HYBRIDIZATION CHARACTERISTICS OF BIOMOLECULAR ADAPTERS, COVALENT DNA STREPTAVIDIN CONJUGATES

Semisynthetic, covalent streptavidin-DNA adducts are versatile molecul ar connectors for the fabrication of both nano-and microstructured pro tein arrays by use of DNA hybridization. In this study, the hybridizat ion characteristics of six adduct species, each containing a different DNA sequence of 21 or 24 bases, have been compared. First, the adduct s were conjugated to biotinylated alkaline phosphatase, and their bind ing to immobilized oligomer complements of similar lengths was quantif ied in a microplate assay. The binding efficiency observed varied to a great extent with the specific sequence of the oligonucleotide attach ed, and could not be predicted from affiliated thermodynamic data of d uplex stability. To further elucidate the hybridization properties, th e hybridization rate constants of association and dissociation (k(assn ) and k(dissn)) have been determined for both unconjugated oligonucleo tides and protein adducts, using a surface plasmon resonance biosensor . The k(assn) values observed for the oligonucleotides are in the rang e of 9 x 10(3) to 2 x 10(5) M-1 s(-1) and correlate with structural pr operties of the probe strands. Up to 3-fold decreased k(assn) values w ere obtained for the corresponding protein adducts. Likewise, values w ere observed for k(dissn) ranging from 1.4 x 10(-4) to 1.9 x 10(-5) s( -1) for the oligonucleotides. The dissociation of the analogous protei n conjugates was reduced by up to 5-fold. The extent of this decrease correlates with the formation of homodimeric or intramolecular aggrega tion of probe strands. A mechanistic model for explaining these data i s based on attractive intramolecular interaction between the nucleic a cid and protein moiety.