posters 5th Asia-Pacific NMR Symposium 2013

A Method for the Ligation of Paramagnetic Lanthanide Binding Tags to DNA Oligonucleotides (#108)

Thomas Carruthers 1 , Tony Herlt 1 , Gottfried Otting 1
  1. Australian National University, Canberra, ACT 0200, Australia

The ligation of a paramagnetic lanthanide ion to a biomolecule is a useful technique that enables an NMR spectroscopist to obtain structural information up to 40 Å from the tag. In a protein, a cysteine residue can be substituted in place of a surface exposed residue by simple mutagenesis, enabling site-specific ligation to the exposed thiol group. Here describes a convenient method for ligating paramagnetic lanthanide tags to a similarly exposed functional group in a DNA oligonucleotide.

Synthetic DNA oligonucleotides with modification such as fluorescent tags are commercially available, however, these are intended for use in small-scale analysis and become prohibitively expensive for quantities suitable for NMR. DNA oligonucleotides with phosphorothioate modifications, however, have long been used in biotechnology as a means to extend the half-life of DNA aptomers or probes and are cheap and easy to incorporate site-specifically between any two bases. They preserve the charge of the DNA, hardly change the mass, are only slightly more hydrophobic, and are resistant to exonuclease degredation. By site specifically incorporating a phosphorothioate linkage between two bases of a synthetic oligonucleotide, a unique functional group is introduced into the DNA strand that can be accessed for modification without disrupting base pairing. As the phosphorus in the phosphodiester linkage is pro-chiral, and the sulfur incorporation is typically not enantiometically selective, purification of the diastereomers produced is necessary prior to analysis. Fortunately, this purification has been found to be simple with a non-chiral C-18 column.

Ligation of a paramagnetic tag to the phosphorothioate modified DNA oligonucleotide is a one step process at room temperature and the completion of reaction can be monitored visually. The ligated DNA oligonucleotide can then be desalted to remove excess tag prior to analysis.