Deuterated nucleotides as chemical probes of RNA structure: a detailed protocol for the enzymatic synthesis of a complete set of nucleotides specifically deuterated at ribose carbons
Abstract We describe here a detailed protocol for the synthesis of ribonucleotides specifically deuterated at each ribose carbon atom. We synthesized 20 specifically deuterated ribonucleotides: ATP, CTP, GTP, and UTP, each of which contained one of five deuterated riboses (either 1′-D, 2″-D, 3′-D,...
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| Format: | Article |
| Language: | English |
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ScienceOpen
2015-04-01
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| Series: | ScienceOpen Research |
| Online Access: | https://www.scienceopen.com/document?vid=eb44f1b0-c408-4336-a2c0-aed203250898 |
| _version_ | 1797896891649753088 |
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| author | Robert Azad Shakti Ingle Tom Tullius |
| author_facet | Robert Azad Shakti Ingle Tom Tullius |
| author_sort | Robert Azad |
| collection | DOAJ |
| description |
Abstract
We describe here a detailed protocol for the synthesis of ribonucleotides specifically deuterated at each ribose carbon atom. We synthesized 20 specifically deuterated ribonucleotides: ATP, CTP, GTP, and UTP, each of which contained one of five deuterated riboses (either 1′-D, 2″-D, 3′-D, 4′-D, or 5′,5″-D2). Our synthetic approach is inspired by the pioneering work of Tolbert and Williamson, who developed a method for the convenient one-pot enzymatic synthesis of nucleotides (Tolbert, T. J. and Williamson, J. R. (1996) J. Am. Chem. Soc. 118, 7929–7940). Our protocol consists of a comprehensive list of required chemical and enzymatic reagents and equipment, detailed procedures for enzymatic assays and nucleotide synthesis, and chromatographic procedures for purification of deuterated nucleotides. As an example of the utility of specifically deuterated nucleotides, we used them to synthesize specifically deuterated sarcin/ricin loop (SRL) RNA and measured the deuterium kinetic isotope effect on hydroxyl radical cleavage of the SRL.
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| first_indexed | 2024-04-10T07:50:01Z |
| format | Article |
| id | doaj.art-8bd30f90e2164b239924113b0264013a |
| institution | Directory Open Access Journal |
| issn | 2199-1006 |
| language | English |
| last_indexed | 2024-04-10T07:50:01Z |
| publishDate | 2015-04-01 |
| publisher | ScienceOpen |
| record_format | Article |
| series | ScienceOpen Research |
| spelling | doaj.art-8bd30f90e2164b239924113b0264013a2023-02-23T10:21:04ZengScienceOpenScienceOpen Research2199-10062015-04-0110.14293/S2199-1006.1.SOR-LIFE.ALCJCN.v1Deuterated nucleotides as chemical probes of RNA structure: a detailed protocol for the enzymatic synthesis of a complete set of nucleotides specifically deuterated at ribose carbonsRobert AzadShakti IngleTom Tullius Abstract We describe here a detailed protocol for the synthesis of ribonucleotides specifically deuterated at each ribose carbon atom. We synthesized 20 specifically deuterated ribonucleotides: ATP, CTP, GTP, and UTP, each of which contained one of five deuterated riboses (either 1′-D, 2″-D, 3′-D, 4′-D, or 5′,5″-D2). Our synthetic approach is inspired by the pioneering work of Tolbert and Williamson, who developed a method for the convenient one-pot enzymatic synthesis of nucleotides (Tolbert, T. J. and Williamson, J. R. (1996) J. Am. Chem. Soc. 118, 7929–7940). Our protocol consists of a comprehensive list of required chemical and enzymatic reagents and equipment, detailed procedures for enzymatic assays and nucleotide synthesis, and chromatographic procedures for purification of deuterated nucleotides. As an example of the utility of specifically deuterated nucleotides, we used them to synthesize specifically deuterated sarcin/ricin loop (SRL) RNA and measured the deuterium kinetic isotope effect on hydroxyl radical cleavage of the SRL. https://www.scienceopen.com/document?vid=eb44f1b0-c408-4336-a2c0-aed203250898 |
| spellingShingle | Robert Azad Shakti Ingle Tom Tullius Deuterated nucleotides as chemical probes of RNA structure: a detailed protocol for the enzymatic synthesis of a complete set of nucleotides specifically deuterated at ribose carbons ScienceOpen Research |
| title | Deuterated nucleotides as chemical probes of RNA structure: a detailed protocol for the enzymatic synthesis of a complete set of nucleotides specifically deuterated at ribose carbons |
| title_full | Deuterated nucleotides as chemical probes of RNA structure: a detailed protocol for the enzymatic synthesis of a complete set of nucleotides specifically deuterated at ribose carbons |
| title_fullStr | Deuterated nucleotides as chemical probes of RNA structure: a detailed protocol for the enzymatic synthesis of a complete set of nucleotides specifically deuterated at ribose carbons |
| title_full_unstemmed | Deuterated nucleotides as chemical probes of RNA structure: a detailed protocol for the enzymatic synthesis of a complete set of nucleotides specifically deuterated at ribose carbons |
| title_short | Deuterated nucleotides as chemical probes of RNA structure: a detailed protocol for the enzymatic synthesis of a complete set of nucleotides specifically deuterated at ribose carbons |
| title_sort | deuterated nucleotides as chemical probes of rna structure a detailed protocol for the enzymatic synthesis of a complete set of nucleotides specifically deuterated at ribose carbons |
| url | https://www.scienceopen.com/document?vid=eb44f1b0-c408-4336-a2c0-aed203250898 |
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