Biophysical and Biological Characterization of Hairpin
and Molecular Beacon RNase H Active Antisense Oligonucleotides
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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 <i>in vitro</i>, 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