Using antisense technologies to modulate noncoding RNA function

Families of noncoding RNA

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.

Using antisense oligonucleotides to regulate noncoding RNAs

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 T_m, 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 our application support scientists at applicationsupport@idtdna.com.