1. Enzymatic Functionalization of RNA Oligonucleotides by Terminal Uridylyl Transferase Using Fluorescent and Clickable Nucleotide Analogs.
- Author
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Dutta S and Srivatsan SG
- Subjects
- Uridine Triphosphate chemistry, Uridine Triphosphate metabolism, Uridine Triphosphate analogs & derivatives, Alkynes chemistry, RNA Nucleotidyltransferases metabolism, RNA Nucleotidyltransferases chemistry, Azides chemistry, Nucleotides chemistry, Nucleotides metabolism, Fluorescent Dyes chemistry, Click Chemistry, RNA chemistry, RNA metabolism, Oligonucleotides chemistry, Oligonucleotides metabolism
- Abstract
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
2+ 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., (© 2024 Wiley-VCH GmbH.)- Published
- 2024
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