Selective Covalent Conjugation of Phosphorothioate DNA Oligonucleotides with Streptavidin 英文参考文献.docVIP

Molecules 2011, 16, 6916-6926; doi:10.3390/molecule OPEN ACCESS molecules ISSN 1420-3049 /journal/molecules Article Selective Covalent Conjugation of Phosphorothioate DNA Oligonucleotides with Streptavidin Kersten S. Rabe and Christof M. Niemeyer * TU Dortmund, Fakult?t Chemie, Biologisch-Chemische Mikrostrukturtechnik, Otto-Hahn Strasse 6, 44227 Dortmund, Germany * Author to whom correspondence should be addressed; E-Mail: christof.niemeyer@tu-dortmund.de; Tel.: +49-231-755-7080; Fax: +49-231-755-7082. Received: 26 June 2011; in revised form: 8 August 2011 / Accepted: 11 August 2011 / Published: 15 August 2011 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