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

Abstract

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 and R 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 than the R 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 (Δχ) tensors, with larger tensors than for the tagged PT oligonucleotides, indicating that the tag is not completely immobilized after ligation with a PT group.

Lanthanide Tagging of Oligonucleotides to Nucleobase for Paramagnetic NMR

“…Lanthanides can either be loaded into a metal‐binding site of a metalloprotein, [9,10] or be site‐specifically attached to a biomolecule via a metal binding tag [11–25] . This has also been demonstrated for oligosaccharides, [26–32] but only in one case so far for oligonucleotides, [33] with attachment of the tag at a phosphorothioate group. Herein, we present a chemical approach developed for lanthanide tagging of oligonucleotides by using the modified nucleobase thymidine that allows to attach the Cys‐Ph‐TAHA tag [34] and the MesS‐Ph‐TAHA tag via a disulfide bond.…”

Design and applications of lanthanide chelating tags for pseudocontact shift NMR spectroscopy with biomacromolecules

“…Subsequently, the development of single-and double-armed lanthanide chelating tags (LCTs) that can be attached to a protein of interest and, thus, yield valuable structural restraints for the determination of the structures of biomacromolecules in solution strongly expanded the field of paramagnetic NMR [9,13,14,24,62,63]. Today, paramagnetic NMR is an established method in structural biology and includes many different applications, such as refining crystal structures of proteins [64], probing solution dynamics of proteins [65], localization of ligands on proteins [20,[66][67][68][69][70][71], study of protein-protein complexes [72], alleviation of signal dispersion in crowded HSQC spectra [73], facilitating analysis of large sized proteins [12], conformational analysis of carbohydrates [74], as well as investigation of the solution structure of nucleic acids [30]. Besides the application of paramagnetic effects in protein NMR spectroscopy, also detailed research studies on solvent dependence as well as pH and temperature effects on the magnetic anisotropy caused by paramagnetic molecules alone and when attached to proteins were performed [28,75].…”

“…In order to prevent motional averaging of the paramagnetic effects observed in affinity binding, the covalent attachment of the LCT incorporating the paramagnetic metal centre directly to the nucleic acids constitutes a highly desirable approach. The covalent labelling of nucleic acids was demonstrated for an EPR application by Song et al [199] and Goldfarb [200] via a copper catalysed azide-alkyne click reaction of a DNA strand incorporating an azide functionality and an LCT bearing an alkyne functionality as well as by Wu et al [30] for the analysis of PCSs via modification of a phosphorothioate group in DNA with an LCT containing an electrophilic bromoacetamide functionality for alkylation. Insights into the solution structure and dynamics of RNA and DNA molecules obtained by paramagnetic NMR are highly desirable not only for fundamental research, but can also give significant inputs for characterization of binding sites of RNA and DNA targeting molecules in future drug discovery.…”

“…Interestingly, the observed PCSs and associated anisotropy parameters at pH 6.5 are 40% smaller than those obtained at a pH of 8.0. Since the two LCTs can be readily synthesized in parallel and two different magnetic susceptibility tensors can be obtained using the same tagging site, the presented LCTs can be conveniently used for studies of protein structure and ligand binding.Besides disulphide, pyridine-derived and iodoacetamide linkers, bromoacetamides were shown by Wu et al to be of significant value for ligation of an LCT to a phosphorothioate group in DNA[30]. The reduction-stable linkage that is formed between the LCT equipped with a bromo leaving group and the phosphorothioate offered the possibility for the authors to detect PCSs on DNA and subsequently fit the corresponding anisotropic ∆χ-parameters.…”

“…The stereogenic phosphorus atom generated by the introduction of a phosphorothioate group leads to different sets of observed PCS. However, it is possible to separate the diastereomers by reverse-phase HPLC on a semipreparative C18 column leading to only one set of signals observed[30], thus opening the avenue of the transfer of the PCS methodology from proteins to DNA and RNA.…”