Alternate-site isotopic labeling of ribonucleotides for NMR studies of ribose conformational dynamics in RNA

Abstract

Heteronuclear NMR spin relaxation studies of conformational dynamics are coming into increasing use to help understand the functions of ribozymes and other RNAs. Due to strong $^{13}\hbox{C}--^{13}\hbox{C}$ magnetic interactions within the ribose ring, however, these studies have thus far largely been limited to ¹³C and ¹⁵N resonances on the nucleotide base side chains. We report here the application of the alternate-site ¹³C isotopic labeling scheme, pioneered by LeMaster for relaxation studies of amino acid side chains, to nucleic acid systems. We have used different strains of E. coli to prepare mononucleotides containing ¹³C label in one of two patterns: Either C1′ or C2′ in addition to C4′, termed (1′/2′,4′) labeling, or nearly complete labeling at the C2′ and C4′ sites only, termed (2′,4′) labeling. These patterns provide isolated $^{13}\hbox{C}--^{1}$H spin systems on the labeled carbon atoms and thus allow spin relaxation studies without interference from $^{13}\hbox{C}--^{13}\hbox{C}$ scalar or dipolar coupling. Using relaxation studies of AMP dissolved in glycerol at varying temperature to produce systems with correlation times characteristic of different size RNAs, we demonstrate the removal of errors due to $^{13}\hbox{C}--^{13}\hbox{C}$ interaction in T ₁ measurements of larger nucleic acids and in T _1ρ measurements in RNA molecules. By extending the applicability of spin relaxation measurements to backbone ribose groups, this technology should greatly improve the flexibility and completeness of NMR analyses of conformational dynamics in RNA.