Tuning Structural Organization via Molecular Design and Hierarchical Assembly to Develop Supramolecular Thermoresponsive Hydrogels.

The cellular microenvironment is composed of a dynamic hierarchical fibrillar architecture providing a variety of physical and bioactive signals to the surrounding cells. This dynamicity, although common in biology, is a challenge to control in synthetic matrices. Here, responsive synthetic supramolecular monomers were designed that are able to assemble into hierarchical fibrous structures, combining supramolecular fiber formation via hydrogen bonding interactions, with a temperature-responsive hydrophobic collapse, resulting in cross-linking and hydrogel formation. Therefore, amphiphilic molecules were synthesized, composed of a hydrogen bonding ureido-pyrimidinone (UPy) unit, a hydrophobic alkyl spacer, and a hydrophilic oligo(ethylene glycol) tail. The temperature responsive behavior was introduced by functionalizing these supramolecular amphiphiles with a relatively short poly(N-isopropylacrylamide) (PNIPAM) chain (M n ∼ 2.5 or 5.5 kg/mol). To precisely control the assembly of these monomers, the length of the alkyl spacer between the UPy moiety and PNIPAM was varied in length. A robust sol-gel transition, with the dodecyl UPy-PNIPAM molecule, was obtained, with a network elasticity enhancing over 2000 times upon heating above room temperature. The UPy-PNIPAM compounds with shorter alkyl spacers were already hydrogels at room temperature. The sol-gel transition of the dodecyl UPy-PNIPAM hydrogelator could be tuned by the incorporation of different UPy-functionalized monomers. Furthermore, we demonstrated the suitability of this system for microfluidic cell encapsulation through a convenient temperature sol-gel transition. Our results indicate that this novel thermoresponsive supramolecular system offers a modular platform to study and guide single-cell behavior.

The effect of charge and albumin on cellular uptake of supramolecular polymer nanostructures.

Intracellular delivery of functional biomolecules by using supramolecular polymer nanostructures has gained significant interest. Here, various charged supramolecular ureido-pyrimidinone (UPy)-aggregates were designed and formulated via a simple "mix-and-match" method. The cellular internalization of these UPy-aggregates in the presence or absence of serum proteins by phagocytic and non-phagocytic cells, i.e., THP-1 derived macrophages and immortalized human kidney cells (HK-2 cells), was systematically investigated. In the presence of serum proteins the UPy-aggregates were taken up by both types of cells irrespective of the charge properties of the UPy-aggregates, and the UPy-aggregates co-localized with mitochondria of the cells. In the absence of serum proteins only cationic UPy-aggregates could be effectively internalized by THP-1 derived macrophages, and the internalized UPy-aggregates either co-localized with mitochondria or displayed as vesicular structures. While the cationic UPy-aggregates were hardly internalized by HK-2 cells and could only bind to the membrane of HK-2 cells. With adding and increasing the amount of serum albumin in the cell culture medium, the cationic UPy-aggregates were gradually taken up by HK-2 cells without anchoring on the cell membranes. It is proposed that the serum albumin regulates the cellular internalization of UPy-aggregates. These results provide fundamental insights for the fabrication of supramolecular polymer nanostructures for intracellular delivery of therapeutics.

DNA-Functionalized Supramolecular Polymers: Dynamic Multicomponent Assemblies with Emergent Properties.

Recent years have witnessed an increasing interest in hybrid molecular systems in which the programmability of DNA hybridization is used to introduce enhanced molecular control in synthetic systems. The first examples of DNA-functionalized supramolecular polymers have been reported only recently, but have already revealed structural and functional properties that are not easily obtained in either synthetic supramolecular polymers or DNA-only based systems. In this Topical Review, we provide an overview of the various forms of additional control offered by DNA hybridization for different types of supramolecular polymers and discuss how orthogonal supramolecular interactions in these hybrid systems can give rise to emergent structural and functional properties.

Polymorphism in Benzene-1,3,5-tricarboxamide Supramolecular Assemblies in Water: A Subtle Trade-off between Structure and Dynamics.

In biology, polymorphism is a well-known phenomenon by which a discrete biomacromolecule can adopt multiple specific conformations in response to its environment. The controlled incorporation of polymorphism into noncovalent aqueous assemblies of synthetic small molecules is an important step toward the development of bioinspired responsive materials. Herein, we report on a family of carboxylic acid functionalized water-soluble benzene-1,3,5-tricarboxamides (BTAs) that self-assemble in water to form one-dimensional fibers, membranes, and hollow nanotubes. Interestingly, one of the BTAs with the optimized position of the carboxylic group in the hydrophobic domain yields nanotubes that undergo reversible temperature-dependent dynamic reorganizations. SAXS and Cryo-TEM data show the formation of elongated, well-ordered nanotubes at elevated temperatures. At these temperatures, increased dynamics, as measured by hydrogen-deuterium exchange, provide enough flexibility to the system to form well-defined nanotube structures with apparently defect-free tube walls. Without this flexibility, the assemblies are frozen into a variety of structures that are very similar at the supramolecular level, but less defined at the mesoscopic level.

