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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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Method for the site-directed spin labeling approach. (A) Schematic representation of the synthesis of site-specific spin labeled RNA. 6-thioguanosine-5′-O-monophosphate (pG6T = 6-T-GMP) is incorporated at a specific position. (B) The proxyl-RNA coupling reaction involves the addition of 3-(2-Iodoacetamido)-proxyl on 6-thioguanosine-5′-O-monophosphate. (C) Secondary structure of the 55-nt (tyrS) RNA molecule corresponding to the specifier loop domain and K-turn sequence motif of the tyrS leader RNA. The Specifier Loop Domain and the tetraloop are boxed. The arrow indicates the chosen segmentation site and the circle indicates the thio-modified site.
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Figure 1: Method for the site-directed spin labeling approach. (A) Schematic representation of the synthesis of site-specific spin labeled RNA. 6-thioguanosine-5′-O-monophosphate (pG6T = 6-T-GMP) is incorporated at a specific position. (B) The proxyl-RNA coupling reaction involves the addition of 3-(2-Iodoacetamido)-proxyl on 6-thioguanosine-5′-O-monophosphate. (C) Secondary structure of the 55-nt (tyrS) RNA molecule corresponding to the specifier loop domain and K-turn sequence motif of the tyrS leader RNA. The Specifier Loop Domain and the tetraloop are boxed. The arrow indicates the chosen segmentation site and the circle indicates the thio-modified site.

Mentions: Here, we propose a general, fully enzymatic method for the introduction of a paramagnetic probe at a specific position in an RNA sequence. Our technique is based on a segmental approach using a ligation protocol with T4 RNA ligase 2 (36–38). Two RNA fragments corresponding each to a part of the full-length RNA are designed and transcribed using T7 RNA polymerase. One transcribed acceptor RNA is ligated to a donor RNA in which a thio-modified nucleotide is introduced at its 5′-end by in vitro transcription with T7 RNA polymerase (Figure 1A). The ligation product corresponding to the full-length RNA contains a single thio-modified nucleotide at a specific position (Figure 1C). The paramagnetic spin label is subsequently attached to the RNA containing the thio-modified base at the chosen position. This method yields to a very efficient way of incorporating modified nucleotides in an RNA sequence without size limitation. This enables the study of large size RNA molecules using spectroscopic tools. The high incorporation efficiency of the paramagnetic probe will be of interest to measure distances with EPR methods. For NMR, it offers the possibility to combine segmental and selective isotopic labeling with PRE measurements, opening a novel perspective to study the dynamics and the structure of RNA molecules.


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)

Method for the site-directed spin labeling approach. (A) Schematic representation of the synthesis of site-specific spin labeled RNA. 6-thioguanosine-5′-O-monophosphate (pG6T = 6-T-GMP) is incorporated at a specific position. (B) The proxyl-RNA coupling reaction involves the addition of 3-(2-Iodoacetamido)-proxyl on 6-thioguanosine-5′-O-monophosphate. (C) Secondary structure of the 55-nt (tyrS) RNA molecule corresponding to the specifier loop domain and K-turn sequence motif of the tyrS leader RNA. The Specifier Loop Domain and the tetraloop are boxed. The arrow indicates the chosen segmentation site and the circle indicates the thio-modified site.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Method for the site-directed spin labeling approach. (A) Schematic representation of the synthesis of site-specific spin labeled RNA. 6-thioguanosine-5′-O-monophosphate (pG6T = 6-T-GMP) is incorporated at a specific position. (B) The proxyl-RNA coupling reaction involves the addition of 3-(2-Iodoacetamido)-proxyl on 6-thioguanosine-5′-O-monophosphate. (C) Secondary structure of the 55-nt (tyrS) RNA molecule corresponding to the specifier loop domain and K-turn sequence motif of the tyrS leader RNA. The Specifier Loop Domain and the tetraloop are boxed. The arrow indicates the chosen segmentation site and the circle indicates the thio-modified site.
Mentions: Here, we propose a general, fully enzymatic method for the introduction of a paramagnetic probe at a specific position in an RNA sequence. Our technique is based on a segmental approach using a ligation protocol with T4 RNA ligase 2 (36–38). Two RNA fragments corresponding each to a part of the full-length RNA are designed and transcribed using T7 RNA polymerase. One transcribed acceptor RNA is ligated to a donor RNA in which a thio-modified nucleotide is introduced at its 5′-end by in vitro transcription with T7 RNA polymerase (Figure 1A). The ligation product corresponding to the full-length RNA contains a single thio-modified nucleotide at a specific position (Figure 1C). The paramagnetic spin label is subsequently attached to the RNA containing the thio-modified base at the chosen position. This method yields to a very efficient way of incorporating modified nucleotides in an RNA sequence without size limitation. This enables the study of large size RNA molecules using spectroscopic tools. The high incorporation efficiency of the paramagnetic probe will be of interest to measure distances with EPR methods. For NMR, it offers the possibility to combine segmental and selective isotopic labeling with PRE measurements, opening a novel perspective to study the dynamics and the structure of RNA molecules.

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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