Bioorthogonal Regulation of Negatively Charged Protein Side Chains in Living Systems

Negatively charged amino acid side chains are essential determinants of protein structure and function, yet their precise temporal regulation in living systems remains challenging. Although coupling the genetic code expansion strategy with bioorthogonal decaging reaction has enabled conditional control over various residues, the regulation of negatively charged aspartic acid and glutamic acid side chains has primarily relied on light-triggered photodecaging, while complementary chemical-decaging methods remain limited for applications involving limited light penetration or concerns regarding potential photodamage. Here, we developed para-azidobenzyl aspartate (PABD) through the combination of computationally guided molecular design and directed evolution, establishing a small molecule-triggered, bioorthogonal approach for site-specific regulation of negative charges across diverse biological contexts. PABD-mediated decaging provides efficient and traceless restoration of native aspartate side chains under physiological conditions at molecular, cellular, and organismal levels. Using this strategy, we demonstrate chemical regulation of Cas9-mediated genome editing and ALKBH1-dependent RNA epigenetic modification in mammalian cells, and even the controlled activation of an enzyme in live animals. This work provides a generalizable bioorthogonal platform for manipulating negatively charged protein side chains, offering a versatile chemical tool for gain-of-function studies in complex biological environments.