PHOTOCHEMICALLY AND CHEMICALLY ACTIVATABLE ANTISENSE OLIGONUCLEOTIDES- COMPARISON OF THEIR REACTIVITIES TOWARDS DNA AND RNA TARGETS

Dodecadeoxyribonucleotides derivatized with 1,10-phenanthroline or pso ralen were targeted to the point mutation (G double right arrow U) in codon 12 of the Ha-ras mRNA. DNA and RNA fragments, 27 nucleotides in length, and containing the complementary sequence of the 12mers, were used to compare tire reactivity of the activatable dodecamers (cleavag e oil the target by the phenanthroline-12mer conjugates; photo-induced cross-linking of psoralen-12mer conjugates to the target). The reacti vity of the RNA with the dodecamers was weaker than that of the DNA ta rget. With psoralen-substituted oligonucleotides, it was possible to o btain complete discrimination between the mutated target (which contai ned a psoralen-reactive T(U) in the 12th codon) and the normal target (which contained G at the same position). When longer Ha-ras RNA fragm ents were used as targets (120 and 820 nucleotides), very little react ivity was observed. Part of the reactivity could be recovered by using 'helper' oligonucleotides that hybridized to adjacent sites on the su bstrate. A 'helper' chain length greater than 13 was required to impro ve the reactivity of dodecamers. However, the dodecanucleotides induce d RNase H cleavage of the target RNA in the absence of 'helper' oligon ucleotide. Therefore, in the absence of the RNase H enzyme, long oligo nucleotides are needed to compete with the secondary structures of the mRNA. In contrast, formation of a ternary complex oligonucleotide-mRN A-RNase H led to RNAT cleavage with shorter oligonucleotides.