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PMID: 10684928 Published · ppublish English Journal Article

Effects of RNA secondary structure on cellular antisense activity.

Nucleic acids research ·Vol. 28 ·No. 6 ·2000-03-15 ·Pages 1340-7

Vickers TA, Wyatt JR, Freier SM

Abstract

The secondary and tertiary structures of a mRNA are known to effect hybridization efficiency and potency of antisense oligonucleotides in vitro. Additional factors including oligonucleotide stability and cellular uptake are also thought to contribute to antisense potency in vivo. Each of these factors can be affected by the sequence of the oligonucleotide. Although mRNA structure is presumed to be a critical determinant of antisense activity in cells, to date little direct experimental evidence has addressed the significance of structure. In order to determine the importance of mRNA structure on antisense activity, oligonucleotide target sites were cloned into a luciferase reporter gene along with adjoining sequence to form known structures. This allowed us to study the effect of target secondary structure on oligonucleotide binding in the cellular environment without changing the sequence of the oligonucleotide. Our results show that structure does play a significant role in determining oligonucleotide efficacy in vivo. We also show that potency of oligonucleotides can be improved by altering chemistry to increase affinity for the mRNA target even in a region that is highly structured.

MeSH Terms
Animals Antigens, CD/genetics B7-2 Antigen Base Composition Base Pairing/genetics Base Sequence Binding Sites COS Cells Genes, Reporter/genetics Humans Inhibitory Concentration 50 Intercellular Adhesion Molecule-1/genetics Membrane Glycoproteins/genetics Nucleic Acid Conformation Nucleic Acid Hybridization/genetics Oligoribonucleotides/chemistry,genetics,metabolism Proto-Oncogene Proteins c-raf/genetics RNA/genetics RNA Stability/genetics RNA, Antisense/chemistry,genetics,metabolism RNA, Messenger/chemistry,genetics,metabolism Ribonuclease H/metabolism Substrate Specificity Thermodynamics Transfection
Chemicals
Antigens, CD B7-2 Antigen CD86 protein, human Membrane Glycoproteins Oligoribonucleotides RNA, Antisense RNA, Messenger RNA, recombinant Intercellular Adhesion Molecule-1 RNA Proto-Oncogene Proteins c-raf Ribonuclease H
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Vickers T A
Isis Pharmaceuticals, Department of Molecular and Structural Biology, 2280 Faraday Avenue, Carlsbad, CA 92008, USA. [email protected]
Wyatt J R
Freier S M
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
1362-4962
DOI
10.1093/nar/28.6.1340
Email
[email protected]
Published
2000-03-15
Pages
1340-7
Language
English
Region
England
NLM ID
0411011
PMCID
PMC111043
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/28.6.1340 PMC Full Text View journal details
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