Antisense oligonucleotides (ASOs) serve as effective tools for suppressing gene expression both in vitro and in vivo. Ongoing advancements in the design and chemistry of antisense compounds have elevated this technology to a commonplace instrument in fundamental research, genomics, target validation, and drug discovery. ASOs are increasingly employed to corroborate phenotypes obtained through RNA interference (RNAi)-mediated gene silencing experiments.
Constituting synthetic oligonucleotides, typically 15–22 bases in length, ASOs incorporate a phosphorothioate-modified DNA segment spanning at least six bases. These sequences are intentionally crafted in an antisense orientation to the target RNA, giving rise to their nomenclature. To induce gene expression inhibition, ASOs are introduced into the cellular or organismal milieu, binding to the target RNA and forming an RNA/DNA heteroduplex, which serves as a substrate for endogenous cellular RNase H (depicted in Figure 1) [2,3]. The resultant reduction in RNA levels can be quantified using techniques such as RT-qPCR or RNA-seq.
Moreover, ASOs can be tailored to investigate the dynamics of alternatively spliced mRNA. Alternative splicing represents one of several mechanisms modulating gene expression in response to environmental changes or developmental cues. These ASOs are specifically designed to target the pre-mRNA sequence, aligning with the exon and intron junction. In contrast to their aforementioned counterparts, these ASOs create a double-stranded region that impedes the binding of splicing factors through steric hindrance. To forestall RNase H activation, these ASOs are crafted with 2' modifications on the sugar moiety across the entire sequence length.
Chimeric oligos and Phosphorothioates
Although unmodified oligodeoxynucleotides may exhibit some level of antisense activity, they are susceptible to swift degradation by both endo- and exo-nucleases. Many 2′-O-modified RNA species, such as 2′OMe RNAs and locked nucleic acid bases, also face sensitivity to exonuclease degradation. For antisense applications, the most straightforward and widely utilized approach to confer nuclease resistance is through phosphorothioate (PS) modification. In phosphorothioates, a sulfur atom substitutes a non-bridging oxygen in the oligo phosphate backbone. Within the IDT ordering system, the presence of a phosphorothioate internucleoside linkage is denoted by an asterisk. Notably, PS oligos may exhibit elevated non-specific protein binding compared to unmodified phosphodiester (PO) oligos, potentially inducing toxicity or other artifacts at high concentrations.
Our recommendation advocates for the incorporation of phosphorothioate modification into ASO sequences to enhance stability. Additionally, phosphorothioate linkages foster binding to serum proteins, thereby augmenting the bioavailability of the ASO and facilitating effective cellular uptake.
Chimeric antisense designs using chemically modified RNA and DNA bases
Cutting-edge antisense design incorporates chimeric structures comprising both DNA and modified RNA bases [1]. Integration of modified RNA, such as 2′-O-methyl (2′OMe) RNA, or locked nucleic acid bases, into chimeric antisense constructs enhances both nuclease stability and the affinity (Tm) of the antisense oligonucleotide for the target RNA [4–6]. However, it's crucial to note that these modifications do not activate RNase H cleavage. The optimal antisense strategy involves a "gapmer" design, which incorporates 2′-O-modified RNA or locked nucleic acid bases into chimeric antisense oligos while retaining an RNase H activating domain. Given the sensitivity of many 2′-O-modified RNA species (such as 2′OMe RNAs and locked nucleic acid bases) to exonuclease degradation, we recommend introducing phosphorothioate modification into the ASO sequence to confer stability (refer to the "Phosphorothioates and chimeric oligos" section above).
Substituting 5-methyl-dC for dC within CpG motifs can be advantageous. This substitution slightly elevates the Tm of the antisense oligo and concurrently diminishes the likelihood of an adverse immune response to Toll-like receptor 9 (TLR9) in vivo. For most antisense applications, standard desalt purification is recommended. However, when deploying oligos in live animals, higher purity may be necessary. In such instances, we advise employing HPLC purification coupled with Na+ salt exchange, followed by end-user ethanol precipitation of the antisense oligo. This approach helps mitigate potential toxicity arising from residual chemicals that may persist from the synthesis process.