Two-step synthesis of 5′-deoxy-5′-thioguanosine-5′-monophosphorothioate and its incorporation efficiency into 5′-terminus of RNA for preparation of thiol-functionalized RNA

Several 5′-modifications of RNA molecules have been shown to have broad applications in studying RNA structures, mapping RNA-protein interactions, and in vitro selection of catalytic RNAs. While phosphorothioate modification is one of the most popular methods for functionalizing the 5′-terminus of RNA by a transcription or kinase reaction, conjugation of terminal phosphorothioates with fluorophores has been achieved only with a low efficiency. To overcome this limitation, we have developed a two-step synthetic method for 5′-deoxy-5′-thioguanosine-5′-monophosphorothioate by combining two known reactions and measured its incorporation efficiency into the 5′-terminus of RNA by in vitro transcription using T7 RNA polymerase that requires guanosine to efficiently initiate transcription, followed by treatment of alkaline phosphatase, yielding a terminal sulfhydryl group at the 5′-termini of RNA molecules. Since the sulfhydryl group can be used as an alternative to phosphorothioates, our method may provide a useful route to efficiently introduce reporters, such as fluorophores, into the 5′-terminus of RNA via a stable thio-linker, or to tether the oligomer to a solid support.