SYNTHESIS OF ISOTOPE LABELED OLIGONUCLEOTIDES AND THEIR USE IN AN NMR-STUDY OF A PROTEIN-DNA COMPLEX

The synthesis of an oligonucleotide labeled with C-13 at the thymine methyls and N-15 at the exocyclic amino groups of the cytosines is described. (CH3I)-C-13 and (NH4OH)-N-15 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.