Efficient enzymatic synthesis of 13C,15N-labeled DNA for NMR studies.
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 isotope-labeled DNA for NMR studies that is as easy as that for isotope labeling of RNA. The method was used to synthesize a uniformly 13C,15N-labeled 32-nucleotide DNA that binds to human basic fibroblast growth factor with high affinity and specificity. Isotope-edited experiments were applied to the 13C,15N-labeled DNA bound to unlabeled protein, and the 13C,15N-labeled DNA was also examined in complex with 15N-labeled protein. The NMR experiments show that the DNA adopts a well-defined stable structure when bound to the protein, and illustrate the potential of 13C,15N-labeled DNA for structural studies of DNA-protein complexes.
Smith
D E
DE
NeXstar Pharmaceuticals Inc., Boulder, CO 80301, USA.
Su
J Y
JY
Jucker
F M
FM
D002247:Carbon Isotopes; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D009838:Oligodeoxyribonucleotides; D016222:Fibroblast Growth Factor 2; D004247:DNA