Hybridization Characteristics of Biomolecular Adaptors, Covalent DNA−Streptavidin Conjugates

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Abstract
Semisynthetic, covalent streptavidin−DNA adducts are versatile molecular connectors for the fabrication of both nano- and microstructured protein arrays by use of DNA hybridization. In this study, the hybridization characteristics of six adduct species, each containing a different DNA sequence of 21 or 24 bases, have been compared. First, the adducts were conjugated to biotinylated alkaline phosphatase, and their binding to immobilized oligomer complements of similar lengths was quantified in a microplate assay. The binding efficiency observed varied to a great extent with the specific sequence of the oligonucleotide attached, and could not be predicted from affiliated thermodynamic data of duplex stability. To further elucidate the hybridization properties, the hybridization rate constants of association and dissociation (kassn and kdissn) have been determined for both unconjugated oligonucleotides and protein adducts, using a surface plasmon resonance biosensor. The kassn values observed for the oligonucleotides are in the range of 9 × 103 to 2 × 105 M-1 s-1 and correlate with structural properties of the probe strands. Up to 3-fold decreased kassn values were obtained for the corresponding protein adducts. Likewise, values were observed for kdissn ranging from 1.4 × 10-4 to 1.9 × 10-5 s-1 for the oligonucleotides. The dissociation of the analogous protein conjugates was reduced by up to 5-fold. The extent of this decrease correlates with the formation of homodimeric or intramolecular aggregation of probe strands. A mechanistic model for explaining these data is based on attractive intramolecular interaction between the nucleic acid and protein moiety.