Antisense oligonucleotides
A chimeric oligo with a central window of 8–10 DNA residues flanked by 2′-modified wings, usually on a phosphorothioate backbone. The wings give affinity and nuclease resistance; the DNA gap makes the oligo/RNA duplex a substrate for endogenous RNase H1, which cuts the RNA and releases the oligo to act again. This is the format used for knockdown, and the one that reaches nuclear transcripts RNAi cannot.
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Gapmers
A chimeric oligo with a central window of 8–10 DNA residues flanked by 2′-modified wings, usually on a phosphorothioate backbone. The wings give affinity and nuclease resistance; the DNA gap makes the oligo/RNA duplex a substrate for endogenous RNase H1, which cuts the RNA and releases the oligo to act again. This is the format used for knockdown, and the one that reaches nuclear transcripts RNAi cannot.
How it differs from the standard method
- Needs about five contiguous deoxyribose residues to engage RNase H1; a uniformly 2′-modified oligo of the same sequence will bind its target and do nothing.
- Works in the nucleus, where RNase H1 is abundant, so pre-mRNAs, intron-retained transcripts and nuclear-retained lncRNAs are all accessible.
- One oligo destroys many transcript copies, so potency does not require stoichiometric occupancy.
- Affinity is a liability as well as an asset: LNA wings give single-digit nanomolar potency and promiscuous cleavage of imperfectly matched transcripts, the mechanism behind high-affinity gapmer hepatotoxicity.
- Typical design screens 10–20 positions along the transcript, because activity varies by orders of magnitude with site in a way that is still not predictable.
1One strand, and a mechanism that depends on its chemistry
An antisense oligonucleotide is a short single strand, usually 16–25 bases, complementary to a sequence in an RNA. It finds its site by Watson–Crick pairing, and then one of two quite different things happens, decided not by the sequence but by the chemistry of the backbone and sugars.
Gapmer
2′-modified wings for affinity and nuclease resistance, 8–10 DNA residues in the middle so RNase H1 recognises the hybrid. Works in nucleus and cytoplasm, catalytic in the sense that one oligo destroys many copies.
Steric block
Uniform 2′-O-methyl, 2′-MOE or a morpholino. No cleavage at all. Used to mask a splice site or a silencer, block an upstream open reading frame, or displace a protein from an RNA.
Phosphorothioate
One non-bridging oxygen replaced by sulphur. Buys nuclease resistance and avid protein binding, which is what lets the oligo enter cells and persist in tissue, and also what makes it toxic at high concentration.
2′ modifications
2′-O-methyl, 2′-MOE and LNA raise the melting temperature, so the oligo can be shorter and more potent. Higher affinity also means more duplexes with imperfect matches, which for a gapmer means more off-target cleavage.
2Chemistry is the subject, not a detail
An unmodified phosphodiester DNA oligo has a lifetime of minutes in serum and hours in a cell, so it cannot be used for anything. Replacing a backbone oxygen with sulphur gives a phosphorothioate, which is nuclease-resistant and, because it binds avidly to proteins, is taken up by cells from the medium with no transfection reagent at all: slowly, at micromolar concentrations, by a route that resembles what happens in an animal.
- Gapmer
- An oligo with a central DNA window flanked by 2′-modified wings, designed so that the hybrid it forms is an RNase H1 substrate. The standard format for knockdown.
- RNase H1
- An endogenous enzyme that cleaves the RNA strand of a DNA/RNA duplex. It needs about five contiguous deoxyribose residues to engage, and it is abundant in the nucleus.
- Phosphorothioate (PS)
- A backbone in which one non-bridging oxygen is replaced by sulphur. Gives nuclease resistance, protein binding, free cellular uptake and dose-limiting toxicity.
- 2′-MOE
- 2′-O-(2-methoxyethyl): the second-generation sugar modification. Raises affinity and stability, and is the chemistry in several approved drugs.
- LNA / BNA
- Locked (bridged) nucleic acid: a methylene bridge fixes the sugar pucker, giving the highest affinity per residue, and the highest risk of off-target RNase H cleavage.
- Morpholino (PMO)
- A morpholine ring and an uncharged phosphorodiamidate backbone. Completely nuclease-resistant, cannot recruit RNase H, and needs electroporation or a conjugate to get into cultured cells.
- Gymnosis
- Free uptake of a phosphorothioate oligo from the medium without any transfection reagent. Slow and inefficient, and the format that best predicts behaviour in tissue.
- Exon skipping / inclusion
- Changing whether an exon ends up in the mature message, by masking a splice site or a splicing silencer with a steric-block oligo.
3Getting it into the cell
Three routes, and they answer different questions. Cationic lipid gives nanomolar potency and a clean dose–response, but it bypasses the uptake pathway the oligo would actually use, so potency measured this way does not rank designs for in vivo work.
