[NMR paper] Chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations.
Chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations.
Related ArticlesChemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations.
Nucleic Acids Res. 2016 Apr 7;44(6):e52
Authors: Longhini AP, LeBlanc RM, Becette O, Salguero C, Wunderlich CH, Johnson BA, D'Souza VM, Kreutz C, Dayie TK
Abstract
Stable isotope labeling is central to NMR studies of nucleic acids. Development of methods that incorporate labels at specific atomic positions within each nucleotide promises to expand the size range of RNAs that can be studied by NMR. Using recombinantly expressed enzymes and chemically synthesized ribose and nucleobase, we have developed an inexpensive, rapid chemo-enzymatic method to label ATP and GTP site specifically and in high yields of up to 90%. We incorporated these nucleotides into RNAs with sizes ranging from 27 to 59 nucleotides using in vitro transcription: A-Site (27 nt), the iron responsive elements (29 nt), a fluoride riboswitch from Bacillus anthracis(48 nt), and a frame-shifting element from a human corona virus (59 nt). Finally, we showcase the improvement in spectral quality arising from reduced crowding and narrowed linewidths, and accurate analysis of NMR relaxation dispersion (CPMG) and TROSY-based CEST experiments to measure ?s-ms time scale motions, and an improved NOESY strategy for resonance assignment. Applications of this selective labeling technology promises to reduce difficulties associated with chemical shift overlap and rapid signal decay that have made it challenging to study the structure and dynamics of large RNAs beyond the 50 nt median size found in the PDB.
[NMR paper] Improving dipolar recoupling for site-specific structural and dynamics studies in biosolids NMR: windowed RN-symmetry sequences.
Improving dipolar recoupling for site-specific structural and dynamics studies in biosolids NMR: windowed RN-symmetry sequences.
Related Articles Improving dipolar recoupling for site-specific structural and dynamics studies in biosolids NMR: windowed RN-symmetry sequences.
Phys Chem Chem Phys. 2016 Jan 18;
Authors: Lu X, Zhang H, Lu M, Vega AJ, Hou G, Polenova T
Abstract
Experimental characterization of one-bond heteronuclear dipolar couplings is essential for structural and dynamics characterization of molecules by solid-state...
nmrlearner
Journal club
0
01-19-2016 07:37 PM
[NMR paper] Preparation of Multiple Site-Specific Mutant Proteins for NMR Studies by PCR-Directed Cell-Free Protein Synthesis.
Preparation of Multiple Site-Specific Mutant Proteins for NMR Studies by PCR-Directed Cell-Free Protein Synthesis.
Preparation of Multiple Site-Specific Mutant Proteins for NMR Studies by PCR-Directed Cell-Free Protein Synthesis.
Methods Mol Biol. 2014;1118:169-87
Authors: Ozawa K, Qi R
Abstract
Cell-free protein synthesis (CFPS) offers a fast and inexpensive approach to selectively label proteins with isotopes that can then be detected by nuclear magnetic resonance (NMR) spectroscopy directly in the translation mixture. We describe...
nmrlearner
Journal club
0
01-08-2014 11:23 AM
[NMR paper] Asymmetry of 13C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy.
From Mendeley Biomolecular NMR group:
Asymmetry of 13C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy.
Journal of biomolecular NMR (2012). Volume: 52, Issue: 1. Pages: 65-77. Chandar S Thakur, T Kwaku Dayie et al.
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 (13)C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides using Escherichia coli...
nmrlearner
Journal club
0
01-21-2013 02:09 PM
[NMR paper] Asymmetry of 13C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy.
From Mendeley Biomolecular NMR group:
Asymmetry of 13C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy.
Journal of biomolecular NMR (2012). Volume: 52, Issue: 1. Pages: 65-77. Chandar S Thakur, T Kwaku Dayie et al.
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 (13)C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides using Escherichia coli...
nmrlearner
Journal club
0
11-22-2012 11:49 AM
[NMR paper] Asymmetry of 13C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy.
From Mendeley Biomolecular NMR group:
Asymmetry of 13C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy.
Journal of biomolecular NMR (2012). Volume: 52, Issue: 1. Pages: 65-77. Chandar S Thakur, T Kwaku Dayie et al.
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 (13)C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides using Escherichia coli...
nmrlearner
Journal club
0
10-12-2012 09:58 AM
[NMR paper] Asymmetry of 13C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy.
From Mendeley Biomolecular NMR group:
Asymmetry of 13C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy.
Journal of biomolecular NMR (2012). Volume: 52, Issue: 1. Pages: 65-77. Chandar S Thakur, T Kwaku Dayie et al.
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 (13)C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides using Escherichia coli...
nmrlearner
Journal club
0
08-24-2012 08:01 PM
[NMR paper] Efficient enzymatic synthesis of 13C,15N-labeled DNA for NMR studies.
Efficient enzymatic synthesis of 13C,15N-labeled DNA for NMR studies.
Related Articles Efficient enzymatic synthesis of 13C,15N-labeled DNA for NMR studies.
J Biomol NMR. 1997 Oct;10(3):245-53
Authors: Smith DE, Su JY, Jucker FM
The power of heteronuclear NMR spectroscopy to study macromolecules and their complexes has been amply demonstrated over the last decade. The obstacle to routinely applying these techniques to the study of DNA has been the synthesis of 13C,15N-labeled DNA. Here we present a simple and efficient method to generate...
nmrlearner
Journal club
0
08-22-2010 05:08 PM
Site-specific labeling of nucleotides for making RNA for high resolution NMR studies
Abstract 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...
Chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations.
Linear Mode
