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James E. Summerton, Ph.D.

[5] Antisense Oligos Compared

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Antisense oligos compared:

Structural basis for functional properties

Antisense oligos act to block the function of their targeted RNA transcripts. To be useful as research tools and therapeutics they need to be stable in biological systems for at least hours, but preferably days to weeks or longer. While short strands of DNA or RNA may appear to be the obvious choice for use as antisense oligos, because they are degraded by enzymes in the blood and within cells in a few minutes it is necessary to design into the antisense oligo’s structure a means to substantially slow, or preferably prevent, their enzymatic degradation.

The four antisense types shown below dominate the antisense therapeutics field. Drugs of three of these structural types ( S-DNAs, 2′-modified S-RNAs, and Morpholinos ) have been approved by the FDA for clinical use. Drugs of the fourth type, siRNAs, are now undergoing clinical trials.

Four antisense types

It is important to realize that the means by which resistance to enzymatic degradation is achieved in a given structural type strongly affects key functional properties, including: stability in biological systems; sequence specificity; targeting predictability; targeting versatility; and, confounding off-target effects. The strategies by which resistance to degradation is achieved in these antisense structural types, and the corresponding impact on their antisense function, is described below (reviewed in: Current Topics in Medicinal Chem Chemistry 7: 651-660 (2007)).

S-DNAs & 2′-mod. S-RNAs(phosphorothioates)
One popular way to achieve moderate stability (hours to days) is to replace the anionic oxygen with an anionic sulfur in some or all of the phosphodiester inter-subunit linkages in DNA and 2′-modified RNA antisense oligos – as shown below.

S-DNA and 2-mod S-RNA

The elements of phosphothioates (S-DNAs and 2′-modified S-RNAs) which make them moderately stable in biological systems are their anionic sulfurs which serve to slow enzymatic degradation. However, those sulfurs also cause strong binding to a wide range of proteins in the blood, on cell surfaces, and within cells. Binding to proteins in the blood does slow excretion of S-DNAs by the kidneys, but such protein binding is also responsible for multiple non-antisense effects that plague this structural type. A few of the confounding off-target effects common to phosphorothioates are:

  • activation of the complement cascade, which can cause convulsions and death within minutes;
  • CpG activation of the innate immune system, which can cause inadvertent interferon induction; and,
  • formation of G-quartet complexes, which can cause multiple effects that can be mistaken for true antisense effects.

The anionic sulfurs in the backbones of S-DNAs also substantially reduce the binding affinities of such antisense oligos for their targeted RNA transcripts. This reduced binding affinity limits the ability of S-DNAs to invade RNA secondary structures, which are ubiquitous in nearly all RNA transcripts. Because S-DNA cannot readily invade RNA secondary structures, it is difficult to reliably predict suitable target sequences for S-DNAs.

Because S-DNA oligos closely resemble DNA oligos, an S-DNA oligo paired to a complementary RNA transcript is recognized and cleaved by RNase H within cells. The problem this presents is that RNase H recognizes and cleaves paired S-DNA/RNA duplexes as short as 7 or 8 base-pairs. Thus, while an S-DNA antisense oligo can often recognize and mediate cleavage of its targeted sequence in a selected RNA transcript, that same S-DNA will also typically recognize and mediate cleavage of thousands of RNA sequences one does not wish to cleave and is generally unaware of having inadvertently cleaved. Such off-target effects can massively confound experimental results, and in a therapeutic context, they can result in severe and sometimes lethal adverse events.

2′-modified S-RNAs
Like S-DNAs, the anionic sulfurs of 2′-modified S-RNAs also bind to a wide range of proteins in the blood, on cell surfaces, and within cells. As with S-DNAs, such binding to proteins in the blood slows excretion of the antisense oligo by the kidneys, but also slows passage of the antisense oligo from the vascular bed to the extra-vascular interstitial space where most cells reside.

In contrast to the case for S-DNAs, the 2′-modified S-RNAs apparently bind proteins with a lower affinity than do S-DNAs and so cause fewer confounding off-target effects due to protein binding. Probably this lower-affinity binding to proteins is due to greater steric crowding around the anionic sulfurs of the backbone phosphates by the 2′-modifying group of the ribose.

2′-Modified S-RNAs have greater sequence specificity than S-DNAs because the 2′-modified S-RNAs do not mediate RNase H cleavage of duplexes formed between 2′-modified S-RNAs and their complementary and partially complementary sequences in RNA transcripts. As a consequence, 2′-modified S-RNAs are not plagued by the thousands of undesired RNase H-mediated cleavages of partially-complementary sequences that are inadvertently cleaved by most S-DNAs.

The main application of 2′-modified S-RNAs has been for altering splicing patterns of pre-mRNAs. They can do this because they do not mediate cleavage of their targeted transcript – which allows the splice-modified mRNA product to pass into the cytosol and undergo normal translation by ribosomes.


siRNAs (short-interfering RNAs)
A “natural” way to achieve moderate stability (hours to days) of a strand of RNA in biological systems is to pair two complementary RNA strands to form a short duplex siRNA structure with specially-designed overhanging ends. After delivery of that short duplex into a cell, the antisense strand of that duplex will associate with several proteins to form the RISC complex, while the sense RNA strand of the introduced duplex will be degraded. This is illustrated in the following figure.

