title: Biophysical and biological characterization of hairpin and molecular beacon RNase H active antisense oligonucleotides.
abstract: Antisense oligonucleotides (ASOs) are single stranded, backbone modified nucleic acids, which mediate cleavage of complementary RNA by directing RNase H cleavage in cell culture and in animals. It has generally been accepted that the single stranded state in conjunction with the phosphorothioate modified backbone is necessary for cellular uptake and transport to the active compartment. Herein, we examine the effect of using hairpin structured ASOs to (1) determine if an ASO agent requires a single stranded conformation for efficient RNA knock down, (2) use a fluorophore-quencher labeled ASO to evaluate which moieties the ASO interacts with in cells and examine if cellular distribution can be determined with such probes, and (3) evaluate if self-structured ASOs can improve allele selective silencing between closely related huntingtin alleles. We show that hairpin shaped ASOs can efficiently down-regulate RNA in vitro, but potency correlates strongly negatively with increasing stability of the hairpin structure. Furthermore, self-structured ASOs can efficiently reduce huntingtin mRNA in the central nervous system of mice.
authors: †Isis Pharmaceuticals, 2855 Gazelle Court, Carlsbad, California, United States.
authors: Michael E
authors: ME
authors: Østergaard
authors: †Isis Pharmaceuticals, 2855 Gazelle Court, Carlsbad, California, United States.
authors: George
authors: G
authors: Thomas
authors: †Isis Pharmaceuticals, 2855 Gazelle Court, Carlsbad, California, United States.
authors: Erich
authors: E
authors: Koller
authors: ‡Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia V5Z 4H4, Canada.
authors: Amber L
authors: AL
authors: Southwell
authors: ‡Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia V5Z 4H4, Canada.
authors: Michael R
authors: MR
authors: Hayden
authors: †Isis Pharmaceuticals, 2855 Gazelle Court, Carlsbad, California, United States.
authors: Punit P
authors: PP
authors: Seth
mesh_terms: D000483:Alleles; D055592:Biophysical Phenomena; D002460:Cell Line; D055785:Gene Knockdown Techniques; D006801:Humans; D000071058:Huntingtin Protein; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D016914:Ribonuclease H / Q000235:genetics*
chemical_list: C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D016376:Oligonucleotides, Antisense; D016914:Ribonuclease H