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Selective Covalent Conjugation of Phosphorothioate DNA Oligonucleotides with Streptavidin

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 strep...

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Autores principales: Rabe, Kersten S., Niemeyer, Christof M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264524/
https://www.ncbi.nlm.nih.gov/pubmed/21844841
http://dx.doi.org/10.3390/molecules16086916
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author Rabe, Kersten S.
Niemeyer, Christof M.
author_facet Rabe, Kersten S.
Niemeyer, Christof M.
author_sort Rabe, Kersten S.
collection PubMed
description 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.
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spelling pubmed-62645242018-12-10 Selective Covalent Conjugation of Phosphorothioate DNA Oligonucleotides with Streptavidin Rabe, Kersten S. Niemeyer, Christof M. Molecules Article 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. MDPI 2011-08-15 /pmc/articles/PMC6264524/ /pubmed/21844841 http://dx.doi.org/10.3390/molecules16086916 Text en © 2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
spellingShingle Article
Rabe, Kersten S.
Niemeyer, Christof M.
Selective Covalent Conjugation of Phosphorothioate DNA Oligonucleotides with Streptavidin
title Selective Covalent Conjugation of Phosphorothioate DNA Oligonucleotides with Streptavidin
title_full Selective Covalent Conjugation of Phosphorothioate DNA Oligonucleotides with Streptavidin
title_fullStr Selective Covalent Conjugation of Phosphorothioate DNA Oligonucleotides with Streptavidin
title_full_unstemmed Selective Covalent Conjugation of Phosphorothioate DNA Oligonucleotides with Streptavidin
title_short Selective Covalent Conjugation of Phosphorothioate DNA Oligonucleotides with Streptavidin
title_sort selective covalent conjugation of phosphorothioate dna oligonucleotides with streptavidin
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264524/
https://www.ncbi.nlm.nih.gov/pubmed/21844841
http://dx.doi.org/10.3390/molecules16086916
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