Functionalization of covalent DNA-streptavidin conjugates by means of biotinylated modulator components.
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.
Niemeyer
C M
CM
Universität Bremen, FB 2-UFT, Biotechnology and Molecular Genetics, Leobener Strasse, D-28359 Bremen, Germany. CMN@biotec.uni-bremen.de
Ceyhan
B
B
Blohm
D
D
D019921:Biotinylation; D003432:Cross-Linking Reagents / Q000737:chemistry; D004247:DNA / Q000138:chemical synthesis / Q000737:chemistry*; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D007202:Indicators and Reagents / Q000737:chemistry / Q000302:isolation & purification; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D019809:Streptavidin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry
D003432:Cross-Linking Reagents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D009841:Oligonucleotides; D004247:DNA; D019809:Streptavidin