Enzymatic Functionalization of RNA Oligonucleotides by Terminal Uridylyl Transferase Using Fluorescent and Clickable Nucleotide Analogs

Abstract

AbstractWe 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 (Mg2+ and Mn2+ ions) to restrict the processivity of the enzyme, which gave a very good control on the incorporation of clickable nucleotide analogs. In the second approach, borate that complexes with 2′ and 3′ oxygen atoms of a ribose sugar was used as a reversibly binding chelator to block repeated addition of nucleotide analogs. Notably, in the presence of heterocycle‐modified fluorescent UTPs, we obtained single‐nucleotide incorporated RNA products in reasonable yields, while with clickable nucleotides yields were very good. Further, 3′‐end azide‐ and alkyne‐labeled RNA ONs were post‐enzymatically functionalized by CuAAC and SPAAC reactions with fluorescent probes. These strategies broaden the scope of TUTase in site‐specifically installing modifications of different types onto RNA for various applications.