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Stability-inducing modifications in oligonucleotides

Oligonucleotides are short sequences of nucleic acids that are widely used in several biotechnological applications, such as gene editing, diagnostics, and therapeutics. However, these molecules are inherently prone to degradation, mainly due to the action of numerous nucleases (more than 600 different nucleases and DNA/RNA-modifying enzymes have been identified in humans), which limits their stability and effectiveness in various applications, especially therapeutics. To overcome this challenge, researchers have developed several stability-inducing modifications that can enhance the stability and performance of oligonucleotides. This post will go over the most commonly encountered ones, with examples of current clinical compounds, and give a brief description of their respective advantages over non-modified oligos.

One of the most common stability-inducing modifications in oligonucleotides is the introduction of phosphorothioate (PS) linkages. In phosphorothioate-modified oligonucleotides, one of the non-bridging oxygen atoms in the phosphate backbone is replaced by a sulfur atom (ex. Vitravene). This modification enhances the stability of oligonucleotides by increasing their resistance to nuclease degradation. Additionally, phosphorothioate linkages can also improve the binding affinity of oligonucleotides to their target sequences, thereby enhancing their therapeutic efficacy. Phosphorothioate-modified oligonucleotides are much more lipophilic than their phosphodiester counterparts, which leads to increased protein binding and may lead to an increase in non-specific binding. Since phosphorothioates have a chiral center, PS-modified oligonucleotides are obtained as diastereomeric mixtures, although techniques for diastereoselective PS synthesis are emerging. Other phosphodiester isosteres include phosphorodiamidates, phosphate triesters, methyl phosphonates, and borophosphates, although these have been used much less often than PS-oligomers.

phosphorothioate linkages

Another common stability-inducing modification in oligonucleotides is the incorporation of 2'-substituents on the sugar portion of the nucleoside, such as 2'-O-methyl (OMe, ex. Macugen), 2'-O-methoxyethyl (OMOM, ex. Spinraza), or 2'-fluoro modifications. These modifications alter the sugar backbone's conformation and increase oligonucleotides' stability by reducing their susceptibility to nucleases. Moreover, 2'-O-methyl and 2'-O-methoxyethyl modifications can also enhance the specificity and binding affinity of oligonucleotides to their target sequences, making them more effective in gene silencing and other applications.

Ribose 2'-modifications

In addition to phosphorothioate linkages and sugar modifications, several other stability-inducing modifications have been developed for oligonucleotides. For example, introducing locked nucleic acids (LNAs) or peptide nucleic acids (PNAs) can improve oligonucleotides' stability, specificity, and binding affinity. LNAs contain a rigid, locked sugar conformation (C 3'-endo) that enhances the stability of oligonucleotides, while PNAs are synthetic oligonucleotide analogs that form stronger duplex structures with DNA or RNA targets. Both LNAs and PNAs have duplex-stabilizing properties (a Tm increase in the order of 5 oC per LNA residue is typical), with PNAs being notoriously less tolerant of mismatches. Phosphorodiamidate morpholino oligomers (PMO, ex. Eteplirsen) are another type of residue that can be introduced to improve stability and have been used extensively in exon-skipping oligonucleotides. Since PNAs and PMOs feature a neutral backbone, they tend to be poor protein binders, which reduces off-target binding but increases metabolic clearance. Inverted bases, which feature a 3'-3' phosphodiester linkage, can also be used to reduce enzymatic degradation from the 3' end.

Non-ribose nucleotides analogs

Overall, stability-inducing modifications play a crucial role in enhancing the stability and efficacy of oligonucleotides in various biotechnological applications. By incorporating these modifications, researchers can improve the performance of oligonucleotides and unlock their full potential as therapeutics. As the field of oligonucleotide-based technologies continues to advance, the development of novel stability-inducing modifications will be essential for improving the stability and applicability of oligonucleotides and to help fine-tune their pharmacokinetic properties for therapeutic applications.


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  • Xiong, H.; Veedu, R.N.; Diermeier, S.D; "Recent Advances in Oligonucleotides Therapeutics in Oncology", Intl. J. Mol. Sci., 2021, 22:3295.

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