One of our most important cellular defenses against RNA viruses is an innate immune sensor known as retinoic acid inducible gene-I (RIG-I). RIG-I belongs to a family of double-stranded RNA (dsRNA) binding proteins known as RIG-I-like receptors (RLRs) which recognize and respond to various types of viral RNA, providing protection against infection. Studies have shown that both 5'-triphosphate (p3dsRNA) and 5'-diphosphate (p2dsRNA) are potent agonists for the RIG-I receptor, with p2dsRNA being the primary RNA requirement for activation of RIG-I. Until recently, RIG-I had never been captured in complex with p2dsRNA, most likely due to the lack of commercially available 5'-diphophate RNA ligands. The generation of 5'pp-RNA from chemically synthesized 5'ppp-RNA has previously been described, involving incubation of the 5'ppp-RNA with vaccinia virus capping enzyme in the absence of GTP and S-adenosyl methionine. Recently, non-structural protein 12 (nsp12), one of the factors responsible for RNA synthesis in SARS-CoV-2, was used to generate diphosphorylated RNA in the presence of 5mM MgCl2 with a similar efficiency as vaccinia virus capping enzyme. The procedure described herein allows for removal of this intermediate step, enabling direct chemical synthesis of 5'pp-RNA. This protocol was adapted from methods published by Zlatev, et al. and optimized for the MerMade 12 RNA-DNA synthesizer (BioAutomation).