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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. 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

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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 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

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. It is becoming increasingly popular to confirm phenotypes seen using 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 and facilitates productive cellular uptake.

2′-O-methoxy-ethyl (MOE), Affinity Plus Locked Nucleic Acid, 2′-O-methyl RNA, and 5-methyl dC

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 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 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.

Technical reports

Frequently asked questions

What types of modifications are routinely used in antisense experiments?

Phosphothioate bonds are added to antisense oligos to protect them from nuclease degradation. Antisense oligos can include a full phosphothioate backbone; however, the Tm decreases with each phosphothioate bond added. This effect can be decreased by 'capping' the oligo—including 2–5 phosphothioate bonds to each end of your oligo rather than fully thioating the oligo backbone.

5-Me dC and 2' O-Me RNA bases and C5-propyne pyrimidine modifications are sometimes added to increase nuclease resistance and binding affinity of antisense oligos, but these modifications are not critical. You can start your experiments without them and add them to subsequent oligos if you require increased stability. Antisense oligos are generally 18–24 bases in length. The concentrations used will need to be determined on a case-by-case basis as they vary across organisms. Usually using 2µM (10 µg/mL) antisense oligo is a good starting point. For more information on antisense oligo design and usage, see the technical report, Introducing antisense oligonucleotides into cells.


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