Three novel phosphoramidites, each bearing a phthaloyl-protected aminooxy tail, were prepared and applied in automated oligonucleotide synthesis. After chain assembly, the phthaloyl protection was removed with hydrazinium acetate. Normal succinyl linker turned to be stable under these conditions, and hence the support-bound oligonucleotide could be converted to a pyrene oxime conjugates by reacting with pyrene carbaldehyde or cis-retinal. Standard ammonolytic deprotection then released the deprotected conjugate in solution. Alternatively, the crude aminooxy-tethered oligonucleotide was immobilized to microscopic polymer particles by reacting the aminooxy function with the particle-bound aldehyde or epoxide groups. These immobilized oligonuceotides were shown to serve properly as probes in a mixed phase hybridization assay.

Several alternative methods have been described for the immobilization of oligodeoxyribonucleotides to uniformly sized glycidyl methacrylate/ethylene dimethacrylate particles. Hybridization of complementary oligodeoxyribonucleotides labeled with photoluminescent europium(III) chelates to these particle-bound oligonucleotide probes was followed by subjecting a single microparticle to a time-resolved fluorescence measurement. The hybridization was further quantified by releasing the europium ion to a fluorescence enhancement solution and determining its concentration against europium(III) chloride standards. Both the efficiency and kinetics of the hybridization were observed to depend markedly on the linker employed to tether the oligonucleotide probes to the particles. These effects and those of the experimental conditions, such as oligonucleotide concentration in solution, oligonucleotide density on particles, and number of particles in a given volume of assay solution, are discussed.

Oligodeoxyribonucleotides were assembled by conventional phosphoramidite chemistry on uniformly sized (50 microns) porous glycidyl methacrylate/ethylene dimethacrylate (SINTEF) and compact polystyrene (Dynosphere) particles, the aminoalkyl side chains of which were further derivatized with DMTrO-acetyl groups. The linker was completely resistant toward ammonolytic deprotection of the base moieties. The quality of oligonucleotides was assessed by repeating the synthesis on the same particles derivatized with a cleavable ester linker. The ability of the oligonucleotide-coated particles to bind complementary sequences via hybridization was examined by following the attachment of oligonucleotides bearing a photoluminescent europium(III) chelate to the particles. The fluorescence emission was measured directly on a single particle. The effects of the following factors on the kinetics and efficiency of hybridization were studied: number of particles in a given volume of the assay solution, loading of oligonucleotide on the particle, concentration of the target oligonucleotide in solution, length of the hybridizing sequence, presence of noncomplementary sequences, and ionic strength. The fluorescence signal measured on a single particle after hybridization was observed to be proportional to the concentration of the target oligonucleotide in solution over a concentration range of 5 orders of magnitude.

[reaction: see text] Novel nucleosidic phosphoramidite blocks were synthesized by a Mitsunobu reaction between 2'-deoxy-5'-O-(4,4'-dimethoxytrityl)uridine and a primary alcohol containing a conjugate group in its structure (a protected functional group, an organic dye, or a precursor of a lanthanide(III) chelate) followed by phosphitylation. They were used in machine-assisted DNA synthesis in the standard manner. A slightly modified deprotection procedure was used for the preparation of oligonucleotide conjugates tethered to lanthanide(III) chelates. For the latter application one non-nucleosidic block was also synthesized.