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Scientific Fundamentals Explained

Using antisense technologies to modulate noncoding RNA function

Subfamilies of RNA are being increasingly recognized as having important roles in gene regulation. Some of these RNAs have similar lengths and splicing structures to mRNA, but do not encode proteins. Already thousands of these noncoding RNAs, which include long intergenic noncoding RNAs (lincRNAs), long noncoding RNAs (lncRNAs), and small nucleolar RNAs (snoRNAs), have been discov­ered, but relatively few have been function­ally characterized. Most noncoding RNAs are localized to the nucleus, limiting the use of RNAi for loss-of-function studies due to the low prevalence of the requisite enzymes in the nucleus [1]. However, RNase H, which is present in the nucleus, can be engaged to mediate RNA degradation, enabling the use of chemically modified antisense DNA oligonucleotides to downregulate targeted nuclear noncoding RNAs.

Antisense oligonucleotides

Antisense oligonucleotides (ASOs) are short, synthetic 14–22 nt oligonucleotides that lo­calize to the nucleus. They include phospho­rothioate (PS) linkages that confer nuclease resistance, thus enhancing intracellular stability. A PS bond is created by replacing one of the non-bridging oxygen atoms in the phosphate backbone of the oligo with a sulfur atom (Figure 1). Inclusion of PS bonds increases oligonucleotide half-life in human serum by up to 10 hours compared to approximately 1 hour for an unmodified oligonucleotide with the same nucleotide sequence [2]. However, the introduction of PS bonds lowers the binding affinity of the oligonucleotides and can cause cytotoxicity.

Figure 1. Phosphorothioate bond.


2’-O-alkyl groups increase antisense effectiveness

Further modifications to PS-modified oligos help to increase their binding affinity and reduce toxicity. A commonly used modifica­tion is introduction of 2’-O-methyl (2’OMe; Figure 2) RNA to create a “gapmer” ASO, a chimeric oligo comprising a DNA sequence with flanking 2’OMe nucleotides that help offset the reduction in binding affinity from the PS linkages. ASOs containing these 2’OMe modifications display enhanced nu­clease resistance in addition to lower toxicity and increased hybridization affinities.

Figure 2. 2'-O-Methyl RNA.


Enabling RNase H cleavage in antisense applications

RNase H, an endogenous enzyme found in all cells, specifically cleaves RNA linkages in a double-stranded RNA:DNA heteroduplex [3,4,5], making RNase H cleavage of the target the most effective mechanism for antisense activity. 2'OMe nucleotides are not recognized by RNase H; therefore, ASOs de­signed for RNase H–mediated degradation cannot be comprised entirely of 2'OMe nu­cleotides. For RNase H–mediated antisense activation, the gapmer ASO, comprising a central block of 10 DNA nucleotides flanked by blocks of 4–5 Tm-enhancing 2'OMe nucleotides [6] (Figure 3), is the favored design, allowing RNase H to cleave target RNA bound to the DNA core of the gapmer ASO.

Figure 3. Structure of IDT Standard Negative Control Gapmer ASO Sequence. A central core of phosphorothioate-modified DNA is flanked by 2’-O-methyl (2’OMe) modified RNA bases. [* = phosphorothioate linkage; m = 2’OMe modification.]

Designing effective antisense oligonucleotides

There are currently no predictive algorithms for ASO design as the inability to predict tar­get accessibility makes it difficult to develop such algorithms. Targets could be inacces­sible due to protein binding or inherent RNA secondary structure. IDT recommends empirical testing by performing an initial screen using 5–10 PS-modified, standard, desalted DNA oligos to establish target ac­cessibility. Once an accessible site is located, the PS-modified DNA ASO can be substi­tuted with a 2’OMe gapmer ASO to reduce toxicity and, importantly, increase Tm, which correlates with increased ASO potency.

RNase H–dependent antisense silencing using gapmer ASOs is a powerful tool for specific modulation of nuclear noncoding RNAs. The technique also holds promise as a therapeutic tool for correcting RNA gain-of-function effects in RNA-dominant diseases [7]. For help with designing your antisense experiments, contact IDT Technical Support at techsupport@idtdna.com.


  1. Guttman M and Rinn JL (2012) Modular regula­tory principles of large non-coding RNAs. Nature, 482:339–346.
  2. Campbell JM, Bacon TA, and Wickstrom E (1990) Oligodeoxynucleoside phosphorothioate stabili­ty in subcellular extracts, culture media, sera, and cerebrospinal fluid. J Biochem Biophys Methods, 20:259−267.
  3. 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.
  4. Eder PS and Walder JA (1991) Ribonuclease H from K562 human erythroleulemia cells. Purifica­tion, characterization, and substrate specificity. J Biol Chem, 266:6472−6479.
  5. Eder PS, Walder RY, and Walder JA (1993) Sub­strate specificity of human RNase H1 and its role in excision repair of ribose residues misincorpo­rated in DNA. Biochimie, 75:123−126.
  6. Lennox KA, Sabel JL, et al. (2006) Characteri­zation of modified antisense oligonucleotides in Xenopus laevis embryos. Oligonucleotides, 16(1):26–42.
  7. Wheeler TM, Leger AJ, et al. (2012) Targeting nuclear RNA for in vivo correction of myotonic dystrophy. Nature, 488:111–115.

Product focus

The Unafold Tool—a free, online, secondary structure prediction software. The program calculates the probability of single-stranded RNA or DNA folding, or hybridization between 2 single strands.

Access the UNAFold Tool here.

ASOs (antisense oligonucleotides)—short, synthetic 15–25mer oligonucleotides that localize to the nucleus. They are ordered as desalted DNA oligos, and include phospho¬rothioate (PS) linkages that confer nuclease resistance, thus enhancing intracellular stability. 2′-O-methyl RNA bases can be added in addition to the PS linkages to decrease toxicity and increase ASO efficacy.

Learn more about ASOs and order them from the Antisense Oligonucleotides product page of the IDT website.

DsiRNA (Dicer-substrate RNAs)—chemically synthesized 27mer duplex RNAs that have increased potency in RNAi experiments compared to traditional siRNAs.

Read additional details about DsiRNAs and order them from the DsiRNA product page of the IDT website.

Additional reading

A new renaissance for antisense in the era of lncRNA—Read about the importance of long, noncoding RNAs (lncRNAs), and the pros and cons of antisense oligonucleotides (ASO) and RNAi strategies for lncRNA knockdown.

Tips for successful lncRNA knockdown: Design, delivery, and analysis of antisense and RNAi reagents—IDT research scientist Kim Lennox has been optimizing effective lncRNA knockdown with antisense and RNAi reagents. Read her tips for successful lncRNA knockdown.

RNAi and DsiRNA: Pathway, mechanism, and design—An overview of the RNA interference (RNAi) pathway, its applications, and design considerations for gene silencing experiments.

Advantageous properties of Dicer-substrate interfering RNA (DsiRNA) enable improved RNAi-based gene silencing—Compare canonical siRNA designs to DsiRNAs for performance in siRNA processing and RISC assembly, and learn which lead to stronger silencing efficacy. 

Authors: Nicola Brookman-Amissah, PhD, is a Scientific Writer and Kim Lennox, MSc, is a Senior Research Assistant at IDT.

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