That RISC complex then serves to position its component antisense strand on a complementary sequence of an RNA transcript.

If the RNA transcript is perfectly, or near-perfectly complementary to the antisense strand of the RISC complex then that transcript is generally cleaved by an associated nuclease component of the RISC complex – which leads to full degradation of that RNA transcript.

In addition, the RISC complex commonly also positions its component antisense strand on a variety of partially-complementary sequences of many different RNA transcripts (typically several hundred), which results in the function of those partially-complementary transcripts being blocked to varying degrees – but without cleavage of the involved transcripts. These confounding off-target blockages of partially-complementary transcripts by RISC complexes are difficult to predict, difficult to recognize, and the level of such blockages vary widely and unpredictably. Most notably, those many off-target blockages can become completely befuddling when a cocktail of siRNAs are targeted against multiple sequences in the same transcript – in an attempt to identify which effects are due to blockage of the actual intended target transcript.

It is noteworthy that this blockage-without-cleavage mode of action of siRNAs closely resembles the activity of natural microRNAs, where each microRNA commonly affects the function of hundreds of different transcripts having partial complementarity to the microRNA.

While inadvertent blocking of some RNA transcripts by this microRNA-type activity of siRNAs can be partially reduced by a massive informatics search of the transcriptome for each prospective target sequence, even with such a search there typically are still dozens to a hundred or more “non-targeted” RNA transcripts which will be blocked to some extent by any given siRNA. Alternatively, blocking of “non-targeted” RNA transcripts by microRNA-type activity of siRNAs can be reduced by lowering the concentration of the original siRNA – but this comes at the risk of reducing the potency of that siRNA to the point of failing to achieve the desired biological result.


Complete resistance to enzymatic degradation can be achieved by designing an antisense oligo with non-ionic linkages between the subunits. The most successful non-ionic antisense oligos are the Morpholinos, which have non-ionic phosphorodiamidate inter-subunit linkages instead of the anionic phosphodiester inter-subunit linkages of conventional antisense oligos and natural DNA and RNA. Morpholinos also have morpholine backbone rings instead of the conventional ribose or deoxyribose rings of natural nucleic acids. A short segment of a Morpholino oligo is shown below.

Key structural elements of Morpholinos are their non-ionic inter-subunit linkages and their novel morpholine backbone ring structures. Because proteins interact with nucleic acids in large part via ionic interactions, the lack of ionic charges on the Morpholino backbone avoids significant interactions with proteins. This lack of interaction provides complete resistance to degradative enzymes in blood and within cells.

The lack of interaction with proteins probably also accounts for why Morpholinos do not activate the complement cascade, are not involved in CpG-mediated activation of the innate immune system, do not induce interferon, and do not form biologically active G-quartet complexes – all of which are problems that often plague the more conventional poly-anionic antisense oligos.

Their lack of ionic charge probably also accounts for why Morpholinos freely pass between the cytosol and nucleus of the cell – which allows their versatile use for both splice modification in the nucleus and translational blocking in the cytosol. 

Because Morpholinos are not dependent on cellular machinery (such as RNase H for S-DNAs, or the RISC complex for siRNAs) in order to achieve blocking of their targeted RNA transcripts, Morpholino antisense activity tends to be fast, straight-forward, and reliable.

Also because their antisense activity is independent of cellular machinery, and only a function of their high affinity for complementary RNAs, they are quite effective for invading RNA secondary structures. That facile invasion of RNA secondary structures results in the Morpholinos’ unmatched targeting predictability.

Finally, an unusually long run of 14 to 15 contiguous bases of a Morpholino must bind to a complementary sequence in its targeted RNA transcript in order for the Morpholino to achieve its antisense effect. Because of this stringent high-information binding requirement, Morpholinos provide by far the highest sequence specificity of all antisense types. 

Morpholinos’ exquisite sequence specificity, combined with their other exceptional properties, have made them the preferred antisense tools for use in the most demanding of all antisense applications – the study of developing embryos wherein intricate cascades of gene activations and deactivations are precisely controlled with respect to both time and position in the rapidly maturing embryo. Since the year 2000 Morpholinos have been the essential tools for most researchers in developmental biology, and their use has revolutionized that very technically demanding field. As of 2017 scientists have published over 8,400 research papers wherein custom-sequence Morpholinos were their key tools (searchable at: ), with over 5,000 of those publications being in the very challenging field of developmental biology.


The following table provides a qualitative comparison of the four dominant antisense structural types in regard to properties needed for high-specificity and long-duration therapeutic activity.

Property S-DNA 2′-mod S-RNA siRNA Morpholino
Stability in blood & cells limited limited limited Complete
Sequence specificity lowest medium medium Highest
Predictable targeting lowest medium medium Highest
Alter splicing no Yes no Yes
Block translation Yes Yes Yes Yes
General lack of off-target effects no no no Yes