The secondary and tertiary structures of a mRNA are known to effect hybridi
zation efficiency and potency of antisense oligonucleotides in vitro. Addit
ional factors including oligonucleotide stability and cellular uptake are a
lso thought to contribute to antisense potency in vivo. Each of these facto
rs can be affected by the sequence of the oligonucleotide. Although mRNA st
ructure is presumed to be a critical determinant of antisense activity in c
ells, to date little direct experimental evidence has addressed the signifi
cance of structure. In order to determine the importance of mRNA structure
on antisense activity, oligonucleotide target sites were cloned into a luci
ferase reporter gene along with adjoining sequence to form known structures
. This allowed us to study the effect of target secondary structure on olig
onucleotide binding in the cellular environment without changing the sequen
ce of the oligonucleotide. Our results show that structure does play a sign
ificant role in determining oligonucleotide efficacy in vivo. We also show
that potency of oligonucleotides can be improved by altering chemistry to i
ncrease affinity for the mRNA target even in a region that is highly struct
ured.