ABSTRACT
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.
Subject(s)
Mammals , AnimalsABSTRACT
Though the biomaterials community has widely utilized near-ultraviolet (UV) light to make and modify scaffolds for 3D cell culture, thorough examination of the downstream effects of such light on cell function has not been performed. Here, we investigate the global effects of common light treatments on NIH3T3 fibroblasts and human mesenchymal stem cells (hMSCs), cell types regularly employed in tissue engineering. Unchanged proliferation rates, an absence of apoptotic induction, and an unaltered proteome following low-dose 365 nm light exposure are observed, implying that near-UV-based radical-free photochemistries can be exploited in biomaterial systems without deleteriously affecting cell fate.
ABSTRACT
Stimuli-responsive biomaterials are useful platforms for environmentally triggered drug delivery. By varying the molecular architecture of orthogonal stimuli-labile linkages between small molecules and non-degradable materials, we demonstrate the Boolean logic-based release of model therapeutics from gels. Programmable responses are demonstrated for materials sensitive to input combinations involving enzymes, chemical reductants, and light via YES, OR, and AND logic gates.
Subject(s)
Biocompatible Materials/chemistry , Drug Delivery Systems/methods , Hydrogels/chemistry , Logic , Small Molecule Libraries/chemistryABSTRACT
This work introduces light-activated bioorthogonal noncanonical amino acid tagging (laBONCAT) as a method to selectively label, isolate, and identify proteins newly synthesized at user-defined regions in tissue culture. By photocaging l-azidohomoalanine (Aha), metabolic incorporation into proteins is prevented. The caged compound remains stable for many hours in culture, but can be photochemically liberated rapidly and on demand with spatial control. Upon directed light exposure, the uncaged amino acid is available for local translation, enabling downstream proteomic interrogation via bioorthogonal conjugation. Exploiting the reactive azide moiety present on Aha's amino acid side chain, we demonstrate that newly synthesized proteins can be purified for quantitative proteomics or visualized in synthetic tissues with a new level of spatiotemporal control. Shedding light on when and where proteins are translated within living samples, we anticipate that laBONCAT will aid in understanding the progression of complex protein-related disorders.
