Antisense oligonucleotides

Antisense oligonucleotides (ASOs) are short oligonucleotides that enable gene silencing by the RNase H pathway. Chemical modifications to the ASOs enhance binding affinity and potency, while also helping to mitigate DNA toxicity.  The following standard modifications are available for ordering:

  • 2’-O-Methoxyethyl (2’MOE)—increases binding affinity, providing high potency and nuclease resistance
  • Affinity Plus—locked nucleic acids that confer the highest binding affinity modification available
  • 2’-O-Methyl—provides resistance to endonucleases as well as increase binding affinity
  • %-methyl dC—used to prevent or limit undesired immune responses when substituted into DNA CpG motifs, especially when the ASOs are to be administered in vivo

Phosphorothioate (PS) linkages are available to confer nuclease resistance and promote nuclear uptake.

  • 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.


ProductPhosphorothioate BondDNA bases2' O-Methyl RNA bases5' 5-Methyl dCHPLCNa+ Salt Exchange
100 nmole DNA Oligo€ 2,50 EUR€ 0,46 EUR / base€ 6,50 EUR€ 38,00 EUR€ 32,00 EUR€ 57,00 EUR
250 nmole DNA oligo€ 2,50 EUR€ 0,85 EUR / base€ 11,00 EUR€ 45,00 EUR€ 50,00 EUR€ 57,00 EUR
1 umole DNA Oligo€ 3,50 EUR€ 1,95 EUR / base€ 15,50 EUR€ 68,00 EUR€ 75,00 EUR€ 57,00 EUR
5 umole DNA Oligo€ 15,00 EUR€ 8,00 EUR / base€ 60,00 EUR€ 135,00 EUR€ 185,00 EUR€ 170,00 EUR
10 umole DNA Oligo€ 21,00 EUR€ 13,50 EUR / base€ 110,00 EUR€ 270,00 EUR€ 275,00 EUR€ 170,00 EUR

Antisense oligonucleotides (ASOs) are DNA oligos, typically 15–25 bases long, designed in antisense orientation to the RNA of interest. Hybridization of the antisense oligo to the target RNA results in RNase H cleavage of the RNA. Targeting non-coding (ncRNA) thus prevents its action, and targeting mRNA prevents protein translation, thereby blocking gene expression. To increase nuclease resistance, we recommend adding phosphorothioate (PS) modifications in oligo [1]. In the IDT ordering system, use an asterisk to indicate the the position of a phosphorothioate internucleoside linkage. Consider adding modified bases, such as 2′-O- methoxy-ethyl (2′-MOE), or locked nucleic acids such as Affinity Plus bases, in chimeric antisense designs to increase nuclease stability and affinity (Tm) of the antisense oligo to the target RNA. Substitution of 5-Methyl dC for dC will in CpG motifs will slightly increase the Tm of the antisense oligo [1].

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′
DNA, all PS
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' +T+C+C+T+GC*G*A*A*A*T*G*T*C*C+A+T+C+G+T 3′
Affinity Plus/DNA PS/PO chimera
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

2MOErN = MOE RNA base

+N = Affinity Plus base

mN = 2′-O-Me RNA base

For assistance, contact

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 routinely used tool in basic research, genomics, target validation, and drug discovery. A nucleic acid sequence, usually 15–25 bases long, is designed in antisense orientation to the RNA of interest; the sequence is made as a synthetic oligonucleotide and is introduced into the cell or organism. ASOs containing a phosphorothioate-modified DNA segment of at least 6 bases long will bind the target RNA and form an RNA/DNA heteroduplex, which is a substrate for endogenous cellular RNase H [2–3]. RNase H cleavage of the target RNA can inhibit the fuction of non-coding RNAs or prevent protein translation. The decrease in RNA levels can be measured using RT-qPCR or RNA-seq.

Figure 1. Antisense oligo (ASO)–mediated cleavage of a mRNA target by RNase H. ASOs can also be used to target non-coding RNAs (ncRNAs).

Phosphorothioates and chimeric oligos

While unmodified oligodeoxynucleotides can display some antisense activity, they are subject to rapid degradation by nucleases and are therefore of limited utility. 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.

Phosphorothioate linkages also promote binding to serum proteins. This increases the bioavailability of the ASO and facilitates productive cellular uptake.

2′-O- methoyx-ethyl (MOE), Affinity Plus, 2′-O-methyl RNA, and 5-methyl dC

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

It can also 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.


  1. Lennox K, Behlke MA (2016) Mini-review on curernt strategies to knockdown long non-coding RNAs. J rare Dis Res Treat. 1(3):66–70.
  2. Walder RY and Walder JA (1988) Role of RNase H in hybrid-arrested translation by antisense oligonucleotides. Proc Natl Acad Sci USA, 85:5011–5015.
  3. Dagle JM, Walder JA, and Weeks DL (1990) Targeted degradation of mRNA in Xenopus oocytes and embryos directed by modified oligonucleotides: studies of An2 and cyclin in embryogenesis. Nucleic Acids Res, 18:4751–4757.
  4. Braasch DA, Liu Y, and Corey DR (2002) Antisense inhibition of gene expression in cells by oligonucleotides incorporating locked nucleic acids: effect of mRNA target sequence and chimera design. Nucleic Acids Res, 30:5160–5167.
  5. Kurreck J, Wyszko E, et al. (2002) Design of antisense oligos stabilized by locked nucleic acids. Nucleic Acids Res, 30:1911–1918.
  6. Grunweller A, Wyszko E, et al. (2003) Comparison of different antisense strategies in mammalian cells using locked nucleic acids, 2'-O-methyl RNA, phosphorothioates and small interfering RNA. Nucleic Acids Res, 31:3185–3193.

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