Mixed-phase hybridization of short Oligodeoxyribonucleotides on microscopic polymer particles:: Effect of one-base mismatches on duplex stability

Hybridization of short oligonucleotides (10- and 11-mers) to complementary probes immobilized to microscopic polymer particles was quantified by a sandwich type mixed-phase hybridization assay based on a time-resolved fluorometric measurement of a photoluminescent europium(III) chelate from the surface of a single particle. Among the 54 sequences that were studied, 21 were fully complementary to the particle-bound probes, while 33 contained an internal one-base mismatch. The observed affinities were compared to those predicted by the nearest-neighbor model. In addition, various factors, such as the pore size of the particle, the linker structure, the charge type of the probe, and the efficiency of agitation, that might be expected to affect the kinetics of mixed-phase hybridization have been examined.