Photochemically and chemically activatable antisense oligonucleotides: comparison of their reactivities towards DNA and RNA targets.
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.
Godard
G
G
Laboratoire de Biophysique, INSERM U 201-CNRS UA 481, Paris, France.
François
J C
JC
Duroux
I
I
Asseline
U
U
Chassignol
M
M
Nguyen
T
T
Hélène
C
C
Saison-Behmoaras
T
T
D001483:Base Sequence; D003062:Codon; D003432:Cross-Linking Reagents / Q000737:chemistry; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D018736:DNA Adducts / Q000378:metabolism*; D011564:Furocoumarins / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000138:chemical synthesis / Q000378:metabolism*; D010618:Phenanthrolines / Q000737:chemistry; D010777:Photochemistry; D017354:Point Mutation / Q000502:physiology; D011089:Polydeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D011131:Polyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D016914:Ribonuclease H; D018631:ras Proteins / Q000235:genetics
D003062:Codon; D003432:Cross-Linking Reagents; D018736:DNA Adducts; D011564:Furocoumarins; D016376:Oligonucleotides, Antisense; D010618:Phenanthrolines; D011089:Polydeoxyribonucleotides; D011131:Polyribonucleotides; D012333:RNA, Messenger; C056465:psoralen-DNA adduct; D012313:RNA; D004247:DNA; D016914:Ribonuclease H; D018631:ras Proteins; C025205:1,10-phenanthroline