Antisense oligonucleotides (ASOs) can be used to inhibit gene expression both in vitro and in vivo. Improvements in the design and chemistry of antisense compounds have enabled this technology to become a routine tool in basic
research, genomics, target validation, and drug discovery. ASOs are increasingly used to confirm phenotypes obtained from RNAi-mediated gene silencing experiments, also.
ASOs are nucleic acid sequences made as synthetic oligonucleotides, usually 15–22–bases
long, containing a phosphorothioate-modified DNA segment of at least six bases. They are designed in antisense orientation to the RNA of interest, hence the name. To inhibit gene expression, they are introduced into the cell or organism, where they
bind the target RNA to form an RNA/DNA heteroduplex, which is a substrate for endogenous cellular RNase H (Figure 1) [2,3]. The resulting decrease in RNA levels can be measured using RT-qPCR or RNA-seq.
ASOs can also be designed to investigate the role of alternatively spliced mRNA. Alternative splicing is one of many ways gene expression is modulated to respond to changing environmental conditions or developmental cues. ASOs are designed to the pre-mRNA
sequence, and therefore, are complementary to the exon and intron junction. Unlike above, these ASOs create a double-stranded region that sterically blocks splicing factors from binding. These ASOs are designed with 2' modifications on the sugar moiety throughout the length of the sequence in order to prevent RNase H activation.