44235-G7
Self-Assembling, Dynamic Hydrogels Cross-Linked via Complementary DNA Interactions
Summary: Development of hydrogel materials that respond to specific stimuli has been of significant interest in the design of modern functional materials. A variety of previous studies have used the ligand-binding capability of proteins to design hydrogels that change their crosslinking density in response to stimuli. However, these materials generally undergo relatively small dynamic response, with limited control over response characteristics. We have developed an alternative approach that exploits the ability of proteins to undergo nanometer-scale conformational changes in response to stimuli. We report a class of novel protein-based hydrogel materials that undergo tunable, reversible dynamic responses with a wide dynamic range (volume decreases to 25–90% of initial volume). These materials also undergo tunable, reversible changes in optical transparency
(optical transparency decreases to 35–100% of initial optical transparency), and this phenomenon can be used as a mechanism for label-free biosensing.
Approach: To exploit molecular motions in a macroscopic material we have utilized the well-characterized conformational dynamics of the protein calmodulin (CaM). Calmodulin has two distinct conformational states. In the presence of calcium, CaM is in an extended conformation and is a dumbbell-shaped protein. Calcium-bound CaM undergoes a rapid transition from an extended dumbbell to a collapsed conformation in response to binding of ligands, which include the small molecule drug trifluoperazine (TFP). We hypothesized that appropriate incorporation of CaM into a hydrogel network would enable its motion to be translated into a spatially defined structural change in a polymer network, resulting in a protein-based, dynamic material. To assemble protein-based materials using a photochemical approach, we first generated photo-reactive protein units using an engineered protein and a Michael-type addition reaction. We synthesized and purified an engineered version of CaM that includes cysteine residues in place of threonine residues at the ends of the dumbbell-shaped protein (CaM (T34C, T110C)), and reacted this engineered protein with a 10X molar excess of poly(ethylene glycol) diacrylate (PEGDA) chains with varying molecular weight. The resulting conjugates were photocrosslinked to form tunable dynamic, protein-based hydrogels.
Results: Michael-type addition of a cysteine sulfhydryl group to an acrylate group resulted in formation of PEG-CaM-PEG conjugates, and a subset of these conjugates could be efficiently photo-crosslinked into hydrogel networks.
CaM-PEG302, PEG400-CaM-PEG400, PEG575-CaM-PEG575, PEG1000-CaM-PEG1000, and PEG8000-CaM-PEG8000 respectively) was confirmed by SDS-PAGE, and the full reaction of
The PEG molecular weight and φ dictated the dynamic properties of CaM-based networks, and variation in these parameters enabled tuning of network dynamics. When exposed to the well-characterized CaM-binding ligand TFP, the volume of these hydrogels decreased significantly and the magnitude of the change depended on the PEG chain length and φ. PEG400-CaM-PEG400 hydrogels underwent the highest magnitude of volume change at all three φs studied, with a maximum decrease to 25±14% of the initial hydrogel volume in the 12wt% PEG400-CaM-PEG400 condition, a 4-fold volume decrease. PEG8000-CaM-PEG8000 hydrogels demonstrated a more modest dynamic response (volume decreases to ~90% of initial volume). Dynamic changes in volume upon CaM-ligand binding were highly reversible in nearly all experimental conditions. When hydrogels were rinsed with EGTA to disrupt the Ca-dependent TFP-CaM binding, each of the hydrogels recovered to greater than 90% of their initial volume.
CaM-containing hydrogels also underwent substantial and reversible changes in optical properties when exposed to TFP, and the changes were strongly dependent on PEG molecular weight and φ. These changes in optical transparency followed identical trends to those of the corresponding volume shifts described in the previous paragraph. Upon removal of the TFP ligand and incubation in a Ca++ buffer, all hydrogels recovered to over 80% of their initial optical transparency. The reversibility of these dynamic hydrogel systems was further tested by repeatedly cycling the hydrogels between collapsed and expanded states. Both the reversibility of the volume change and the optical transparency change were well maintained up to at least 5 cycles. Importantly, changes in optical transparency in CaM-based hydrogels can be used as a mechanism to create high throughput, label-free biosensing platforms. As a simple demonstration, two types of PEG-CaM-PEG hydrogels (PEG8000-CaMPEG8000 and PEG575-CaM-PEG575) were formed in wells of a 1536-well plate and exposed to TFP. Microscopic images demonstrated a sharp contrast between the optical transparency of the two hydrogel types, and it is therefore possible to detect TFP in a label-free manner.
