ASO Chemistry Comparison Chart
Complete reference for backbone modifications, sugar chemistries, ASO architectures, and mechanism matching — all in one place.
Backbone Modifications
Phosphodiester (PO)
Natural DNA/RNA
Advantages
Considerations
FDA examples: Research reagents only
Phosphorothioate (PS)
Sulfur replaces non-bridging oxygen
Advantages
Considerations
FDA examples: Nusinersen, Inotersen, Mipomersen
Morpholino (PMO)
Morpholine ring + neutral backbone
Advantages
Considerations
FDA examples: Eteplirsen, Golodirsen, Casimersen
Sugar Modifications (2' Position)
Modifications at the 2' position increase binding affinity and nuclease resistance. 2'-modified nucleotides are not compatible with RNase H — used in wings only for gapmers.
| Modification | Full Name | Tm Boost | RNase H | Nuclease Resistance | Notes |
|---|---|---|---|---|---|
| 2'-H (DNA) | Deoxyribose | Baseline | ✓ Yes | Low | Used in gapmer central region |
| 2'-OMe | 2'-O-Methyl | +1°C/nt | ✗ No | Moderate | Cost-effective; moderate affinity |
| 2'-MOE | 2'-O-Methoxyethyl | +1-2°C/nt | ✗ No | High | Gold standard; FDA-validated |
| 2'-F | 2'-Fluoro | +1.5°C/nt | ✗ No | High | Often combined with 2'-OMe |
| LNA | Locked Nucleic Acid | +3-6°C/nt | ✗ No | Very High | Highest affinity; hepatotoxicity risk |
| cEt | Constrained Ethyl | +3-5°C/nt | ✗ No | Very High | Improved safety vs LNA |
ASO Architectures
Gapmer
RNase H-Mediated KnockdownModified wings protect from degradation and increase affinity. The central DNA gap recruits RNase H to cleave the target RNA.
Typical design: 5-10-5 or 3-10-3 (wing-gap-wing)
FDA examples: Inotersen (TTR), Mipomersen (ApoB), Tofersen (SOD1)
Mixmer
Steric Blocking (Splice Modulation)Alternating pattern prevents RNase H recruitment. Works by steric hindrance — physically blocking cellular machinery.
Typical design: Alternating modified/unmodified
FDA examples: Nusinersen (SMN2 splice switch)
Fully Modified (Uniform)
Steric Blocking with Maximum StabilityMaximum nuclease resistance. Purely steric mechanism. Often used with PS backbone.
Typical design: All positions carry the same 2' modification
FDA examples: Splice modulation, miRNA inhibition
Mechanism → Chemistry Matching
Quick reference for choosing the right architecture and chemistry based on your therapeutic goal.
| Therapeutic Goal | Architecture | Backbone | Wings / Sugar | Gap |
|---|---|---|---|---|
| mRNA Knockdown | Gapmer | PS | 2'-MOE o cEt | DNA |
| Exon Skipping | Mixmer o Uniform | PS o PMO | LNA/MOE mix | None |
| Exon Inclusion | Mixmer o Uniform | PS | 2'-MOE | None |
| Translation Block | Uniform o Mixmer | PS | 2'-MOE | None |
| miRNA Inhibition | Uniform (anti-miR) | PS | LNA | None |
FDA-Approved ASO Drugs
| Drug | Target | Mechanism | Chemistry | Route | Year |
|---|---|---|---|---|---|
| Nusinersen (Spinraza) | SMN2 | Splice inclusion | 2'-MOE PS uniform | IT | 2016 |
| Eteplirsen (Exondys 51) | DMD exon 51 | Exon skipping | PMO | IV | 2016 |
| Inotersen (Tegsedi) | TTR | RNase H knockdown | 2'-MOE PS gapmer | SC | 2018 |
| Golodirsen (Vyondys 53) | DMD exon 53 | Exon skipping | PMO | IV | 2019 |
| Casimersen (Amondys 45) | DMD exon 45 | Exon skipping | PMO | IV | 2021 |
| Tofersen (Qalsody) | SOD1 | RNase H knockdown | 2'-MOE PS gapmer | IT | 2023 |
Decision Tree: Choosing the Right Chemistry
Reduce target mRNA/protein levels?
→ Use GAPMER (MOE-DNA-MOE or cEt-DNA-cEt with PS backbone)
Modulate splicing (skip or include exon)?
→ CNS target → 2'-MOE PS uniform (Nusinersen-like) · Muscle target → PMO (Eteplirsen-like)
Block translation without degradation?
→ Use UNIFORM 2'-MOE or MIXMER design
Inhibit microRNA?
→ Use ANTI-MIR design (LNA-containing, short)
References
1. Crooke ST et al. (2021) Nat Rev Drug Discov 20:427-453. PMID: 33762737
2. Khvorova A, Watts JK. (2017) Nat Biotechnol 35:238-248. PMID: 28244990
3. Shen X, Bhattacharya D. (2019) Nucleic Acid Ther 29:141-152. PMID: 31329511
4. Hagedorn PH et al. (2018) Drug Discov Today 23:101-114. PMID: 28890197
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