Synthesis of isotope labeled oligonucleotides and their use in an NMR study of a protein-DNA complex.
The synthesis of an oligonucleotide labeled with 13C at the thymine methyl and 15N at the exocyclic amino groups of the cytosines is described. 13CH3I and 15NH4OH were used as sources of the labels. The labeled oligonucleotide was characterized by several NMR techniques. The duplex possesses a labeled functional group in the major groove at every base pair which makes it a very suitable probe for the study of sequence-specific protein-DNA interaction. The labeled thymine methyl group facilitates the detection of hydrophobic contacts with aliphatic side-chains of proteins. This is demonstrated in an NMR study of a complex between the glucocorticoid receptor DNA-binding domain and the labeled oligomer, which revealed a hydrophobic contact between a thymine methyl group and the methyl groups of a valine residue. There are indications for small differences between the solution structure the X-ray structure of the complex.
Kellenbach
E R
ER
Department of Chemistry, University of Utrecht, The Netherlands.
Remerowski
M L
ML
Eib
D
D
Boelens
R
R
van der Marel
G A
GA
van den Elst
H
H
van Boom
J H
JH
Kaptein
R
R
D000818:Animals; D001483:Base Sequence; D002247:Carbon Isotopes; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis* / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism*
D002247:Carbon Isotopes; D004268:DNA-Binding Proteins; D009587:Nitrogen Isotopes; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011965:Receptors, Glucocorticoid; D004247:DNA