Accelerating DNA-Based Computing on a Supramolecular Polymer.

Dynamic DNA-based circuits represent versatile systems to perform complex computing operations at the molecular level. However, the majority of DNA circuits relies on freely diffusing reactants, which slows down their rate of operation substantially. Here we introduce the use of DNA-functionalized benzene-1,3,5-tricarboxamide (BTA) supramolecular polymers as dynamic scaffolds to template DNA-based molecular computing. By selectively recruiting DNA circuit components to a supramolecular BTA polymer functionalized with 10-nucleotide handle strands, the kinetics of strand displacement and strand exchange reactions were accelerated 100-fold. In addition, strand exchange reactions were also favored thermodynamically by bivalent interactions between the reaction product and the supramolecular polymer. The noncovalent assembly of the supramolecular polymers enabled straightforward optimization of the polymer composition to best suit various applications. The ability of supramolecular BTA polymers to increase the efficiency of DNA-based computing was demonstrated for three well-known and practically important DNA-computing operations: multi-input AND gates, Catalytic Hairpin Assembly and Hybridization Chain Reactions. This work thus establishes supramolecular BTA polymers as an efficient platform for DNA-based molecular operations, paving the way for the construction of autonomous bionanomolecular systems that confine and combine molecular sensing, computation, and actuation.

Controlling protein activity by dynamic recruitment on a supramolecular polymer platform.

Nature uses dynamic molecular platforms for the recruitment of weakly associating proteins into higher-order assemblies to achieve spatiotemporal control of signal transduction. Nanostructures that emulate this dynamic behavior require features such as plasticity, specificity and reversibility. Here we introduce a synthetic protein recruitment platform that combines the dynamics of supramolecular polymers with the programmability offered by DNA-mediated protein recruitment. Assembly of benzene-1,3,5-tricarboxamide (BTA) derivatives functionalized with a 10-nucleotide receptor strand into µm-long supramolecular BTA polymers is remarkably robust, even with high contents of DNA-functionalized BTA monomers and associated proteins. Specific recruitment of DNA-conjugated proteins on the supramolecular polymer results in a 1000-fold increase in protein complex formation, while at the same time enabling their rapid exchange along the BTA polymer. Our results establish supramolecular BTA polymers as a generic protein recruitment platform and demonstrate how assembly of protein complexes along the supramolecular polymer allows efficient and dynamic control of protein activity.

Controlling and tuning the dynamic nature of supramolecular polymers in aqueous solutions.

Structural and kinetic exchange properties of supramolecular polymers composed of mono- and bivalent ureidopyrimidinone-based monomers are investigated in aqueous solutions. It is shown that exchange dynamics can be controlled by mixing different types of monomers. This tunability widens the scope in their design as biomaterials.

Cucurbit[8]uril templated supramolecular ring structure formation and protein assembly modulation.

The interplay of Phe-Gly-Gly (FGG)-tagged proteins and bivalent FGG-tagged penta(ethylene glycol) as guest molecules with cucurbit[8]uril (Q8) hosts is studied to modulate the supramolecular assembly process. Ring structure formation of the bivalent guest molecule with Q8 leads to enhanced binding properties and efficient inhibition of protein assemblies.

Quantifying stickiness: thermodynamic characterization of intramolecular domain interactions to guide the design of förster resonance energy transfer sensors.

The introduction of weak, hydrophobic interactions between fluorescent protein domains (FPs) can substantially increase the dynamic range (DR) of Förster resonance energy transfer (FRET)-based sensor systems. Here we report a comprehensive thermodynamic characterization of the stability of a range of self-associating FRET pairs. A new method is introduced that allows direct quantification of the stability of weak FP interactions by monitoring intramolecular complex formation as a function of urea concentration. The commonly used S208F mutation stabilized intramolecular FP complex formation by 2.0 kCal/mol when studied in an enhanced cyan FP (ECFP)-linker-enhanced yellow FP (EYFP) fusion protein, whereas a significantly weaker interaction was observed for the homologous Cerulean/Citrine FRET pair (ΔG0(o-c) = 0.62 kCal/mol). The latter effect could be attributed to two mutations in Cerulean (Y145A and H148D) that destabilize complex formation with Citrine. Systematic analysis of the contribution of residues 125 and 127 at the dimerization interface in mOrange.linker.mCherry fusion proteins yielded a toolbox of new mOrange-mCherry combinations that allowed tuning of their intramolecular interaction from very weak (ΔG0(o-c) = .0.39 kCal/mol) to relatively stable (ΔG0(o-c) = 2.2 kCal/mol). The effects of these mutations were also studied by monitoring homodimerization of mCherry variants using fluorescence anisotropy. These mutations affected intramolecular and intermolecular domain interactions similarly, although FP interactions were found to be stronger in the latter. The knowledge thus obtained allowed successful construction of a red-shifted variant of the bile acid FRET sensor BAS-1 by replacement of the self-associating Cerulean-Citrine pair by mOrange.mCherry variants with a similar intramolecular affinity. Our findings thus allow a better understanding of the subtle but important role of intramolecular domain interactions in current FRET sensors and help guide the construction of new sensors using modular design strategies.