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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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Effect of site-specific spin-labeled RNA analyzed by NMR. (A) Bar graph of intensity ratio between the spin-labeled RNA (IproxylRNA) and the free form RNA (IRNA), normalized to C54, extracted from MLEV experiments, versus the primary sequence of the RNA. The quantification of residues C18, U22, C35, U39, C41 and C43 was not feasible due to the disappearance of the corresponding resonances. The corresponding ratios were estimated by measuring values at the same frequencies as the resonances observable in the wild-type RNA. (B) Sphere representation of the RNA structure colored according to the intensity ratios extracted from MLEV experiments. The spin-labeled G24 residue is highlighted in light blue. (C) Bar graph of intensity ratio between the (G1-A23)-(G246TGproxyl–C55) RNA and the free form RNA, normalized to A50, extracted from HSQC experiment, versus the primary sequence of the RNA. (D) Sphere representation of the RNA structure colored according to the intensity ratios extracted from HSQC experiments. The spin-labeled G24 residue is highlighted in light blue.
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Figure 6: Effect of site-specific spin-labeled RNA analyzed by NMR. (A) Bar graph of intensity ratio between the spin-labeled RNA (IproxylRNA) and the free form RNA (IRNA), normalized to C54, extracted from MLEV experiments, versus the primary sequence of the RNA. The quantification of residues C18, U22, C35, U39, C41 and C43 was not feasible due to the disappearance of the corresponding resonances. The corresponding ratios were estimated by measuring values at the same frequencies as the resonances observable in the wild-type RNA. (B) Sphere representation of the RNA structure colored according to the intensity ratios extracted from MLEV experiments. The spin-labeled G24 residue is highlighted in light blue. (C) Bar graph of intensity ratio between the (G1-A23)-(G246TGproxyl–C55) RNA and the free form RNA, normalized to A50, extracted from HSQC experiment, versus the primary sequence of the RNA. (D) Sphere representation of the RNA structure colored according to the intensity ratios extracted from HSQC experiments. The spin-labeled G24 residue is highlighted in light blue.

Mentions: The impact of the spin-labeled guanosine on the RNA 3D structure was then further investigated using NMR. The pattern of imino-proton frequencies observed for the wild-type and the spin-labeled RNA was very similar, except for few residues that displayed reduced intensities, such as G24, indicating that the global fold of the RNA is not affected by the presence of the spin label (Figure 5A). In particular, the unperturbed resonance of G31 imino proton indicates that the UUCG tetraloop fold is not altered in the spin-labeled RNA. A semi-quantitative analysis of the enhanced relaxation due to the paramagnetic proxyl was conducted by monitoring intensities of the cross-peaks between non-exchangeable H5 and H6 protons in MLEV experiments (Figure 5B). Semi-quantitative analysis of PRE is in agreement with the global RNA 3D structure (Figure 6A and B). Indeed, residues C18, U22, C27, U33 and C35 display low values of PRE (IproxylRNA/IRNA), consistent with their positions in the vicinity of the spin label while C54 and C55, which are located more than 50 Å away from the labeling site, were not affected (Figures 5B and 6A). Noteworthy, larger PRE values are found for residues U39, C41 and C43 located in the Specifier Loop Domain of the RNA, opposite to the tetraloop, suggesting that the proxyl is oriented toward this region. It should be noted that dynamical behaviors of specific regions of an RNA molecule may considerably affect the paramagnetic relaxation, as reported for the paramagnetic 5′-end labeled HIV-1 TAR RNA study where significant differences between experimental and modeled distances were observed (25). Interestingly, PRE values measured using homonuclear H5-H6 correlations are larger than those measured using heteronuclear experiments (compare PRE values of C6-H6 with H5-H6 of C27 in Figure 6A and C). Further experimental and modeling studies will be necessary to establish accurate quantification procedures enabling accurate distances to be obtained from PRE data in RNA. Our efficient and selective RNA spin labeling protocol provides unprecedented perspectives in this direction.


