DNA hybridization on microparticles: determining capture-probe density andequilibrium dissociation constants

Many DNA-probe assays utilize oligonucleotide-coated microparticles for cap ture of complementary nucleic acids from solution. During development of th ese assays, as well as in other particle-based nucleic acid applications, i t is useful to know both the amount of duplex formation expected under vari ous experimental conditions and the coating density of the capture oligonuc leotide on the particle surface. We examined the simplest form of a DNA-pro be microparticle assay: hybridization of a particle-bound capture oligonucl eotide to its solution-phase complement. Fluorescein-labeled solution-phase oligonucleotide was hybridized to varying amounts of particles, and the am ount of labeled oligonucleotide remaining in solution at equilibrium was me asured. We present a simple two-state, all-or-none model for bimolecular hy bridization of non-self-complementary sequences that can be used to calcula te the equilibrium dissociation constant (Kd) from hybridization data. With experimental conditions where both the Kd value and the concentration of c apture probe in the reaction are small relative to the concentration of lab eled complementary oligonucleotide in the reaction, density of the capture probe on the particle's surface can also be determined. Kd values for parti cle-based hybridization were different from those obtained from solution-ph ase thermodynamic parameters. At higher temperatures, hybridization on part icles was more efficient than hybridization in solution.