Details

Autor(en) / Beteiligte

• Niemeyer, Christof M
• Ceyhan, Bülent
• Blohm, Dietmar

Titel

Functionalization of Covalent DNA−Streptavidin Conjugates by Means of Biotinylated Modulator Components

Ist Teil von

• Bioconjugate chemistry, 1999-09, Vol.10 (5), p.708-719

Ort / Verlag

United States: American Chemical Society

Erscheinungsjahr

1999

Quelle

MEDLINE

Beschreibungen/Notizen

• Covalent DNA−streptavidin conjugates are versatile biomolecular coupling reagents, since they have binding capacity for both a complementary nucleic acid and four molecules of biotin. The DNA−streptavidin hybrid molecules have been investigated for their capabilities to bind two different types of biotinylated components. Thus, (i) a functional biomolecule, e.g., a single-stranded DNA fragment or an enzyme and (ii) low-molecular weight biotin derivatives (“modulators”) were coupled stepwise with the hybrid molecules. Modulators were d-biotin, aminobiotin, and biotin-fluorescein conjugate as well as a lysine-rich 10mer peptide, containing a biotin and a fluorescein substituent. These modulators were chosen to affected the hybridization properties of the DNA−streptavidin conjugates. As investigated by surface-plasmon resonance and microplate solid-phase hybridization measurements, d-biotin, biotin-fluorescein, and aminobiotin decreased the efficiency of hybridization with complementary, surface-bound oligonucleotides to a varying extent. The basic peptide increased the conjugate's hybridization efficiency. Moreover, it was demonstrated in two examples how modulators can be utilized as additional functional domains of streptavidin-based conjugates. First, fluorescein-containing modulators were used as hapten groups, allowing a sensitive detection by means of specific antibodies directed against the modulator. Second, the biotinylated peptide was used as a carrier molecule to attach multiple fluorogenic lanthanide-chelate groups to the streptavidin conjugate, enabling its sensitive detection by time-resolved fluorometry. The applicability of this kind of bioconjugation strategy to generate sensor-probes for gene detection assays was demonstrated.