A fully enzymatic method for site-directed spin labeling of long 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).
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 firstname.lastname@example.org.Show MeSH
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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.
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 email@example.com.