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)

Effect of site-specific spin-labeled RNA analyzed by NMR. (A) Bar graph of intensity ratio between the spin-labeled RNA (IproxylRNA) and the free form RNA (IRNA), normalized to C54, extracted from MLEV experiments, versus the primary sequence of the RNA. The quantification of residues C18, U22, C35, U39, C41 and C43 was not feasible due to the disappearance of the corresponding resonances. The corresponding ratios were estimated by measuring values at the same frequencies as the resonances observable in the wild-type RNA. (B) Sphere representation of the RNA structure colored according to the intensity ratios extracted from MLEV experiments. The spin-labeled G24 residue is highlighted in light blue. (C) Bar graph of intensity ratio between the (G1-A23)-(G246TGproxyl–C55) RNA and the free form RNA, normalized to A50, extracted from HSQC experiment, versus the primary sequence of the RNA. (D) Sphere representation of the RNA structure colored according to the intensity ratios extracted from HSQC experiments. The spin-labeled G24 residue is highlighted in light blue.
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Figure 6: Effect of site-specific spin-labeled RNA analyzed by NMR. (A) Bar graph of intensity ratio between the spin-labeled RNA (IproxylRNA) and the free form RNA (IRNA), normalized to C54, extracted from MLEV experiments, versus the primary sequence of the RNA. The quantification of residues C18, U22, C35, U39, C41 and C43 was not feasible due to the disappearance of the corresponding resonances. The corresponding ratios were estimated by measuring values at the same frequencies as the resonances observable in the wild-type RNA. (B) Sphere representation of the RNA structure colored according to the intensity ratios extracted from MLEV experiments. The spin-labeled G24 residue is highlighted in light blue. (C) Bar graph of intensity ratio between the (G1-A23)-(G246TGproxyl–C55) RNA and the free form RNA, normalized to A50, extracted from HSQC experiment, versus the primary sequence of the RNA. (D) Sphere representation of the RNA structure colored according to the intensity ratios extracted from HSQC experiments. The spin-labeled G24 residue is highlighted in light blue.
Mentions: The impact of the spin-labeled guanosine on the RNA 3D structure was then further investigated using NMR. The pattern of imino-proton frequencies observed for the wild-type and the spin-labeled RNA was very similar, except for few residues that displayed reduced intensities, such as G24, indicating that the global fold of the RNA is not affected by the presence of the spin label (Figure 5A). In particular, the unperturbed resonance of G31 imino proton indicates that the UUCG tetraloop fold is not altered in the spin-labeled RNA. A semi-quantitative analysis of the enhanced relaxation due to the paramagnetic proxyl was conducted by monitoring intensities of the cross-peaks between non-exchangeable H5 and H6 protons in MLEV experiments (Figure 5B). Semi-quantitative analysis of PRE is in agreement with the global RNA 3D structure (Figure 6A and B). Indeed, residues C18, U22, C27, U33 and C35 display low values of PRE (IproxylRNA/IRNA), consistent with their positions in the vicinity of the spin label while C54 and C55, which are located more than 50 Å away from the labeling site, were not affected (Figures 5B and 6A). Noteworthy, larger PRE values are found for residues U39, C41 and C43 located in the Specifier Loop Domain of the RNA, opposite to the tetraloop, suggesting that the proxyl is oriented toward this region. It should be noted that dynamical behaviors of specific regions of an RNA molecule may considerably affect the paramagnetic relaxation, as reported for the paramagnetic 5′-end labeled HIV-1 TAR RNA study where significant differences between experimental and modeled distances were observed (25). Interestingly, PRE values measured using homonuclear H5-H6 correlations are larger than those measured using heteronuclear experiments (compare PRE values of C6-H6 with H5-H6 of C27 in Figure 6A and C). Further experimental and modeling studies will be necessary to establish accurate quantification procedures enabling accurate distances to be obtained from PRE data in RNA. Our efficient and selective RNA spin labeling protocol provides unprecedented perspectives in this direction.

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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