{"title":"使用荧光和可点击核苷酸类似物,通过末端尿苷酸转移酶对 RNA 寡核苷酸进行酶功能化。","authors":"Swagata Dutta, Seergazhi G Srivatsan","doi":"10.1002/asia.202400475","DOIUrl":null,"url":null,"abstract":"<p><p>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<sup>2+</sup> and Mn<sup>2+</sup> 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.</p>","PeriodicalId":145,"journal":{"name":"Chemistry - An Asian Journal","volume":null,"pages":null},"PeriodicalIF":3.5000,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Enzymatic Functionalization of RNA Oligonucleotides by Terminal Uridylyl Transferase Using Fluorescent and Clickable Nucleotide Analogs.\",\"authors\":\"Swagata Dutta, Seergazhi G Srivatsan\",\"doi\":\"10.1002/asia.202400475\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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<sup>2+</sup> and Mn<sup>2+</sup> 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.</p>\",\"PeriodicalId\":145,\"journal\":{\"name\":\"Chemistry - An Asian Journal\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.5000,\"publicationDate\":\"2024-07-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Chemistry - An Asian Journal\",\"FirstCategoryId\":\"1\",\"ListUrlMain\":\"https://doi.org/10.1002/asia.202400475\",\"RegionNum\":3,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"CHEMISTRY, MULTIDISCIPLINARY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Chemistry - An Asian Journal","FirstCategoryId":"1","ListUrlMain":"https://doi.org/10.1002/asia.202400475","RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"CHEMISTRY, MULTIDISCIPLINARY","Score":null,"Total":0}
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 (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.
期刊介绍:
Chemistry—An Asian Journal is an international high-impact journal for chemistry in its broadest sense. The journal covers all aspects of chemistry from biochemistry through organic and inorganic chemistry to physical chemistry, including interdisciplinary topics.
Chemistry—An Asian Journal publishes Full Papers, Communications, and Focus Reviews.
A professional editorial team headed by Dr. Theresa Kueckmann and an Editorial Board (headed by Professor Susumu Kitagawa) ensure the highest quality of the peer-review process, the contents and the production of the journal.
Chemistry—An Asian Journal is published on behalf of the Asian Chemical Editorial Society (ACES), an association of numerous Asian chemical societies, and supported by the Gesellschaft Deutscher Chemiker (GDCh, German Chemical Society), ChemPubSoc Europe, and the Federation of Asian Chemical Societies (FACS).