"Uniformly" modified phosphodiester or phosphorothioate oligonucleotides incorporating 2'-deoxy-2'-fluoroadenosine, -guanosine, -uridine, and -cytidine, reported herein for the first time, when hybridized with RNA afforded consistent additive enhancement of duplex stability without compromising base-pair specificity. CD spectra of the 2'-deoxy-2'-fluoro-modified oligonucleotides hybridized with RNA indicated that the duplex adopts a fully A-form conformation. The 2'-deoxy-2'-fluoro-modified oligonucleotides in phosphodiester form were not resistant to nucleases; however, the modified phosphorothioate oligonucleotides were highly nuclease resistant and retained exceptional binding affinity to the RNA targets. The stabilizing effects of the 2'-deoxy-2'-fluoro modifications on RNA-DNA duplexes were shown to be superior to those of the 2'-O-methylribo substitutions. RNA hybrid duplexes with uniformly 2'-deoxy-2'-fluoro-modified oligonucleotides did not support HeLa RNase H activity; however, incorporation of the modifications into "chimeric" oligonucleotides has been shown to activate mammalian RNase H. "Uniformly" modified 2'-deoxy-2'-fluoro phosphorothioate oligonucleotides afforded antisense molecules with (1) high binding affinity and selectivity for the RNA target and (2) stability toward nucleases.

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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.