Designing biological light from scratch - a new paradigm for bioimaging and biosensing - Project Summary An ideal bioimaging technique should allow real-time visualization (temporal) across a wide range of scales (spatial) while being non-invasive. During the past decades, fluorescent proteins have undergone extensive engineering and unlocked the monitoring of cellular events at the micro- scale, but their use for in vivo macro-scale imaging is limited by the need for external excitation light. On the other hand, bioluminescence technologies produce photons via a luciferase- catalyzed chemiluminescence reaction that does not require external light, making them more suitable for in vivo imaging. However, naturally occurring luciferases are either faint, ATP- dependent, or poorly folded, limiting broad biomedical use. Since bioluminescent proteins are scarce in nature and both the enzyme (a.k.a. luciferase) and the substrate must be engineered simultaneously to improve the light emission efficiency, the development of bioluminescent probes has lagged behind that of well-established fluorescent protein families. Here, we present an integrated and interdisciplinary approach, combining de novo protein design, synthetic chemistry, and deep learning methods, to create a family of new-to-nature luciferase- synthetic substrate pairs from scratch, dubbed “neoluminescence”. On the chemistry side, we will create synthetic substrates with high quantum yield, distinct emission colors, and biocompatible properties. In parallel, we will develop new deep learning models to guide the design of compact, thermostable de novo luciferases tailored to each substrate, achieving high catalytic activity, high substrate specificity, cofactor independence, and orthogonality across pairs. Together, these advances will deliver a broadly applicable neoluminescent toolkit for multiplex monitoring of biological processes across micro (nm) to macro (cm) scales. Our goal is to harness the power of AI-guided de novo protein design to boldly transform the luminescent probes for vast biomedical applications beyond what fluorescent proteins have achieved, paving the way for the utilities in preclinical studies, clinical diagnostics, and beyond to improve human health.
