Abstract
M.Tech.
RNA interference (RNAi) is a gene silencing mechanism that uses short RNA duplexes to block
gene expression. This mechanism has been widely explored to determine functions of genes.
Furthermore, this phenomenon has been used to silence unwanted genes such as viral genes.
RNAi has been successfully employed in non-mammalian organisms such as plants, where long
dsRNAs (more than 30 bp) have been used without inducing non-specific effects. However, in
mammalian cells, cytoplasmic dsRNAs of more than 30 bp trigger non-specific induction of
many genes, which may result from the activation of dsRNA-dependent protein kinase (PKR)
and 2’,5’-oligoadenylate synthetase (2’,5’-OAS), via the interferon response pathway. In this
study, we describe a novel and simple strategy to overcome nonspecific effects induced by
longer RNA duplexes. This strategy uses sodium bisulphite which is a mutagen that deaminates
cytosine residue to uracil residues in order to introduce mutations in the sense strand of the
duplex. Introduction of these mutations results in the formation of G:U pairings between the
sense and antisense strands of the long hairpin RNA. RNA duplexes with mismatches have been
shown to be able to prevent interferon induction in mammalian cells. According to the obtained
results, long hairpins RNA with and without mismatches were unable to inhibit the expression of
the target region, which was the U5 region of the HIV-1 subtype C LTR. The U5 region of the
LTR is actively involved in the reverse transcription of HIV-1. Therefore silencing of this region
would have led to the inhibition or reverse transcription blockage. Furthermore, data showed that
the interferon response was induced when using these long hairpin RNA duplexes. Due to the
sensitivity of mammalian cells, the action of sodium bisulphite could have stimulated certain
genes of the interferon pathway. Even though hairpins constructed in this study were unable to
prevent the induction of the interferon response pathway and also could not silence the target,
this strategy of using sodium bisulphite has a great potential as shown by its ability to induce
changes in cytosine residues and leaving other nucleotides unchanged.