Isotope-Labeled RNA Building Blocks for NMR Structure and Dynamics Studies

Olenginski, Lukasz T.; Taiwo, Kehinde M.; LeBlanc, Regan M.; Dayie, T. Kwaku

doi:10.3390/molecules26185581

Cited by 10 publications

(15 citation statements)

References 124 publications

(256 reference statements)

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“…multidimensional experiments or selective magnetization transfer [5][6][7]), the issue can be tackled on the sample preparation side. Site-specific isotope labeling is one way to overcome the signal overlap that has been addressed extensively in the literature [8,9].…”

Section: Introductionmentioning

confidence: 99%

“…The most common method for site-specific incorporation of 13 C or 15 N-labeled nucleotides is solid-phase oligonucleotide synthesis (SPOS). Since these building blocks are often synthesized in-house, they can be modified, atom-specifically labeled or both [9,10]. While SPOS has unique strengths, the most important one being simple incorporation of chemical modifications or isotope-labeled residues, the yield drops rapidly for larger constructs (>50 nucleotides (nt)).…”

Section: Introductionmentioning

confidence: 99%

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Enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR

et al. 2022

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Solution NMR spectroscopy is a well-established tool with unique advantages for structural studies of RNA molecules. However, for large RNA sequences, the NMR resonances often overlap severely. A reliable way to perform resonance assignment and allow further analysis despite spectral crowding is the use of site-specific isotope labeling in sample preparation. While solid-phase oligonucleotide synthesis has several advantages, RNA length and availability of isotope-labeled building blocks are persistent issues. Purely enzymatic methods represent an alternative and have been presented in the literature. In this study, we report on a method in which we exploit the preference of T7 RNA polymerase for nucleotide monophosphates over triphosphates for the 5’ position, which allows 5’-labeling of RNA. Successive ligation to an unlabeled RNA strand generates a site-specifically labeled RNA. We show the successful production of such an RNA sample for NMR studies, report on experimental details and expected yields, and present the surprising finding of a previously hidden set of peaks which reveals conformational exchange in the RNA structure. This study highlights the feasibility of site-specific isotope-labeling of RNA with enzymatic methods.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR

et al. 2022

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“…The most common method for site-specific incorporation of 13 C or 15 N-labeled nucleotides is solid-phase oligonucleotide synthesis (SPOS). Since these building blocks are often synthesized in-house, they can be modified, atom-specifically labeled or both (9,10). While SPOS has unique strengths, the most important one being simple incorporation of chemical modifications or isotope-labeled residues, the yield drops rapidly for larger constructs (>50 nucleotides (nt)).…”

Section: Introductionmentioning

confidence: 99%

“…multidimensional experiments or selective magnetization transfer(5-7)), the issue can be tackled on the sample preparation side. Site-specific isotope labeling is one way to overcome the signal overlap that has been addressed extensively in the literature (8,9). The most common method for site-specific incorporation of 13 C or 15 N-labeled nucleotides is solid-phase oligonucleotide synthesis (SPOS).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Purely enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR

Feyrer

Gurdap

Marušič

et al. 2022

Preprint

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Solution NMR spectroscopy is a well-established tool with unique advantages for structural studies of RNA molecules. However, for large RNA sequences, the NMR resonances often overlap severely. A reliable way to perform resonance assignment and allow further analysis despite spectral crowding is the use of site-specific isotope labeling in sample preparation. While solid-phase oligonucleotide synthesis has several advantages, RNA length and availability of isotope-labeled building blocks are persistent issues. Purely enzymatic methods pose as an alternative and have been presented in the literature. In this study, we report on a method in which we exploit the preference of T7 RNA polymerase for nucleotide monophosphates over triphosphates for the 5’ position, which allows 5’-labeling of RNA. Successive ligation to an unlabeled RNA strand generates a site-specifically labeled RNA. We show the successful production of such an RNA sample for NMR studies, report on experimental details and expected yields, and present the surprising finding of a previously hidden set of peaks which reveals conformational exchange in the RNA structure. This study highlights the feasibility of site-specific isotope-labeling of RNA with enzymatic methods.

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Syntheses of Specifically ¹⁵N‐Labeled Adenosine and Guanosine

Jones

Gaffney

2022

Current Protocols

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This unit describes the specific incorporation of 15 N into the N7 and amino positions of adenosine (Basic Protocol 1), and conversion of the adenosine to guanosine labeled at the N1, N7, and amino positions (Basic Protocol 2). Two variations of the procedures are also presented that include either 12 C or 13 C at the C8 position of adenosine, and 13 C at either the C8 or C2 position of guanosine. These 13 C tags permit the incorporation of two 15 N-labeled nucleosides into an RNA strand while ensuring that their nuclear magnetic resonance (NMR) signals can be distinguished from each other by the presence or absence of C-N coupling. While the major application of these specifically 15 N-labeled nucleosides is NMR, the additional mass makes them useful in mass spectrometry (MS) as well. The procedures can also be adapted to synthesize the labeled deoxynucleosides. The Support Protocol describes the synthesis of 7-methylguanosine. CAUTION:The procedures in this unit use a number of highly toxic and dangerous reagents. Raney nickel is pyrophoric when dry and may burst into flames if not kept wet. Phosphorous oxychloride (POCl 3 ) is very reactive and hydrolyzes to hydrochloric and phosphoric acids, which are both highly corrosive to skin and tissue. Cyanogen bromide is very dangerous; improper use of this reagent has led to deaths in the laboratory. All reactions must be done with great care in an appropriate chemical fume hood.

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Isotope-Labeled RNA Building Blocks for NMR Structure and Dynamics Studies

Cited by 10 publications

References 124 publications

Enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR

Enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR

Purely enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR

Syntheses of Specifically ¹⁵N‐Labeled Adenosine and Guanosine

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Isotope-Labeled RNA Building Blocks for NMR Structure and Dynamics Studies

Cited by 10 publications

References 124 publications

Enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR

Enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR

Purely enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR

Syntheses of Specifically 15N‐Labeled Adenosine and Guanosine

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Syntheses of Specifically ¹⁵N‐Labeled Adenosine and Guanosine