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An assessment of the antisense properties of RNase H-competent and steric-blocking oligomers
M A Bonham1, S Brown, A L Boyd
1Department of Molecular Cell Biology, Glaxo Research Institute, Research Triangle Park, NC 27709, USA.
Abstract:
The antisense activity and gene specificity of two classes of oligonucleotides (ONs) were directly compared in a highly controlled assay. One class of ONs has been proposed to act by targeting the degradation of specific RNAs through an RNase H-mediated mechanism and consists of C-5 propynyl pyrimidine phosphorothioate ONs (propyne-S-ON). The second class of antisense agents has been proposed to function by sterically blocking target RNA formation, transport or translation and includes sugar modified (2'-O-allyl) ONs and peptide nucleic acids (PNAs). Using a CV-1 cell based microinjection assay, we targeted antisense agents representing both classes to various cloned sequences localized within the SV40 large T antigen RNA. We determined the propyne-S-ON was the most potent and gene-specific agent of the two classes which likely reflected its ability to allow RNase H cleavage of its target. The PNA oligomer inhibited T Ag expression via an antisense mechanism, but was less effective than the propyne-S-ON; the lack of potency may have been due in part to the PNAs slow kinetics of RNA association. Interestingly, unlike the 2'-O-allyl ON, the antisense activity of the PNA was not restricted to the 5' untranslated region of the T Ag RNA. Based on these findings we conclude that PNAs could be effective antisense agents with additional chemical modification that will lead to more rapid association with their RNA target.
Insights
C-5 propynyl pyrimidine phosphorothioate oligonucleotides (propyne-S-ONs) demonstrated superior potency and gene specificity compared to peptide nucleic acids (PNAs) and 2'-O-allyl oligonucleotides in targeting SV40 large T antigen RNA.
Area of Science:
- Molecular Biology
- Antisense Technology
- Oligonucleotide Therapeutics
Background:
- Oligonucleotides (ONs) are investigated for antisense activity, targeting RNA degradation or steric blocking.
- Two main classes of antisense ONs exist: RNase H-mediated degradation agents (propyne-S-ONs) and steric blocking agents (2 -O-allyl ONs, PNAs).
Purpose of the Study:
- To directly compare the antisense activity and gene specificity of propyne-S-ONs and steric blocking ONs (2 -O-allyl ONs, PNAs).
- To evaluate the efficacy of different antisense oligonucleotide classes against SV40 large T antigen RNA.
Main Methods:
- Utilized a CV-1 cell-based microinjection assay for controlled delivery of antisense agents.
- Targeted cloned sequences within the SV40 large T antigen RNA with representative ONs from each class.
Main Results:
- Propyne-S-ONs exhibited the highest potency and gene specificity, attributed to RNase H-mediated cleavage.
- Peptide nucleic acids (PNAs) inhibited T antigen expression via antisense mechanisms but were less potent than propyne-S-ONs, potentially due to slow RNA association kinetics.
- PNA antisense activity was not limited to the 5 untranslated region, unlike 2 -O-allyl ONs.
Conclusions:
- Propyne-S-ONs are potent and specific antisense agents, likely due to RNase H involvement.
- PNAs show promise as antisense agents but require chemical modifications to enhance RNA association kinetics for improved efficacy.