Naked oligonucleotides are inherently prone to degradation and exhibit poor drug-like properties. The chemical modification is now one of the most promising ways to make oligonucleotide-based drugs deliver, maintain, and perform. The sugar moiety or the phosphate backbone is changed to improve metabolism and function, increase affinity for protein binding, and delay renal excretion. All the changes confer distinct properties on oligonucleotides, some combinations of which provide customised therapeutics.
First-Generation Modifications: Phosphorothioate Backbone
First-generation oligonucleotide changes mainly addressed the phosphate backbone. Adding a non-bridging oxygen atom instead of a sulphur one gives rise to phosphorothioate (PS) oligonucleotides that are resistant to endonucleases and are more bioavailable by suppressing renal clearance. But those modifications usually decrease target affinity and are associated with increased toxicity. However, despite these restrictions, the PS backbone has become the bedrock of therapeutic oligonucleotides and allowed for FDA-approved medicines such as Fomivirsen.
Second-Generation Modifications: 2'-Sugar Derivatives
Second-generation modifications focus on ribose sugar. Changes at the 2' end - for example, 2'-O-methyl (2'-OMe) and 2'-fluoro (2'-F) - make them more binding-specific to target RNA and also more resistant to nucleases. Conformationally restricted derivatives, including locked nucleic acids (LNA) and tricyclo-DNA (tcDNA), impart rigidity. LNAs are built with a methylene bridge between 2' and 4' carbons, and tcDNAs a cyclopropane ring. These modifications are excellent for binding and thermal stability and are useful in RNA therapeutics.
Third-Generation Modifications: Nucleobase and Backbone Innovations
Advanced nucleotide modifications are about nucleobases and backbones. Phosphorodiamidate morpholino oligomers (PMOs) and peptide nucleic acids (PNAs), for example, dispense with the sugar-phosphate backbone in favour of uncharged units. PMOs have a morpholine ring; PNAs have amide bases. These advances increase nuclease resistance and binding specificity without triggering immune activation. PMO therapies such as Eteplirsen for Duchenne muscular dystrophy, approved by the FDA, point to their clinical promise.
Figure 1. Chemical modifications of oligonucleotides[1].
Chemical modifications in ASOs aim to improve stability, efficacy, and delivery. Common strategies include replacing the phosphodiester (PO) backbone with PS linkages, modifying the 2'-ribose position, and incorporating artificial scaffolds such as PMOs and PNAs. PS linkages extend circulation time and enhance serum stability, while 2' modifications, such as 2'-OMe and 2'-MOE, significantly improve binding affinity and nuclease resistance. Additionally, introducing 5-methyl cytosine reduces immunostimulatory effects and enhances stability, exemplified by FDA-approved drugs like Nusinersen and Mipomersen.
Figure 2. The main chemically modified antisense oligonucleotides[2].
Small interfering RNAs (siRNAs) require chemical stabilization to function as effective therapeutics. Modifications at the 2'-ribose position, such as 2'-F and 2'-OMe, enhance nuclease resistance without impairing RNA-induced silencing complex (RISC) assembly. Incorporating PS linkages at terminal nucleotides further improves stability. However, siRNAs demand sophisticated delivery strategies due to limited cellular uptake. Conjugating siRNAs with N-acetylgalactosamine (GalNAc) enables targeted delivery to hepatocytes, leading to FDA-approved treatments for liver-related diseases, including Inclisiran and Vutrisiran.
Figure 3. siRNA chemical modification introduced in the 3' and 5' end[3].
Table 1: Different chemical modifications used in approved oligonucleotide therapy[4].
| Chemical Modification | Drug | Oligonucleotide Class | Indication | Advantages and Disadvantages of Modification |
| PS (DNA) | Fomivirsen | ASO | CMV retinitis (1998 withdrawn) | PS backbone modification:
|
| 2'-OMe/2'-F, PS | Givosiran | siRNA | Acute hepatic porphyria (2019) | 2'-Sugar modification:
|
| Lumasiran | siRNA | Primary hyperoxluria (2020) | ||
| Inclisiran | siRNA | Heterozygous familial hypercholesterolemia (HeFH) or clinical atherosclerotic cardiovascular disease (ASCVD) (2020, EMA), (2021, FDA) | ||
| Vutrisiran | siRNA | Hereditary ATTR (hATTR) (2022) | ||
| 2'-F/2'-OMe | Pegaptanib | Aptamer | Neovascular AMD (2004, withdrawn) | |
| 2'-OMe | Patisiran | siRNA | Hereditary ATTR (hATTR) (2018) | |
| 5'-Me-C, PS, 2'-O-MOE, | Mipomersen | ASO | Homozygous familial hypercholesterolemia (HoFH) (2013 withdrawn) | 5'-Me-C nucleobase modification:
|
| Nusinersen | SSO | Spinal muscular atrophy (SMA) (2016) | ||
| Volanesorsen | ASO | Familial chlylomicronemia syndrome (FCS) (2019) | ||
| Inotersen | ASO | Hereditary ATTR (hATTR) (2018) | ||
| Tofersen | ASO | Amyotrophic lateral sclerosis (ALS) (2023) | ||
| PMO | Eteplirsen | SSO | Duchene muscular dystrophy (DMD) (2016) | PMO bracket modification:
|
| Golodirsen | SSO | Duchene muscular dystrophy (DMD) (2019) | ||
| Viltolarsen | SSO | Duchene muscular dystrophy (DMD) (2020) | ||
| Casimersen | SSO | Duchene muscular dystrophy (DMD) (2021) |
Modifications to a chemical structure are part and parcel of oligonucleotide therapy. First-generation backbone evolution provided the scaffolding for greater stability and bioavailability. Later sugar and nucleobase modifications fixed affinity and immunogenicity, while uncharged backbones and advanced conjugation techniques keep expanding therapeutic options. These discoveries allowed us to create effective drugs for various genetic and liver conditions - an important step in precision medicine.
References
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