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Sequence context of antisense RelA/NF-kappa B phosphorothioates determines specificity
J Y Maltese1, H W Sharma, L Vassilev
1Division of Oncology, Roche Research Center, Hoffmann-La Roche Inc., Nutley, NJ 07110, USA.
Abstract:
The use of antisense oligomers to achieve inhibition of gene expression is complicated by frequent non-specific effects, and even the control oligomers often exhibit sequence-specific effects. We have recently shown that in diverse tumor-derived cell lines, a 24mer phosphorothioate oligomer antisense to the relA subunit of NF-kappa B transcription factor causes a block of cellular adhesion, inhibition of nuclear NF-kappa B and Sp1 DNA-binding activity and inhibition of tumor cell growth in vitro and in vivo. In this study we use the same model to attempt to define the limits of antisense specificity. We demonstrate that single base pair substitution can virtually abolish the antisense activity. The relative position of mismatches within the antisense sequence is critical to the loss of activity. Our results further indicate that antisense specificity is determined not only by the content of the sequence but also by its occurrence with reference to the surrounding sequences.
Insights
Antisense oligomers targeting gene expression can have non-specific effects. This study shows that even single base changes in antisense sequences significantly impact gene silencing, highlighting the importance of sequence context for specificity.
Area of Science:
- Molecular Biology
- Gene Expression Regulation
- Cancer Therapeutics
Background:
- Antisense oligomers offer a strategy for gene expression inhibition.
- Non-specific effects and sequence-specific activity in control oligomers complicate their use.
- Previous work demonstrated a phosphorothioate oligomer targeting relA (NF-kappa B subunit) inhibits tumor cell growth.
Purpose of the Study:
- To define the limits of antisense specificity in gene silencing.
- To investigate the impact of sequence variations on antisense activity.
- To understand the role of sequence context in antisense efficacy.
Main Methods:
- Utilized a previously established model using a 24mer phosphorothioate oligomer antisense to the relA subunit of NF-kappa B.
- Introduced single base pair substitutions within the antisense sequence.
- Assessed the impact of these substitutions on cellular adhesion, NF-kappa B and Sp1 DNA-binding activity, and tumor cell growth.
Main Results:
- Single base pair substitutions were found to virtually abolish antisense activity.
- The position of mismatches within the antisense sequence critically influenced the loss of activity.
- Antisense specificity depends on both the sequence content and its surrounding sequence context.
Conclusions:
- Antisense specificity is highly sensitive to minor sequence alterations.
- The precise location of mismatches is a key determinant of antisense efficacy.
- Effective antisense therapy design must consider sequence context and potential off-target effects.