Enzymatic functionalization of RNA oligonucleotides by terminal uridylyl transferase using fluorescent and clickable nucleotide analogs

We report a systematic study on controlling the enzyme activity of a terminal uridylyl transferase (TUTase) called SpCID1, which provides methods to effect site-specific incorporation of a single modified nucleotide analog at the 3'-end of an RNA oligonucleotide (ON). Responsive heterocycle-modified fluorescent UTP probes that are useful in analyzing non-canonical nucleic acid structures and azide- and alkyne-modified UTP analogs that are compatible for chemoenzymatic functionalization were used as study systems. In the first strategy, we balanced the concentration of essential metal ion cofactors (Mg