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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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Synthesis of site-specific spin-labeled RNA. (A) Native gel electrophoresis on 10% polyacrylamide. RNA segments and DNA splint were loaded in the T4 RNA ligase 2 buffer: lane 1, (pG246T-C55); lane 2, (pppG1-G23); lane 3, DNA splint; lane 4, (pppG1-G23) + (pG246T-C55) + DNA splint. (B) Denaturing 12% polyacrylamide gel. Lane 1: RNA fragment (G1-G23) acceptor; lane 2: RNA fragment (pG246T-C55) donor; lane 3: DNA splint (43-nt); lane 4: preparative ligation; lane 5: purified ligation product. (C) Imino-proton region of 1D spectra recorded at 20°C of the wild-type RNA full-length (top) and the ligation product (bottom). All imino protons were assigned via sequential Nuclear Overhauser Effects (NOEs) observed in 2D-NOESY experiments, with the exception of the resonances at 10.37, 11.09 and 11.43 p.p.m. that could not be identified unambiguously.
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Figure 2: Synthesis of site-specific spin-labeled RNA. (A) Native gel electrophoresis on 10% polyacrylamide. RNA segments and DNA splint were loaded in the T4 RNA ligase 2 buffer: lane 1, (pG246T-C55); lane 2, (pppG1-G23); lane 3, DNA splint; lane 4, (pppG1-G23) + (pG246T-C55) + DNA splint. (B) Denaturing 12% polyacrylamide gel. Lane 1: RNA fragment (G1-G23) acceptor; lane 2: RNA fragment (pG246T-C55) donor; lane 3: DNA splint (43-nt); lane 4: preparative ligation; lane 5: purified ligation product. (C) Imino-proton region of 1D spectra recorded at 20°C of the wild-type RNA full-length (top) and the ligation product (bottom). All imino protons were assigned via sequential Nuclear Overhauser Effects (NOEs) observed in 2D-NOESY experiments, with the exception of the resonances at 10.37, 11.09 and 11.43 p.p.m. that could not be identified unambiguously.

Mentions: The RNA fragment (G1-A23) was therefore synthesized by classical in vitro transcription whereas (G24-C55) RNA was modified at its 5′-end with the incorporation of a 6-T-GMP nucleoside as described (31). After purification of RNA transcripts, optimization experiments were performed for the ligation by T4 RNA ligase 2, which displays better activity on double-stranded nucleic acids (38). Optimal conditions for the hybridization of the RNA fragments to the complementary DNA splint were first investigated to avoid the formation of side-products. After heating an equimolar mixture of DNA and RNAs at 95°C and snap cooling at 4°C, the formation of the DNA/RNA hybrids was examined by electrophoresis on a native gel (Figure 2A). The experiments revealed that the optimal ligation yield was obtained after a minimal reaction time of 5 h at 37°C, with oligonucleotides concentrations ranging from 10 to 30 μM (Figure 2B). After purification and dialysis against the buffer used for NMR and EPR studies, the ligation product containing one thio-modified guanosine (6-T-GMP), was analyzed by NMR. Figure 2C compares the imino proton region of 1D spectra of the wild-type full-length RNA with the corresponding spectrum of the (G1-A23)-(G246TG–C55) ligation product. The two spectra are nearly identical, suggesting that the incorporation of the modified guanine does not alter the hydrogen-bonding pattern. In conclusion, our NMR data clearly show that the thio-modified residue was introduced into the RNA without altering the global fold of the RNA.


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)

Synthesis of site-specific spin-labeled RNA. (A) Native gel electrophoresis on 10% polyacrylamide. RNA segments and DNA splint were loaded in the T4 RNA ligase 2 buffer: lane 1, (pG246T-C55); lane 2, (pppG1-G23); lane 3, DNA splint; lane 4, (pppG1-G23) + (pG246T-C55) + DNA splint. (B) Denaturing 12% polyacrylamide gel. Lane 1: RNA fragment (G1-G23) acceptor; lane 2: RNA fragment (pG246T-C55) donor; lane 3: DNA splint (43-nt); lane 4: preparative ligation; lane 5: purified ligation product. (C) Imino-proton region of 1D spectra recorded at 20°C of the wild-type RNA full-length (top) and the ligation product (bottom). All imino protons were assigned via sequential Nuclear Overhauser Effects (NOEs) observed in 2D-NOESY experiments, with the exception of the resonances at 10.37, 11.09 and 11.43 p.p.m. that could not be identified unambiguously.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Synthesis of site-specific spin-labeled RNA. (A) Native gel electrophoresis on 10% polyacrylamide. RNA segments and DNA splint were loaded in the T4 RNA ligase 2 buffer: lane 1, (pG246T-C55); lane 2, (pppG1-G23); lane 3, DNA splint; lane 4, (pppG1-G23) + (pG246T-C55) + DNA splint. (B) Denaturing 12% polyacrylamide gel. Lane 1: RNA fragment (G1-G23) acceptor; lane 2: RNA fragment (pG246T-C55) donor; lane 3: DNA splint (43-nt); lane 4: preparative ligation; lane 5: purified ligation product. (C) Imino-proton region of 1D spectra recorded at 20°C of the wild-type RNA full-length (top) and the ligation product (bottom). All imino protons were assigned via sequential Nuclear Overhauser Effects (NOEs) observed in 2D-NOESY experiments, with the exception of the resonances at 10.37, 11.09 and 11.43 p.p.m. that could not be identified unambiguously.
Mentions: The RNA fragment (G1-A23) was therefore synthesized by classical in vitro transcription whereas (G24-C55) RNA was modified at its 5′-end with the incorporation of a 6-T-GMP nucleoside as described (31). After purification of RNA transcripts, optimization experiments were performed for the ligation by T4 RNA ligase 2, which displays better activity on double-stranded nucleic acids (38). Optimal conditions for the hybridization of the RNA fragments to the complementary DNA splint were first investigated to avoid the formation of side-products. After heating an equimolar mixture of DNA and RNAs at 95°C and snap cooling at 4°C, the formation of the DNA/RNA hybrids was examined by electrophoresis on a native gel (Figure 2A). The experiments revealed that the optimal ligation yield was obtained after a minimal reaction time of 5 h at 37°C, with oligonucleotides concentrations ranging from 10 to 30 μM (Figure 2B). After purification and dialysis against the buffer used for NMR and EPR studies, the ligation product containing one thio-modified guanosine (6-T-GMP), was analyzed by NMR. Figure 2C compares the imino proton region of 1D spectra of the wild-type full-length RNA with the corresponding spectrum of the (G1-A23)-(G246TG–C55) ligation product. The two spectra are nearly identical, suggesting that the incorporation of the modified guanine does not alter the hydrogen-bonding pattern. In conclusion, our NMR data clearly show that the thio-modified residue was introduced into the RNA without altering the global fold of the RNA.

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