As one of its goals,s ynthetic biology seeks to increase the number of building blocks in nucleic acids.While efforts towards this goal are well advanced for DNA, they have hardly begun for RNA. Herein, we present ac rystal structure for an RNAr iboswitch where as tem C:Gp air has been replaced by ap air between two components of an artificially expanded genetic-information system (AEGIS), Z and P,( 6-amino-5-nitro-2(1H)-pyridone and 2-aminoimidazo[1,2-a]-1,3,5-triazin-4-(8H)-one). The structure shows that the Z:P pair does not greatly change the conformation of the RNAmolecule nor the details of its interaction with ahypoxanthine ligand. This was confirmed in solution by in-line probing, whicha lso measured a3 .7 nm affinity of the riboswitch for guanine.These data showthat the Z:P pair mimics the natural Watson-Crickg eometry in RNAi nt he first example of ac rystal structure of an RNAm olecule that contains an orthogonal added nucleobase pair.