Cyclic dinucleotides (CDNs) trigger the innate immune response in eukaryotic cells through the stimulator of interferon genes (STING) signaling pathway. To decipher this complex cellular process, a better correlation between structure and downstream function is required. Herein, we report the design and immunostimulatory effect of a novel group of c-di-GMP analogues. By employing an "atomic mutagenesis" strategy, changing one atom at a time, a class of gradually modified CDNs was prepared. These c-di-GMP analogues induce type-I interferon (IFN) production, with some being more potent than c-di-GMP, their native archetype. This study demonstrates that CDN analogues bearing modified nucleobases are able to tune the innate immune response in eukaryotic cells.