STING agonist profiling by nucleotide library defines structural determinants of cyclic dinucleotide recognition.

Stimulator of interferon genes (STING) is the principal mammalian receptor for cyclic dinucleotides (CDNs) and a central hub of innate immune signaling. CDN analogues and small-molecule STING agonists are in active development for cancer immunotherapy and as vaccine adjuvants, although clinical translation has proven challenging. Here, we systematically profile STING nucleotide preferences by treating THP-1 monocytes and pancreatic cancer cells with a structurally diverse nucleotide library. We identify 27 CDNs that activate STING, while non-CDN nucleotides failed to elicit detectable responses, underscoring the selectivity of STING for its ligands. We demonstrate that STING's preference for 2'3'-linked CDNs underlies its capacity to accommodate purine-pyrimidine hybrid nucleobases. Molecular dynamics simulations reveal that nucleobase position relative to the 2'3' linkage is a critical determinant of STING engagement. In addition, we show STING activation by diverse non-hydrolysable c-di-AMP and cGAMP isomers, thereby expanding the opportunities for designing STING agonists for therapeutic applications.