Irreversible light-activated SpyLigation mediates split-protein assembly in 4D.

The conditional assembly of split-protein pairs to modulate biological activity is commonly achieved by fusing split-protein fragments to dimerizing components that bring inactive pairs into close proximity in response to an exogenous trigger. However, current methods lack full spatial and temporal control over reconstitution, require sustained activation and lack specificity. Here light-activated SpyLigation (LASL), based on the photoregulation of the covalent SpyTag (ST)/SpyCatcher (SC) peptide-protein reaction, assembles nonfunctional split fragment pairs rapidly and irreversibly in solution, in engineered biomaterials and intracellularly. LASL introduces an ortho-nitrobenzyl(oNB)-caged lysine into SC's reactive site to generate a photoactivatable SC (pSC). Split-protein pairs of interest fused to pSC and ST are conditionally assembled via near-ultraviolet or pulsed near-infrared irradiation, as the uncaged SC can react with ST to ligate appended fragments. We describe procedures for the efficient synthesis of the photocaged amino acid that is incorporated within pSC (<5 days) as well as the design and cloning of LASL plasmids (1-4 days) for recombinant protein expression in either Escherichia coli (5-6 days) or mammalian cells (4-6 days), which require some prior expertise in protein engineering. We provide a chemoenzymatic scheme for appending bioorthogonal reactive handles onto E. coli-purified pSC protein (<4 days) that permits LASL component incorporation and patterned protein activation within many common biomaterial platforms. Given that LASL is irreversible, the photolithographic patterning procedures are fast and do not require sustained light exposure. Overall, LASL can be used to interrogate and modulate cell signaling in various settings.

Spatiotemporal functional assembly of split protein pairs through a light-activated SpyLigation.

Proteins provide essential functional regulation of many bioprocesses across all scales of life; however, new techniques to specifically modulate protein activity within living systems and in engineered biomaterials are needed to better interrogate fundamental cell signalling and guide advanced decisions of biological fate. Here we establish a generalizable strategy to rapidly and irreversibly activate protein function with full spatiotemporal control. Through the development of a genetically encoded and light-activated SpyLigation (LASL), bioactive proteins can be stably reassembled from non-functional split fragment pairs following brief exposure (typically minutes) to cytocompatible light. Employing readily accessible photolithographic processing techniques to specify when, where and how much photoligation occurs, we demonstrate precise protein activation of UnaG, NanoLuc and Cre recombinase using LASL in solution, biomaterials and living mammalian cells, as well as optical control over protein subcellular localization. Looking forward, we expect that these photoclick-based optogenetic approaches will find tremendous utility in probing and directing complex cellular fates in both time and three-dimensional space.

Dynamic Hydrogels from Host-Guest Supramolecular Interactions.

Hydrogel biomaterials are pervasive in biomedical use. Applications of these soft materials range from contact lenses to drug depots to scaffolds for transplanted cells. A subset of hydrogels is prepared from physical cross-linking mediated by host-guest interactions. Host macrocycles, the most recognizable supramolecular motif, facilitate complex formation with an array of guests by inclusion in their portal. Commonly, an appended macrocycle forms a complex with appended guests on another polymer chain. The formation of poly(pseudo)rotaxanes is also demonstrated, wherein macrocycles are threaded by a polymer chain to give rise to physical cross-linking by secondary non-covalent interactions or polymer jamming. Host-guest supramolecular hydrogels lend themselves to a variety of applications resulting from their dynamic properties that arise from non-covalent supramolecular interactions, as well as engineered responsiveness to external stimuli. These are thus an exciting new class of materials.