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.