Antisense oligonucleotides

Antisense oligonucleotides

We offer chemically synthesized and modified antisense oligonucleotides (ASOs) at different scales with different purification options in tube or plate formats. ASOs are ideal for inhibiting gene expression in vitro, in cellulo, and in vivo studies.

Custom ASOs can include non-standard lengths or modifications, as well as, designs for splice switching or steric blocking.

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Product details

Antisense oligonucleotides (ASOs) are DNA oligos, typically 15–22 bases long designed in antisense orientation to the RNA of interest. Hybridization of the ASO to the target RNA triggers RNase H cleavage of the RNA, which can inhibit the function of non-coding RNAs (e.g., miRNAs, siRNAs, piRNAs, snoRNAs, snRNAs, exRNAs, scaRNAs and lncRNAs) or prevent protein translation of mRNAs. To increase nuclease resistance, we recommend adding phosphorothioate (PS) modifications to the oligo. In the IDT ordering system, use an asterisk to indicate the position of a phosphorothioate internucleoside linkage. Also consider adding modified bases, such as 2′-O-methyl (2′-OMe) or Affinity Plus™ locked nucleic acid bases, in chimeric antisense designs to increase nuclease stability and affinity (T_m) of the antisense oligo to the target mRNA [1]. Substitution of 5-methyl dC for dC in CpG motifs will slightly increase the T_m of the antisense oligo.

* = Phosphorothioate bonds
M = 2'MOE (2'-O-methoxyethyl base) ‡
m = 2'OMe (2'-O-methyl base)
+ = Affinity Plus (locked nucleic acid base)

^‡ IDT's unique 2'MOE modification codes can be found here.

Technical overview

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.

Phosphorothioates and chimeric oligos

Chemically modified RNA and DNA bases for chimeric antisense designs

State-of-the-art antisense design employs chimeras with both DNA and modified RNA bases [1]. The use of modified RNA, such as 2′-O-methoxy-ethyl (2′-MOE) RNA, 2′-O-methyl (2′OMe) RNA, or Affinity Plus locked nucleic acid bases in chimeric antisense designs, increases both nuclease stability and affinity (T_m) of the antisense oligo to the target RNA [4–6]. However, these modifications do not activate RNase H cleavage. The preferred antisense strategy is a "gapmer" design which incorporates 2′-O-modified RNA or Affinity Plus locked nucleic acid bases in chimeric antisense oligos that retain an RNase H activating domain. As many 2′-O-modified RNA (such as 2′OMe RNAs and Affinity Plus locked nucleic acid bases) are sensitive to exonuclease degradation, we recommend phosphorothioate modification of the ASO sequence to provide stability (See the "Phosphorothioates and chimeric oligos" section above).

It can be beneficial to substitute 5-methyl-dC for dC in the context of CpG motifs. Substitution of 5-methyl dC for dC will slightly increase the T_m of the antisense oligo. The use of 5-methyl dC in CpG motifs can also reduce the chance of adverse immune response to Toll-like receptor 9 (TLR9) in vivo. We recommend standard desalt purification for most antisense applications. For use in live animals, higher purity oligos may be required. In these instances, HPLC purification combined with Na⁺ salt exchange followed by end-user ethanol precipitation of the antisense oligo is recommended to mitigate toxicity from residual chemicals that may carry over during synthesis.

Product data

Antisense oligonucleotides with different chemical modifications (2'OMe, 2'MOE and Affinity Plus) exhibit different levels of inhibition of gene expression

Figure 2 demonstrates the differential inhibition effect of three different modifications on seven different targeted sites of the CTNNB1 gene. Affinity Plus ASOs show the highest inhibition among the three modifications, followed by 2’MOE and 2’OME ASOs, respectively. Whereas the Affinity Plus gapmer ASOs show the highest level of inhibition, we recommend screening more sites to mitigate possible toxicity.

Frequently asked questions