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.
Highlights
Almost 30 years ago, the enormous specificity of Watson-Crick base-pairing of synthetic DNA molecules had been recognized by Nadrian Seeman as a powerful means to rationally construct twoand three-dimensional assemblies from this biopolymer [1]
We demonstrate that the novel phosphorothioate DNA (psDNA)-STV conjugates retain their binding capabilities for both biotinylated macromolecules and the complementary nucleic acid
While we had previously optimized this disulfide reduction step with dithiothreitol (DDT) [17], we observed that the psDNA revealed higher coupling efficiency when tris(2-carboxyethyl)phosphine (TCEP) was used as reducing agent
Summary
Almost 30 years ago, the enormous specificity of Watson-Crick base-pairing of synthetic DNA molecules had been recognized by Nadrian Seeman as a powerful means to rationally construct twoand three-dimensional assemblies from this biopolymer [1]. Covalent DNA conjugates of the biotin-binding protein streptavidin (STV) are noteworthy in the respect, because they combine the high-affinity binding of STV for four molecules of D-biotin with an additional binding site for the complementary nucleic acid. Since their initial description [7], these conjugates have been used as versatile adaptors to readily label biotinylated proteins with an oligonucleotide moiety to realize numerous in vitro applications in sensing and biomedical diagnostics [4,6]. The robust psDNA-STV conjugates should find applications in the development of novel biosensing devices
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