functional_genomics

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

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Antisense oligonucleotides

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Antisense oligonucleotides (ASOs) are DNA oligos, typically 15–25 bases long, designed in antisense orientation to the mRNA of interest. 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. 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. 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′
DNA, 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
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/3Phos/3′
Affinity Plus/DNA chimera, PS/PO chimera, 3′-phos

* = Phosphorothioate bonds
mN = 2′-O-methyl RNA base 
+N = Affinity Plus Locked Nucleic Acid base

For assistance, contact euapplicationsupport@idtdna.com.

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 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 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 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/Affinity Plus Locked Nucleic Acid 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.

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. 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 mRNA [3–5]. 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 unmodified DNA is susceptible to rapid degradation by endo- and exo-nucleases and 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 antisense oligo to provide stability. Phosphorothioate linkages also promote binding to serum proteins which increases the bioavailability of the antisense oligo and facilitates productive cellular uptake.

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.

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