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Antisense oligonucleotides

Antisense oligonucleotides (ASOs) are short oligonucleotides that localize to the nucleus and provide a pathway for gene silencing by the RNase H pathway. They are usually single stranded nucleic acids designed to be complementary to a target messenger RNA (mRNA). Phosphorothioate (PS) linkages are available to confer nuclease resistance and, therefore, enhance intracellular stability.


  • Achieve effective inhibition of gene expression in vitro or in vivo
  • Target RNA in the nucleus by using oligos with enhanced intracellular stability
  • Reduce toxicity and artifacts with flexible chimeric designs and useful modifications

Antisense oligonucleotides

Delivered in tubes, dry or resuspended to your specifications.


Product details

Antisense oligonucleotides (ASOs) are DNA oligos, typically 15–25 bases long, designed in antisense orientation to the RNA of interest. Hybridization of the ASO to the target RNA mediates 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. Consider adding modified bases, such as 2′-O-methoxy-ethyl (2′-MOE) or Affinity Plus locked nucleic acid bases, in chimeric antisense designs to increase nuclease stability and affinity (Tm) of the antisense oligo to the target mRNA [1]. Substitution of 5-methyl dC for dC in CpG motifs will slightly increase the Tm of the antisense oligo.

Examples of RNase H active antisense oligos
5′ T*C*C*T*G*C*G*A*A*A*T*G*T*C*C*A*T*C*G*T 3′
5′ /52MOErT/*/i2MOErC/*/i2MOErC/*/i2MOErT/*/i2MOErG/*C*G*A*A*A*T*G*T*C*C* /i2MOErA/*/i2MOErT/*/i2MOErC/*/i2MOErG/*/32MOErT/ 3′2′MOE/DNA, chimera, All PS
5' +C*+T*+G*C*G*A*A*A*T*G*T*C*C*+A*+T*+C 3′
Affinity Plus/DNA chimera, All PS
5′ mU*mC*mC*mU*mG*C*G*A*A*A*T*G*T*C*C*mA*mU*mC*mG*mU 3′2′OMe/DNA chimera, All PS

* = Phosphorothioate bonds
2MOE =O-methoxy-ethyl (MOE) base
+N = Affinity Plus locked nucleic acid base
mN = 2′-O-methyl RNA base


If you require oligos for use in molecular diagnostic applications, or if you are interested in our OEM services, click here.

Product data

Antisense oligonucleotides (ASOs) are used to inhibit gene expression levels both in vitro and in vivo. Recent improvements in design and chemistry of antisense compounds have enabled this technology to become a routine tool in basic research, genomics, target validation, and drug discovery to confirm phenotypes seen with RNAi by gene silencing ASOs. A nucleic acid sequence—made as a synthetic oligonucleotide (usually 15–25 bases long) containing a phosphorothioate-modified DNA segment of at least 6 bases—is designed in antisense orientation to the RNA of interest. The sequence is then introduced into the cell or organism. The ASO will bind the target RNA and 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.

Figure 1. Antisense oligo–mediated cleavage of the target by RNase H.

Phosphorothioates and chimeric oligos

While unmodified oligodeoxynucleotides can display some antisense activity, they are subject to rapid degradation by endo- and exo-nucleases. Many 2′-O-modified RNA (such as 2′OMe RNAs and Affinity Plus locked nucleic acid bases) are sensitive to exonuclease degradation, as well. The simplest and most widely used nuclease-resistant chemistry available for antisense applications is the phosphorothioate (PS) modification. In phosphorothioates, a sulfur atom replaces a non-bridging oxygen in the oligo phosphate backbone. In the IDT ordering system, an asterisk indicates the presence of a phosphorothioate internucleoside linkage. PS oligos can show greater non-specific protein binding than unmodified phosphodiester (PO) oligos, which can cause toxicity or other artifacts when present at high concentrations.

We recommend phosphorothioate modification of ASO sequences to provide stability. Phosphorothioate linkages also promote binding to serum proteins, which increases the bioavailability of the ASO while facilitating productive cellular uptake.

Phosphorothioate linker

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 (Tm) 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 d 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 Tm of the antisense oligo. 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.


Frequently asked questions


  1. Lennox KA, Behlke, MA. Mini-review on current strategies to knockdown long non-coding RNAs. J Rare Dis Res Treat 2016; 1 (3): 66–70.
  2. Walder RY, Walder JA. Role of RNase H in hybrid-arrested translation by antisense oligonucleotides. Proc Natl Acad Sci USA. 1988;85(14):5011–5015. doi:10.1073/pnas.85.14.5011
  3. Dagle JM, Walder JA, Weeks DL. Targeted degradation of mRNA in Xenopus oocytes and embryos directed by modified oligonucleotides: studies of An2 and cyclin in embryogenesis. Nucleic Acids Res.1990;18(16): 4751–4757. doi:10.1093/nar/18.16.4751
  4. Braasch DA, Liu Y, Corey DR. Antisense inhibition of gene expression in cells by oligonucleotides incorporating locked nucleic acids: effect of mRNA target sequence and chimera design. Nucleic Acids Res. 2002;30(23):5160–5167. doi:10.1093/nar/gkf651
  5. Kurreck J, Wyszko E, Gillen C, Erdmann VA. Design of antisense oligos stabilized by locked nucleic acidsNucleic Acids Res. 2002;30(9):1911-1918. doi:10.1093/nar/30.9.1911
  6. Grünweller A, Wyszko E, Bieber B, Jahnel R, Erdmann VA, Kurreck J. Comparison of different antisense strategies in mammalian cells using locked nucleic acids, 2'-O-methyl RNA, phosphorothioates and small interfering RNA. Nucleic Acids Res. 2003;31(12):3185-3193. doi:10.1093/nar/gkg409

*RUO—For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations.