Specific labelling of nucleosides and nucleotides with 13 C and 15 N

2011

Selective isotopic labeling provides an unparalleled window within which to study the structure and dynamics of RNAs by high resolution NMR spectroscopy. Unlike commonly used carbon sources, the asymmetry of 13C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides using Escherichia coli variants, that until now were not feasible. Here we show that an E. coli mutant strain that lacks succinate and malate dehydrogenases (DL323) and grown on [3-13C]-pyruvate affords ribonucleotides with site specific labeling at C5′ (~95%) and C1′ (~42%) and minimal enrichment elsewhere in the ribose ring. Enrichment is also achieved at purine C2 and C8 (~95%) and pyrimidine C5 (~100%) positions with minimal labeling at pyrimidine C6 and purine C5 positions. These labeling patterns contrast with those obtained with DL323 E. coli grown on [1, 3-13C]-glycerol for which the ribose ring is labeled in all but the C4′ carbon position, leading to multiplet splitting of the C1′, C2′ and C3′ carbon atoms. The usefulness of these labeling patterns is demonstrated with a 27-nt RNA fragment derived from the 30S ribosomal subunit. Removal of the strong magnetic coupling within the ribose and base leads to increased sensitivity, substantial simplification of NMR spectra, and more precise and accurate dynamic parameters derived from NMR relaxation measurements. Thus these new labels offer valuable probes for characterizing the structure and dynamics of RNA that were previously limited by the constraint of uniformly labeled nucleotides.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-011-9582-5) contains supplementary material, which is available to authorized users.

Section: Introductionmentioning

confidence: 99%

“…Selective introduction of 13 C isotopes into the ribose ring using chemical synthesis entails difficult regio- and stereo-selective synthesis with low yields (Milecki 2002). The de novo biosynthetic method requires a large number of enzymes of which only a few are commercially available (Schultheisz et al 2008, 2011).…”

Section: Introductionmentioning

confidence: 99%

Asymmetry of 13C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy

2011

“…Nucleic acids and proteins can be labeled with stable isotopes for structural and dynamics studies (Dayie 2008) using E. coli as a common bacterial host (Ponchon and Dardel 2007; Ponchon et al 2009), using enzymes from the pentose phosphate or de novo purine biosynthetic pathways (Gross et al 1983; Parkin et al 1984; Tolbert and Williamson 1996, 1997; Scott et al 2000; Schultheisz et al 2008), or using chemical synthesis (Milecki 2002). …”

Section: Introductionmentioning

confidence: 99%

Site-specific labeling of nucleotides for making RNA for high resolution NMR studies using an E. coli strain disabled in the oxidative pentose phosphate pathway

2010

Escherichia coli (E. coli) is a versatile organism for making nucleotides labeled with stable isotopes (13C, 15N, and/or 2H) for structural and molecular dynamics characterizations. Growth of a mutant E. coli strain deficient in the pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase (K10-1516) on 2-13C-glycerol and 15N-ammonium sulfate in Studier minimal medium enables labeling at sites useful for NMR spectroscopy. However, 13C-sodium formate combined with 13C-2-glycerol in the growth media adds labels to new positions. In the absence of labeled formate, both C5 and C6 positions of the pyrimidine rings are labeled with minimal multiplet splitting due to 1JC5C6 scalar coupling. However, the C2/C8 sites within purine rings and the C1′/C3′/C5′ positions within the ribose rings have reduced labeling. Addition of 13C-labeled formate leads to increased labeling at the base C2/C8 and the ribose C1′/C3′/C5′ positions; these new specific labels result in two- to three-fold increase in the number of resolved resonances. This use of formate and 15N-ammonium sulfate promises to extend further the utility of these alternate site specific labels to make labeled RNA for downstream biophysical applications such as structural, dynamics and functional studies of interesting biologically relevant RNAs.

“…coli variants (Johnson et al 2006; Johnson and Hoogstraten 2008; Dayie and Thakur 2010; Thakur et al 2010a, b) appears to be more general and cost-effective than chemical synthesis (Milecki 2002) or de novo biosynthesis (Schultheisz et al 2008; Schultheisz et al 2011). Selective introduction of 13 C isotopes into the ribose ring using chemical synthesis entails difficult regio- and stereo-selective synthesis with low yields (Milecki 2002). The de novo biosynthetic method requires a large number of enzymes of which only a few are commercially available (Schultheisz et al 2008; Schultheisz et al 2011).…”

Section: Introductionmentioning

confidence: 99%

Asymmetry of 13C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy

2011

Selective isotopic labeling provides an unparalleled window within which to study the structure and dynamics of RNAs by high resolution NMR spectroscopy. Unlike commonly used carbon sources, the asymmetry of 13C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides using E. coli variants, that until now were not feasible. Here we show that an E. coli mutant strain that lacks succinate and malate dehydrogenases (DL323) and grown on [3-13C]-pyruvate affords ribonucleotides with site specific labeling at C5′ (~95%) and C1′ (~42%) and minimal enrichment elsewhere in the ribose ring. Enrichment is also achieved at purine C2 and C8 (~95%) and pyrimidine C5 (~100%) positions with minimal labeling at pyrimidine C6 and purine C5 positions. These labeling patterns contrast with those obtained with DL323 E. coli grown on [1, 3-13C]-glycerol for which the ribose ring is labeled in all but the C4′ carbon position, leading to multiplet splitting of the C1′, C2′ and C3′ carbon atoms. The usefulness of these labeling patterns is demonstrated with a 27-nt RNA fragment derived from the 30S ribosomal subunit. Removal of the strong magnetic coupling within the ribose and base leads to increased sensitivity, substantial simplification of NMR spectra, and more precise and accurate dynamic parameters derived from NMR relaxation measurements. Thus these new labels offer valuable probes for characterizing the structure and dynamics of RNA that were previously limited by the constraint of uniformly labeled nucleotides.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-011-9581-6) contains supplementary material, which is available to authorized users.