Cationic phosphoramidate alpha-oligonucleotides efficiently target single-stranded DNA and RNA and inhibit hepatitis C virus IRES-mediated translation.
A potential means to improve the efficacy of steric-blocking antisense oligonucleotides (ON) is to increase their affinity for a target RNA. The grafting of cationic amino groups to the backbone of the ON is one way to achieve this, as it reduces the electrostatic repulsion between the ON and its target. We have examined the duplex stabilising effects of introducing cationic phosphoramidate internucleoside linkages into ON with a non-natural alpha-anomeric configuration. Cationic alpha-ON bound with high affinity to single-stranded DNA and RNA targets. Duplex stabilisation was proportional to the number of cationic modifications, with fully cationic ON having particularly high thermal stability. The average stabilisation was greatly increased at low ionic strength. The duplex formed between cationic alpha-ON and their RNA targets were not substrates for RNase H. The penalty in T(m) inflicted by a single mismatch, however, was high; suggesting that they are well suited as sequence-specific, steric-blocking, antisense agents. Using a well-described target sequence in the internal ribosome entry site of the human hepatitis C virus, we have confirmed this potential in a cell-free translation assay as well as in a whole cell assay. Interestingly, no vectorisation was necessary for the cationic alpha-ON in cell culture.
Michel
Thibaut
T
Laboratoire de Chimie Organique Biomoléculaire de Synthèse, UMR 5625 CNRS-UMII, CC 008, Université Montpellier II, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France.
Martinand-Mari
Camille
C
Debart
Françoise
F
Lebleu
Bernard
B
Robbins
Ian
I
Vasseur
Jean-Jacques
JJ
D000577:Amides / Q000737:chemistry; D001665:Binding Sites / Q000235:genetics; D002412:Cations / Q000737:chemistry; D045744:Cell Line, Tumor; D003432:Cross-Linking Reagents / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000235:genetics*; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D009691:Nucleic Acid Denaturation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D016376:Oligonucleotides, Antisense / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D010756:Phosphoric Acids / Q000737:chemistry; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D013696:Temperature
D000577:Amides; D002412:Cations; D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D009692:Nucleic Acid Heteroduplexes; D016376:Oligonucleotides, Antisense; D010756:Phosphoric Acids; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D012313:RNA; C011067:phosphoramidic acid; D008156:Luciferases; D016914:Ribonuclease H