New Lanthanide Tag for the Generation of Pseudocontact Shifts in DNA by Site-Specific Ligation to a Phosphorothioate Group

作者：Wu Zuyan; Lee Michael D; Carruthers Thomas J; Szabo Monika; Dennis Matthew L; Swarbrick James D^*; Graham Bim^*; Otting Gottfried^*

来源：Bioconjugate Chemistry, 2017, 28(6): 1741-1748.

DOI：10.1021/acs.bioconjchem.7b00202

Pseudocontact shifts (PCS) generated by paramagnetic lanthanides provide a rich source of long-range structural restraints that can readily be measured by nuclear magnetic resonance (NMR) spectroscopy. Many different lanthanide-binding tags have been designed for site-specific tagging of proteins, but established routes for tagging DNA with a single metal ion rely on difficult chemical synthesis. Here we present a simple and practical strategy for site-specific tagging of inexpensive phosphorothioate (PT) oligonucleotides. Commercially available PT oligonucleotides are diastereomers with S and R stereoconfiguration at the backbone PT site. The respective S-p and R-p diastereomers can readily be separated by HPLC. A new alkylating lanthanide-binding tag, C10, was synthesized that delivered quantitative tagging yields with both diastereomers. PCSs were observed following ligation with the complementary DNA strand to form double-stranded DNA duplexes. The PCSs were larger for the S-p than the R-p oligonucleotide and good correlation between back-calculated and experimental PCSs was observed. The C10 tag can also be attached to cysteine residues in proteins, where it generates a stable thioether bond. Ligated to the A28C mutant of ubiquitin, the tag produced excellent fits of magnetic susceptibility anisotropy (Delta chi) tensors, with larger tensors than for the tagged PT oligonucleotides, indicating that the tag is not completely immobilized after ligation with a PT group.

• 出版日期2017-6