Photochemically and chemically activatable antisense oligonucleotides: comparison of their reactivities towards DNA and RNA targets

Abstract

Dodecadeoxyribonucleotides derivatized with 1,10-phenanthroline or psoralen were targeted to the point mutation (G<-->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 the reactivity of the activatable dodecamers (cleavage of the target by the phenanthroline-12mer conjugates; photo-induced cross-linking of psoralen-12mer conjugates to the target). The reactivity of the RNA with the dodecamers was weaker than that of the DNA target. With psoralen-substituted oligonucleotides, it was possible to obtain complete discrimination between the mutated target (which contained 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 fragments were used as targets (120 and 820 nucleotides), very little reactivity was observed. Part of the reactivity could be recovered by using 'helper' oligonucleotides that hybridized to adjacent sites on the substrate. A 'helper' chain length greater than 13 was required to improve the reactivity of dodecamers. However, the dodecanucleotides induced RNase H cleavage of the target RNA in the absence of 'helper' oligonucleotide. Therefore, in the absence of the RNase H enzyme, long oligonucleotides are needed to compete with the secondary structures of the mRNA. In contrast, formation of a ternary complex oligonucleotide-mRNA-RNase H led to RNAT cleavage with shorter oligonucleotides.

Identification of the Edible Fig `Bianco del Cilento' by Random Amplified Polymorphic DNA Analysis

“…A second treatment of DNA with urea buffer was essential to eliminate impurities, such as tannins, alkaloids, and flavones, present in the extract. These compounds can interact with the double-stranded DNA, interfering with PCR amplification (Bohm, 1987;De Jong, 1991;Godard et al, 1994;Mergny et al, 1992). In a PCR reaction performed with AmpliTaq DNA polymerase (Stoffel Fragment), single extraction with urea buffer of DNA (lane a) resulted in an absence of amplification, while double extraction (lane b) resulted in amplification products (Fig.…”

Furocoumarins in Medicinal Chemistry. Synthesis, Natural Occurrence and Biological Activity

“…The psoralen, in addition to its photoactivity, can increase the ability of oligonucleotides to penetrate cells [226] and the oligo have the possibility of directing psoralen to sites of interest [227]. In this way, it could be possible to obtain a more photochemically active antisense oligonucleotide [228,229] or a psoralen for DNA site-directed damage [230]. A psoralen-linked triplex-forming oligonucleotide targeted to a coding sequence has been reported to produce inhibition of aromatase expression from a mutant cytochrome P450 enzyme aromatase which is an important target in breast cancer treatment [231].…”

DNA and RNA Cleavage Mediated by Phenanthroline-Cuprous Oligonucleotides: From Properties to Applications

“…We have previously shown that octathymidylates tethered to phenanthroline which were able to cleave r(A)10 target in a specific manner, allowed us to determine relative orientation of the binding of IX oligomers on RNA targets (Sun et al 1988). In a more recent study, we have tried to cleave mRNA of Harvey ras oncogene using modified oligonucleotides covalently linked at their extremities to 1,10 phenanthroline (Godard et al 1994). Dodecadeoxyribonucleotides built with p or IX anomers and targeted to the codon 12 region present in human Ha-ras mRNA were unable to cleave RNA targets containing their complementary sequence due to their stable secondary structure.…”

“…Indeed, these phenanthroline-oligomer conjugates induced efficient cleavage of the corresponding DNA targets. We have succeeded in specifically degrading Ha-ras mRNA by addition of a 20-nucleotide-long "helper" oligonucleotide targeted to immediately neighboring sequences (Godard et al 1994). It is likely that cooperative binding of the helper oligonucleotide increases the binding affinity of the phenanthrolinedodecamers towards RNA target, allowing the cleavage reaction to take place.…”