Since their introduction in the 1990s, therapeutic oligonucleotides have become a powerful new approach to treating diseases with high unmet medical need. Today, they are being developed across a broad range of indications, including cancer, neurodegenerative conditions, and genetic disorders.

Modalities such as siRNAs and antisense oligonucleotides (ASOs) work by regulating gene expression at the RNA level, reducing the production of disease-causing proteins. 

However, unlocking their full potential comes with challenges. Oligonucleotides are inherently susceptible to degradation by nucleases and must be delivered efficiently to the right organ and tissue to achieve therapeutic effect.

To overcome these hurdles, modern oligonucleotide therapeutics incorporate a range of chemical modifications. These improve stability in biological environments, extend circulation time, and support targeted delivery. The result is a new generation of molecules with enhanced durability and more precise biological activity.

Commonly used modification strategies:

• Phosphorothioate (PTO) linkages:
  Typically introduced at the 5′ and 3′ ends of an oligonucleotide to protect against exonuclease-mediated degradation. In many designs, three or more PTO linkages at each end are recommended to ensure sufficient stability. Extending PTO modifications throughout the backbone (full PTO design) can further improve resistance to endonucleases, although this may increase the risk of cytotoxic effects.
• 2′-O-Methyl (2′-OMe):
  Enhances target affinity by increasing melting temperature (Tm) and improves resistance to nuclease degradation. However, 2′-OMe modifications do not support RNase H activity. As a result, they are commonly used in chimeric antisense designs that combine modified RNA regions with a central DNA (or phosphorothioate DNA) domain to retain RNase H-mediated cleavage.
• 5-Methyl-deoxycytidine (5-Me-dC):
  Particularly relevant in CpG motifs—cytosine–guanine sequence patterns that can activate the immune system and are used as adjuvants in vaccines and immunotherapies. Incorporation of 5-Me-dC helps reduce unwanted immune stimulation while moderately increasing duplex stability (Tm).
• 2′-O-Methoxyethyl (2′-MOE):
  Increases nuclease resistance and binding affinity (higher Tm), with improved in vivo stability compared to 2′-OMe. Does not support RNase H activity and is therefore commonly used in gapmer designs with a central DNA region to enable cleavage.
• 2′-Fluoro (2′-F):
  Enhances duplex stability and provides strong nuclease resistance. Maintains compatibility with RNAi machinery, making it widely used in siRNA applications.
• Phosphorothioate (PTO) linkages:
  Backbone modification that improves resistance to nuclease degradation and increases plasma protein binding. Commonly added at oligonucleotide ends (≥3 linkages) or throughout the backbone for enhanced stability, though extensive use may increase nonspecific interactions.
• Locked Nucleic Acids (LNA):
  Conformationally restricted ribose that significantly increases binding affinity and Tm while enhancing nuclease resistance. Often used in gapmer designs; excessive incorporation may raise toxicity risk.

Find our complete portfolio of standard therapeutic modifications in the table below. 

metabion recommends purifying all therapeutic oligonucleotides by HPLC, followed by a Na⁺ salt exchange prior to use in cells or in vivo studies, to ensure removal of residual salts from the purification process. In addition, size exclusion chromatography (SEC) can be applied as a polishing step to remove salts and other small molecules that may be cytotoxic to cells if present in sufficient quantity.

Through this purification workflow, metabion therapeutic oligonucleotides are suitable for direct use in in vivo applications.

metabion antisense oligonucleotides have been successfully applied for the correction of a deep intronic splice-site mutation. Find out more in the paper here.