Synthesis of isotope labeled oligonucleotides and their use in an NMR study of a protein-DNA complex

Abstract

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.

15N-Enriched 5-Fluorocytosine as a Probe for Examining Unusual DNA Structures

“…Incorporation of cytosine residues containing 15 N-enriched amino groups has been performed previously with 15 N-enriched precursors (20)(21)(22)(23) and by displacement of the 4-triazole group from substituted 2′-deoxyuridine precursors (24)(25)(26)(27). The 4-Oethyl analogue described here is efficiently displaced by 15 N-enriched ammonia.…”

“…The incorporation of 15 N-enriched cytosine amino groups has allowed selective editing of proton NMR spectra which considerably simplifies examination of base mispairs containing cytosine residues as well as examination of site-specific DNAprotein interactions (21)(22)(23)(24)(25)(26)(27)(28)(29). To date, such studies have been confined to internal, base-paired cytosine residues, and have not yet examined residues at helix ends, model replication forks, or unpaired environments.…”

DNA Duplex Dynamics: NMR Relaxation Studies of a Decamer with Uniformly13C-Labeled Purine Nucleotides

“…If an atom-site specifically enriched sample is available, it will give the most accurate results. Such DNA samples are available with labeling at several positions: C6 of thymidine (24), methyl carbon of thymidine (25), C1Ј (26) and C5Ј (27). Two of them have already been used for oligomer dynamic studies, C6 of thymidine (28) and C1Ј (29).…”

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

“…With the appropriate selection of leaving groups postsynthetic N4 labelling of cytidine and N6 labelling of adenosine was achieved with good yield. 98 Usually the source of 15 N was labelled ammonia (either as a water solution, 70,[98][99][100][101][102][103] or generated in situ from its salt 22,74,75,[104][105][106][107] ). Another possibility is the use of 15 N-labelled phthalimide 108,109 or 15 N-benzylamine 110-114 as a nucleophile (Scheme 10).…”