Boolean Logic‐Based Controlled Release of Bioactive Proteins with Diversified Inputs

Abstract Stimuli‐responsive biomaterials hold great promise in controlled therapeutic delivery, tissue engineering, and biosensing applications. Recently, molecular assembly via autonomous compilation has been employed to create topologically specified protein cargos that can be site‐specifically tethered to and conditionally released from biomaterials following user‐programmable Boolean logic. Prior implementation has been confined to simple fluorescent protein outputs and model protease inputs. In this manuscript, we extend the applicability of this framework by assembling all 7 unique logical operations emanating from a YES/OR/AND 3‐input operator set to deliver bioactive proteins spanning diverse categories: growth factors (epidermal growth factor), model enzymes (β‐lactamase, NanoLuciferase, and thioredoxin A), therapeutic nanobodies (anti‐human epidermal growth factor receptor 2), de novo‐engineered cytokines (Neoleukin), and fluorescent proteins (mGreenLantern). In so doing, we demonstrate programmable biomacromolecule release from material anchors in response to precise combinations of three orthogonal protease actuators while maintaining native bioactivity. Through inclusion of a photocleavable protein motif, we further establish that visible light can be employed as an additional input in specifying logic‐based protein release. We anticipate these methods will powerfully expand opportunities for targeted therapeutic delivery and beyond.