Preparation and characterization of a uniformly 2 H/ 15 N-labeled RNA oligonucleotide for NMR studies

Abstract

An RNA oligonucleotide that contains the binding site for Escherichia coli ribosomal protein S8 was prepared with uniform 15N isotopic enrichment and uniform deuterium enrichment at all non-exchangeable sites using enzymatic methods. The RNA binding site, which contains 44 nt, forms a hairpin in solution and requires Mg2+for proper folding. The longitudinal magnetization recovery rates of the exchangeable protons were compared for the [2H,15N]-enriched RNA molecule and for the corresponding fully [2H,15N]-enriched RNA hairpin. It was found that 1H-1H dipolar relaxation significantly contributes to the recovery of exchangeable proton longitudinal magnetization. The exchangeable proton resonance line widths were less affected by deuteration, indicating that chemical exchange with H2O remains the dominant mechanism of transverse magnetization relaxation. Nevertheless, deuteration of this RNA hairpin was found to enhance the sensitivity of NOE-based experiments relative to the fully protonated hairpin and to simplify 2D NMR spectra. The increased signal-to-noise ratio facilitated the assignment of the cytidine amino resonances and several of the purine nucleotide amino resonances and permitted the identification of NOE crosspeaks that could not be observed in spectra of the fully protonated RNA hairpin.

NMR Methods for Studying the Structure and Dynamics of RNA

“…This perdeuteration procedure also improved the signal-to-noise ratio and reduced spectral overlap in NOESY spectra. A further modification of this procedure allowed back protonation of the H8 atoms on purines and H5 atoms on pyrimidines in an otherwise highly deuterated and 13 C, 15 N-labeled RNA sample. [16] The presence of protonated and 13 C-labeled bases in a mostly perdeuterated sample should improve the relaxation properties and help simplify resonance assignment of larger RNA systems.…”

“…The 15 N, 13 C8-labeled native E. coli tRNA Val sample was obtained by overexpression of E. coli BL21(DE3) cells containing the pVALT7 plasmid. [12,14] Cells were grown with 15 (NH 4 ) 2 SO 4 (2 g L À1 ) as the sole nitrogen source and with 12 C-glucose (2 g L À1 ) and 99 % 13 C-formate (200 mg L À1 ; Cambridge Isotopes Laboratories, Inc., Andover, MA) as the carbon sources. The purification of tRNA Val was performed as described previously.…”

“…Comparison of the X-ray crystal structure model for native E. coli tRNA Val (in red) with the global structure determined from 1 H- 15 N imino RDC data on native E. coli tRNA Val (in green). The structure from the RDC data assumed A-form geometry for the acceptor and anticodon helical arms.…”

“…The aromatic region of 2D 1 H, 13 C R 11 spectra of the 13 C, 15 N-adenosine minimal hammerhead ribozyme A) in the absence of Mg 2 + and B) with 1.0 mm Mg 2 + . The inset shows the sequence and secondary structure of the minimal hammerhead ribozyme where the bold typeface is the 13 C, 15 N-adenosine labeled enzyme strand and the regular typeface is the unlabeled substrate strand with a noncleavable deoxyribose at the cleavage site. Adenosine residues with C8 resonances that have significant power dependence for R 11 in no Mg 2 + conditions are outlined in the secondary structure.…”

The Synthesis of Deuterionucleosides

“…For examples, labelling the prochiral 5'-methylene moiety for stereospecific assignment and through this to measure vicinal 1 H-31 P coupling constants 163 ; to improve the accuracy of coupling data extracted from 1 H-13 C HSQC spectra 222 ; to eliminate crosspeaks completely from crowded regions of 2D spectra 141 or to aid relaxation time measurements 43,46,223,228,229 can be recalled.…”