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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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Characterization of spin-labeled RNA by EPR spectroscopy. EPR spectra of covalently labeled RNA (bottom) compared to free PROXYL (top). Both spectra were recorded in 70 mM sodium phosphate buffer (pH = 6.5), supplemented by 10% v/v D2O, and acquired with identical instrumental parameters. In this case the concentration of spin-labeled RNA was found to be 150 ± 15 μM, whereas the concentration of the free proxyl spectrum corresponds to 50 μM.
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Figure 4: Characterization of spin-labeled RNA by EPR spectroscopy. EPR spectra of covalently labeled RNA (bottom) compared to free PROXYL (top). Both spectra were recorded in 70 mM sodium phosphate buffer (pH = 6.5), supplemented by 10% v/v D2O, and acquired with identical instrumental parameters. In this case the concentration of spin-labeled RNA was found to be 150 ± 15 μM, whereas the concentration of the free proxyl spectrum corresponds to 50 μM.

Mentions: EPR spectroscopy was then used to provide a quantitative assessment of the nitroxide labeling efficiency. The proxyl coupling to the modified RNA resulted into significant changes of the EPR spectrum (Figure 4). While the free unbound PROXYL exhibits three sharp lines of almost equal amplitudes (upper spectrum), larger and heterogeneous line widths were found for the spin-labeled RNA spectrum, indicative of slow and restricted rotational motions (lower spectrum). Further line-shape analysis provided us a correlation time in the range of a few nanoseconds compatible with the RNA molecular size, while it is of ca. 100 picoseconds for the free spin label (43). It is worth noting that no significant trace of free proxyl was found in the spin-labeled RNA spectrum, indicating that all spins are coupled to RNA. The EPR signal was further used to quantify the coupling reaction yield using PROXYL standards of known concentrations. The spin concentration was found to be ca. 150 ± 15 μM from the double integration of the EPR spectrum. This value was in very good agreement with the RNA concentration obtained by ultraviolet absorption (147 ± 15 μM), suggesting that a proxyl-RNA coupling efficiency greater than 95% was achieved. Such a high coupling efficiency results from the separation of the enzymatic RNA ligation and the spin label coupling reactions by a purification step in order to remove the residual DTT. Indeed, while efficient ligation has already been achieved using spin-labeled RNA substrates, significant loss of EPR signal was observed due to the nitroxide reduction under ligation conditions (34).


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)

Characterization of spin-labeled RNA by EPR spectroscopy. EPR spectra of covalently labeled RNA (bottom) compared to free PROXYL (top). Both spectra were recorded in 70 mM sodium phosphate buffer (pH = 6.5), supplemented by 10% v/v D2O, and acquired with identical instrumental parameters. In this case the concentration of spin-labeled RNA was found to be 150 ± 15 μM, whereas the concentration of the free proxyl spectrum corresponds to 50 μM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4150755&req=5

Figure 4: Characterization of spin-labeled RNA by EPR spectroscopy. EPR spectra of covalently labeled RNA (bottom) compared to free PROXYL (top). Both spectra were recorded in 70 mM sodium phosphate buffer (pH = 6.5), supplemented by 10% v/v D2O, and acquired with identical instrumental parameters. In this case the concentration of spin-labeled RNA was found to be 150 ± 15 μM, whereas the concentration of the free proxyl spectrum corresponds to 50 μM.
Mentions: EPR spectroscopy was then used to provide a quantitative assessment of the nitroxide labeling efficiency. The proxyl coupling to the modified RNA resulted into significant changes of the EPR spectrum (Figure 4). While the free unbound PROXYL exhibits three sharp lines of almost equal amplitudes (upper spectrum), larger and heterogeneous line widths were found for the spin-labeled RNA spectrum, indicative of slow and restricted rotational motions (lower spectrum). Further line-shape analysis provided us a correlation time in the range of a few nanoseconds compatible with the RNA molecular size, while it is of ca. 100 picoseconds for the free spin label (43). It is worth noting that no significant trace of free proxyl was found in the spin-labeled RNA spectrum, indicating that all spins are coupled to RNA. The EPR signal was further used to quantify the coupling reaction yield using PROXYL standards of known concentrations. The spin concentration was found to be ca. 150 ± 15 μM from the double integration of the EPR spectrum. This value was in very good agreement with the RNA concentration obtained by ultraviolet absorption (147 ± 15 μM), suggesting that a proxyl-RNA coupling efficiency greater than 95% was achieved. Such a high coupling efficiency results from the separation of the enzymatic RNA ligation and the spin label coupling reactions by a purification step in order to remove the residual DTT. Indeed, while efficient ligation has already been achieved using spin-labeled RNA substrates, significant loss of EPR signal was observed due to the nitroxide reduction under ligation conditions (34).

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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