In this technical note, we systematically describe the chemical synthesis, deprotection, and purification of diphosphorylated RNA oligonucleotides. This process eliminates the need for an enzymatic step after chemical synthesis of a 5’triphosphate RNA, allowing the user to synthesize the 5’diphosphate RNA directly. This protocol can also be applied to other chemically synthesized nucleic acids, including those with modifications.
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