounds show environment-sensitive optical properties in solution, aggregation-induced emission (AIE) and multi-stimuli responsive fluorescence switching in the solid state. Here, a series of 4-azepane-substituted ß-diketone (bdk) ligands (L-H, L-OMe, L-Br) and their corresponding difluoroboron dyes (D-H, D-OMe, D-Br) were synthesized, and various responsive fluorescence properties of the compounds were studied, including solvatochromism, viscochromism, AIE, mechanochromic luminescence (ML) and halochromism. Compared to the ß-diketones, the boron complexes exhibited higher extinction coefficients but lower quantum yields, and red-shifted absorption and emission in CH2Cl2. Computational studies showed that intramolecular charge transfer (ICT) dominated rather than π-π* transitions in all the compounds regardless of boron coordination. In solution, all the bdk ligands and boron dyes showed red-shifted emission in more polar solvents and increased fluorescence intensity in more viscous media. Upon aggregation, the emission of the ß-diketones was quenched, however, the boronated dyes showed increased emission, indicative of AIE. Solid-state emission properties, ML and halochromism, were investigated on spin cast films. For ML, smearing caused a bathochromic emission shift for L-Br, and powder X-ray diffraction (XRD) patterns showed that the "as spun" and thermally annealed states were more crystalline and the smeared state was amorphous. No obvious ML emission shift was observed for L-H or L-OMe, and the boronated dyes were not mechano-active. Trifluoroacetic acid (TFA) and triethylamine (TEA) vapors were used to study halochromism. Large hypsochromic emission shifts were observed for all the compounds after TFA vapor was applied, and reversible fluorescence switching was achieved using the acid/base pair.

Oxygen-Sensing Difluoroboron Thienyl Phenyl ß-Diketonate Polylactides.

Difluoroboron ß-diketonate-polylactides are versatile oxygen-sensing materials. These materials have both fluorescence (F) and oxygen-sensitive, room-temperature phosphorescence (RTP). The fluorescence, being insensitive to oxygen, can act as an internal standard to the changing phosphorescence, and ratiometric sensing of oxygen can be achieved with these simple, single-component materials. To expand the range of colors for this family of fluorophores, a series of thienyl-phenyl-substituted dyes were synthesized with initiator sites for the ring-opening polymerization of lactide. Heavy atoms (Br and I) were added to the dye to modulate the phosphorescence intensity. These halide-substituted thiophene dyes readily aggregated in the poly(lactic acid) (PLA) matrix, generating two fluorescence peaks in air, one for monomer emission and another for aggregate emission. When the dye was dilute in PLA, as a blend, the iodo-thienyl derivative showed impressive singlet-triplet splitting, with blue fluorescence (440 nm) and orange phosphorescence (585 nm), the largest gap recorded for a boron ß-diketonate dye. Nanoparticles fabricated from a mixture of PLA and dye-PLA conjugated polymer benefited from the large singlet-triplet splitting to yield oxygen sensitivity at levels between 0 and 21 %, which can be utilized in biological oxygen-sensing applications.

Blue Thermally Activated Delayed Fluorescence from a Biphenyl Difluoroboron ß-Diketonate.

Optical properties of biphenyl difluoroboron ß-diketonates were studied in poly(lactic acid) (PLA) blends. Increased conjugation lowered the emission energy, decreased the singlet-triplet energy gap and yielded blue thermally activated delayed fluorescence (TADF). The properties of these biphenyl dyes may inform organic light emitting diode (OLED) and bioimaging agent design.