Universal initiator nucleotides for the enzymatic synthesis of 5′-amino- and 5′-thiol-modified RNA

We report the chemical synthesis of 5′-amino- and 5′-thiol-hexaethylene glycol guanosine nucleotides and their enzymatic incorporation into RNA, followed by chemical modifications at their nucleophilic ends. By using two similar routes, the conjugates of guanosine-5′-monophosphate and hexaethylene glycol with attached reactive groups ( SH or NH 2) were synthesized using phosphoramidite chemistry, and characterized by MALDI TOF mass spectrometry. These initiator molecules were efficiently incorporated into RNA at the 5′-end by run-off transcription using T7 RNA polymerase. The potential of these RNA conjugates for a broad reaction range with electrophiles is shown here, thereby enabling their use for diverse biochemical applications.