Antisense Oligonucleotides

Pathway for Gene Silencing by RNase H-Active Antisense Oligonucleotides

Antisense oligonucleotides (ASOs) are short, synthetic 15–25 nt oligonucleotides that localize to the nucleus. They include phosphorothioate (PS) linkages that confer nuclease resistance, thus enhancing intracellular stability.

IDT is exclusively licensed under University of Iowa Research Foundation patents 6,197,944 to manufacture and sell Chimeric Antisense oligos. For further information, see Licenses.

Description100 nmole DNA Oligo250 nmole DNA oligo1 umole DNA Oligo5 umole DNA Oligo10 umole DNA Oligo
Phosphorothioate Bond$3.50 USD$3.50 USD$5.00 USD$20.00 USD$30.00 USD
DNA bases$0.60 USD$1.00 USD$2.10 USD$11.50 USD$20.00 USD
2' O-Methyl RNA bases$9.50 USD$14.50 USD$22.00 USD$85.00 USD$160.00 USD
5' 5-Methyl dC$50.00 USD$60.00 USD$90.00 USD$180.00 USD$360.00 USD
HPLC$42.00 USD$69.00 USD$105.00 USD$240.00 USD$370.00 USD
Na+ Salt Exchange$75.00 USD$75.00 USD$75.00 USD$225.00 USD$225.00 USD

Antisense oligonucleotides have been used for over twenty-five years 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 antisense DNA oligos. A nucleic acid sequence, usually 15–25 bases long, is designed in antisense orientation to the mRNA of interest; the sequence is made as a synthetic oligonucleotide and is introduced into the cell or organism. Hybridization of the antisense oligo to the target mRNA results in RNase H cleavage of the message which prevents protein translation and thereby blocks gene expression. Antisense oligonucleotides containing a native DNA or phosphorothioate-modified DNA segment of at least six bases long will bind the target mRNA and form an RNA/DNA heteroduplex, which is a substrate for endogenous cellular RNases H [1–2]. The decrease in mRNA levels can be measured using real-time PCR.

Figure 1. Antisense Mediated Cleavage of the Targeted Message by RNase H.

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. These problems seem to be worst with PS-DNA and PS-DNA/LNA chimeras. Non-specific protein binding of PS oligos can be minimized by making the oligonucleotide as short as possible (thereby reducing PS content) or using chimeric designs with 2'-O-methyl RNA.

LNA, 2′-O-Methyl RNA, and 5-Methyl dC

State-of-the-art antisense design employs chimeras having both DNA and modified-RNA bases. The use of modified RNA, such as 2′-O-methyl (2'OMe) RNA or LNAs (Exiqon) in chimeric antisense designs, increases both nuclease stability and affinity (Tm) of the antisense oligo to the target mRNA [3–5]. These modifications, however, do not activate RNase H cleavage. The preferred antisense design incorporates 2′-O-modified RNA or LNA in chimeric antisense oligos that retain an RNase H activating domain of DNA (or phosphorothioate DNA). As LNA bases confer significant nuclease resistance, we recommend phosphorothioate modification of only the DNA gap, leaving the LNA flanks as phosphodiester linkages in chimeric LNA antisense oligos. For synthesis reasons, a 3′-phosphate is preferred when an LNA base is at the 3′ end.

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 responses in vivo. IDT recommends that all antisense oligos receive HPLC purification and that oligos undergo a Na+ salt exchange before use in cells or live animals to ensure that salts used in purification are removed. 

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′ 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
5' +T+C+C+T+GC*G*A*A*A*T*G*T*C*C+A+T+C+G+T/3Phos/3′
LNA/DNA chimera, PS/PO chimera, 3′-phos

* = Phosphorothioate Bonds

mN = 2′-O-Me RNA base 

+N = LNA base


  1. 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.
  2. 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.
  3. 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.
  4. Kurreck J, Wyszko E, et al. (2002) Design of antisense oligos stabilized by locked nucleic acids. Nucleic Acids Res, 30:1911–1918.
  5. 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.