4How it fails
- A high-affinity oligo that binds beautifully and changes nothing. Almost always a chemistry that cannot recruit RNase H used for a knockdown experiment. Check that there is a contiguous DNA gap.
- No activity in free uptake at nanomolar concentrations. Gymnosis is 20–50 times less efficient than lipofection: it needs micromolar concentrations and 72–144 h, and the oligo has to carry a phosphorothioate backbone.
- A morpholino that works in an embryo and not in a dish. Uncharged oligos do not complex with cationic lipid. Electroporate, conjugate or scrape-load.
- Knockdown with dead cells. Phosphorothioate protein binding and promiscuous RNase H cleavage both scale with intracellular concentration. Find the lowest concentration that gives the knockdown and report the window.
- A splice switch that lowers the transcript rather than changing its isoform ratio. Either the oligo is a gapmer, or the assay cannot tell the two apart: a primer pair inside the targeted exon falls both when the exon is skipped and when the whole transcript is degraded.
- A dose that was never really the dose. Oligos ship as crude mass and as salts, so quantify by absorbance with the oligo's own extinction coefficient before trusting a nanomolar figure.
Key terms
Practise these as flashcards →A short synthetic single strand complementary to a target RNA, acting either by recruiting RNase H or by occupying a site on the RNA.
An oligo that cannot be cleaved and works by occupancy: masking a splice site, a splicing silencer, a start codon or a protein-binding site.
Using a steric-block oligo to change which exons are included in the mature message, the mechanism behind nusinersen and the exon-skipping oligos for Duchenne muscular dystrophy.
The stability of the oligo/RNA duplex. Sugar modifications raise it, which is how a shorter, more potent oligo is made, and how off-target duplexes become stable too.
RNase H cutting transcripts the gapmer matches imperfectly. Scales with affinity and with intracellular concentration, and is the mechanism behind high-affinity gapmer hepatotoxicity.
The gap between the concentration at which the oligo is active and the concentration at which its chemistry is toxic. The number that decides whether a design is worth pursuing.
A ligand attached to the oligo to direct uptake (GalNAc for hepatocytes, a cell-penetrating peptide for a morpholino) which can improve potency by one to two orders of magnitude.
Screening many oligos tiled along a transcript, because activity varies enormously with site and cannot be predicted reliably.
When to use it
- Depleting a nuclear-retained transcript (a long non-coding RNA, a pre-mRNA, an intron-retained species) where cytoplasmic RNAi machinery cannot reach.
- Changing splicing rather than abundance: skipping an exon that carries a premature stop, or forcing inclusion of one that is being left out.
- Working in cells or tissues that will not transfect, where a phosphorothioate oligo can be added to the medium and taken up unaided.
- Confirming an RNAi phenotype with an entirely different mechanism and a different set of off-targets, which is one of the strongest orthogonal controls available.
- Developing something that could become a drug: antisense chemistry, unlike a transfected duplex, is a format that already works in patients.
Compared with alternatives
The other way to lower a transcript. Prefer RNAi for a cytoplasmic mRNA in an easily transfected line; prefer a gapmer for nuclear-retained RNA, for splicing work, and as an orthogonal check on an RNAi phenotype.
Edits the gene rather than the transcript, so loss is complete and permanent. Prefer antisense when the gene is essential, when you want a dose–response, or when the point is the isoform rather than the gene.
How knockdown and total transcript are quantified. For splicing work it is not enough on its own: the assay has to span the junctions or it cannot tell skipping from degradation.
The delivery step for lipid-mediated work, and the method whose toxicity has to be subtracted. Free uptake avoids it entirely, which is why it is the format that predicts in vivo potency.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
A chimeric oligo with a central window of 8–10 DNA residues flanked by 2′-modified wings, usually on a phosphorothioate backbone. The wings give affinity and nuclease resistance; the DNA gap makes the oligo/RNA duplex a substrate for endogenous RNase H1, which cuts the RNA and releases the oligo to act again. This is the format used for knockdown, and the one that reaches nuclear transcripts RNAi cannot.
- Needs about five contiguous deoxyribose residues to engage RNase H1; a uniformly 2′-modified oligo of the same sequence will bind its target and do nothing.
- Works in the nucleus, where RNase H1 is abundant, so pre-mRNAs, intron-retained transcripts and nuclear-retained lncRNAs are all accessible.
- One oligo destroys many transcript copies, so potency does not require stoichiometric occupancy.
- Affinity is a liability as well as an asset: LNA wings give single-digit nanomolar potency and promiscuous cleavage of imperfectly matched transcripts, the mechanism behind high-affinity gapmer hepatotoxicity.
- Typical design screens 10–20 positions along the transcript, because activity varies by orders of magnitude with site in a way that is still not predictable.