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A fully enzymatic method for site-directed spin labeling of long RNA.

Lebars I, Vileno B, Bourbigot S, Turek P, Wolff P, Kieffer B - Nucleic Acids Res. (2014)

Bottom Line: The paramagnetic thiol-specific reagent is subsequently attached to the RNA ligation product.This novel strategy is demonstrated by introducing a paramagnetic probe into the 55 nucleotides long RNA corresponding to K-turn and Specifier Loop domains from the Bacillus subtilis tyrS T-Box leader RNA.The efficiency of the coupling reaction and the quality of the resulting spin-labeled RNA were assessed by Mass Spectrometry, Electron Paramagnetic Resonance (EPR) and Nuclear Magnetic Resonance (NMR).

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Département de Biologie Structurale, Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé et de la Recherche Médicale (INSERM) U964/Université de Strasbourg, 1 rue Laurent Fries, BP 10142, 67404 Illkirch cedex, France lebars@igbmc.fr.

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Molecular weight determined by MS. (A) ESI-MS spectra of wild-type RNA (top) and of the spin-labeled RNA after MaxEnt deconvolution showing a first approximate of the masses. Exact masses were next calculated manually from the multiply charged species (Table 1). (B) MSMS spectrum of the spin-labeled RNA after RNaseA treatment. The fragment 5′-A23G24SLG25A26C27 p-3′ whose parent mass is 1884.1 Da, contains the thio-modified G bearing the nitroxide modification. Possible fragmentations are indicated by w, x, y and z series.
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Figure 3: Molecular weight determined by MS. (A) ESI-MS spectra of wild-type RNA (top) and of the spin-labeled RNA after MaxEnt deconvolution showing a first approximate of the masses. Exact masses were next calculated manually from the multiply charged species (Table 1). (B) MSMS spectrum of the spin-labeled RNA after RNaseA treatment. The fragment 5′-A23G24SLG25A26C27 p-3′ whose parent mass is 1884.1 Da, contains the thio-modified G bearing the nitroxide modification. Possible fragmentations are indicated by w, x, y and z series.

Mentions: The coupling reaction efficiency as well as the integrity of the resulting spin-labeled RNA were checked using Electro Spray Inject Mass Spectrometry. Exact masses for the wild-type RNA and the spin-labeled RNA were found at 17967.5 ± 0.5 Da and 18180.5 ± 0.9 Da, respectively (Table 1). The corresponding mass difference (213 Da) corresponds to the addition of the proxyl group and the substitution of an oxygen by a sulfur atom on the guanine as expected. Furthermore, as shown in Figure 3A, the spin-labeled RNA was obtained as a major product and no significant signal corresponding to the unmodified RNA could be detected, indicating that the coupling reaction was effective. The modification was further characterized using an RNaseA treatment prior MS analysis in order to confirm the nucleotide sequence and the localization of the modification (Figure 3B). The RNase A cleaves RNA sequence after pyrimidine nucleotides. The fragment 5′-A23G24SLG25A26C27 p -3′ whose monoisotopic mass is 1884.1 Da, contains the thio-modified G bearing the nitroxide modification. MS fragmentation of this modified oligonucleotide leads to typical fragment ions depicted in Figure 3B (Supporting Table S1; 48). Mass values of 329.1, 345.3, 329.1 and 305.0 were found respectively for A23, G25, A26 and C27 while the mass of the spin-labeled G24 nucleotide was found at 558.4 Da, demonstrating that the RNA is fully modified at the desired position.


A fully enzymatic method for site-directed spin labeling of long RNA.

Lebars I, Vileno B, Bourbigot S, Turek P, Wolff P, Kieffer B - Nucleic Acids Res. (2014)

Molecular weight determined by MS. (A) ESI-MS spectra of wild-type RNA (top) and of the spin-labeled RNA after MaxEnt deconvolution showing a first approximate of the masses. Exact masses were next calculated manually from the multiply charged species (Table 1). (B) MSMS spectrum of the spin-labeled RNA after RNaseA treatment. The fragment 5′-A23G24SLG25A26C27 p-3′ whose parent mass is 1884.1 Da, contains the thio-modified G bearing the nitroxide modification. Possible fragmentations are indicated by w, x, y and z series.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150755&req=5

Figure 3: Molecular weight determined by MS. (A) ESI-MS spectra of wild-type RNA (top) and of the spin-labeled RNA after MaxEnt deconvolution showing a first approximate of the masses. Exact masses were next calculated manually from the multiply charged species (Table 1). (B) MSMS spectrum of the spin-labeled RNA after RNaseA treatment. The fragment 5′-A23G24SLG25A26C27 p-3′ whose parent mass is 1884.1 Da, contains the thio-modified G bearing the nitroxide modification. Possible fragmentations are indicated by w, x, y and z series.
Mentions: The coupling reaction efficiency as well as the integrity of the resulting spin-labeled RNA were checked using Electro Spray Inject Mass Spectrometry. Exact masses for the wild-type RNA and the spin-labeled RNA were found at 17967.5 ± 0.5 Da and 18180.5 ± 0.9 Da, respectively (Table 1). The corresponding mass difference (213 Da) corresponds to the addition of the proxyl group and the substitution of an oxygen by a sulfur atom on the guanine as expected. Furthermore, as shown in Figure 3A, the spin-labeled RNA was obtained as a major product and no significant signal corresponding to the unmodified RNA could be detected, indicating that the coupling reaction was effective. The modification was further characterized using an RNaseA treatment prior MS analysis in order to confirm the nucleotide sequence and the localization of the modification (Figure 3B). The RNase A cleaves RNA sequence after pyrimidine nucleotides. The fragment 5′-A23G24SLG25A26C27 p -3′ whose monoisotopic mass is 1884.1 Da, contains the thio-modified G bearing the nitroxide modification. MS fragmentation of this modified oligonucleotide leads to typical fragment ions depicted in Figure 3B (Supporting Table S1; 48). Mass values of 329.1, 345.3, 329.1 and 305.0 were found respectively for A23, G25, A26 and C27 while the mass of the spin-labeled G24 nucleotide was found at 558.4 Da, demonstrating that the RNA is fully modified at the desired position.

Bottom Line: The paramagnetic thiol-specific reagent is subsequently attached to the RNA ligation product.This novel strategy is demonstrated by introducing a paramagnetic probe into the 55 nucleotides long RNA corresponding to K-turn and Specifier Loop domains from the Bacillus subtilis tyrS T-Box leader RNA.The efficiency of the coupling reaction and the quality of the resulting spin-labeled RNA were assessed by Mass Spectrometry, Electron Paramagnetic Resonance (EPR) and Nuclear Magnetic Resonance (NMR).

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Département de Biologie Structurale, Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé et de la Recherche Médicale (INSERM) U964/Université de Strasbourg, 1 rue Laurent Fries, BP 10142, 67404 Illkirch cedex, France lebars@igbmc.fr.

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Related in: MedlinePlus