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

Autor: Dutta S; Department of Chemistry, Indian Institute of Science Education and Research (IISER), Pune Dr. Homi Bhabha Road, Pune, 411008, India., Srivatsan SG; Department of Chemistry, Indian Institute of Science Education and Research (IISER), Pune Dr. Homi Bhabha Road, Pune, 411008, India.
Jazyk: angličtina
Zdroj: Chemistry, an Asian journal [Chem Asian J] 2024 Sep 16; Vol. 19 (18), pp. e202400475. Date of Electronic Publication: 2024 Aug 19.
DOI: 10.1002/asia.202400475
Abstrakt: 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 Mn 2+ 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.)
Databáze: MEDLINE
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