Hybridization Properties of Support-Bound Oligonucleotides: TheEffect of the Site of Immobilization on the Stability and Selectivityof Duplex Formation
Four 12-mer oligodeoxyribonucleotide sequences were immobilized to uniformly sized (50 μm) polymerparticles through C5-tethered thymine and N4-tethered cytosine bases at four different sites in eachsequence. The effect of the site of immobilization on the efficiency and selectivity of hybridization ofthe particle-bound probes was quantified by a sandwich-type assay based on a time-resolvedfluorometric measurement of an oligonucleotide probe labeled with a photoluminescent europium(III) chelate directly from the surface of a single particle. Immobilization through a base in the centralpart of the sequence was observed to destablize the duplex more markedly than tethering through aterminal base. The effect of a one-base mismatch on the duplex stability increased with the increasingdistance from the site of immobilization.