title: Selective covalent conjugation of phosphorothioate DNA oligonucleotides with streptavidin.
abstract: Protein-DNA conjugates have found numerous applications in the field of diagnostics and nanobiotechnology, however, their intrinsic susceptibility to DNA degradation by nucleases represents a major obstacle for many applications. We here report the selective covalent conjugation of the protein streptavidin (STV) with phosphorothioate oligonucleotides (psDNA) containing a terminal alkylthiolgroup as the chemically addressable linking unit, using a heterobifunctional NHS-/maleimide crosslinker. The psDNA-STV conjugates were synthesized in about 10% isolated yields. We demonstrate that the terminal alkylthiol group selectively reacts with the maleimide while the backbone sulfur atoms are not engaged in chemical conjugation. The novel psDNA-STV conjugates retain their binding capabilities for both biotinylated macromolecules and the complementary nucleic acid. Moreover, the psDNA-STV conjugate retained its binding capacity for complementary oligomers even after a nuclease digestion step, which effectively degrades deoxyribonucleotide oligomers and thus the binding capability of regular DNA-STV conjugates. The psDNA-STV therefore hold particular promise for applications e.g. in proteome research and novel biosensing devices, where interfering endogenous nucleic acids need to be removed from analytes by nuclease digestion.
authors.affiliation: TU Dortmund, Fakultät Chemie, Biologisch-Chemische Mikrostrukturtechnik, Otto-Hahn Strasse 6, 44227 Dortmund, Germany.
authors.forename: Kersten S
authors.initials: KS
authors.lastname: Rabe
authors.forename: Christof M
authors.initials: CM
authors.lastname: Niemeyer
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003432:Cross-Linking Reagents / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004720:Endonucleases / Q000378:metabolism; D004926:Escherichia coli; D008301:Maleimides / Q000737:chemistry; D008958:Models, Molecular; D015335:Molecular Probes / Q000032:analysis / Q000138:chemical synthesis; D036103:Nanotechnology / Q000379:methods*; D009693:Nucleic Acid Hybridization; D054735:Phosphorothioate Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D040901:Proteomics / Q000379:methods; D013050:Spectrometry, Fluorescence; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D008301:Maleimides; D015335:Molecular Probes; D054735:Phosphorothioate Oligonucleotides; C043592:maleimide; D001710:Biotin; D004247:DNA; D019809:Streptavidin; D004720:Endonucleases; D000469:Alkaline Phosphatase