Modified Oligonucleotides
Unmodified RNA or DNA oligonucleotides are rapidly degraded in biological matrices and thus have limited utility as therapeutic agents. To enhance the drug-like properties of oligonucleotides, chemical modifications are utilized. The figure below shows the structures and key attributes of the different oligonucleotide chemical modifications currently used in clinical studies. The positions where oligonucleotides are commonly modified include the phosphate backbone, the 2'-position on the sugar, the 5-position of pyrimidine bases, and the addition of targeting ligands such as GalNAc sugars.
Figure 1. Common chemical modifications used in oligonucleotides and their properties.
Sites for Modification
There are several sites on an oligonucleotide which are amenable for chemical modification without interfering with the base-pairing ability of the antisense oligonucleotides (ASOs). For example, modifications to the nucleobases can enhance or modulate base-pairing specificity. Modifications to the phosphodiester backbone can modulate nuclease stability and pharmacokinetic properties. Modifications to the furanose sugar moiety can enhance binding affinity and modulate nuclease stability and/or interactions with cellular proteins which can enhance or decrease ASO activity. Conjugation of lipophilic or cell targeting moieties can modulate protein binding and tissue targeting properties.
Nucleobase Modification
Modifications to the nucleobase impact base-pairing properties of the ASO, which can reduce binding affinity and/or specificity for the targeted mRNA. There is one naturally occurring nucleobase modification which is used extensively in nucleic acid medicinal chemistry to improve duplex stability and nuclease resistance as well as mitigate the immunostimulatory properties of ASOs. This modification is the C-5 methyl substitution on pyrimidine nucleobases, which in turn inspired the design of new classes of heterocycle modifications for use in nucleic acid medicinal chemistry.
Backbone Modification
Some of the most common backbone modifications replace one of the nonbridging oxygen atoms of the PO linkage with sulfur, carbon, nitrogen, or boron. These changes can have profound consequences on the physicochemical and biological properties of the resulting oligonucleotides. For example, replacing the nonbridging oxygen atom with sulfur provides the phosphorothioate linkage which is negatively charged, enhances nuclease stability, and exhibits chemical stability similar to the PO linkage. Replacing the oxygen with a carbon results in the alkylphosphonate linkage, which is charge-neutral, reduces affinity for RNA and is unstable in basic conditions used to deprotect the oligonucleotide after synthesis.
In contrast, replacing the oxygen with nitrogen provides the phosphoramidite linkage which is also charge-neutral, reduces affinity for RNA, and is unstable under the acidic conditions used for removal of the dimethoxytrityl protecting group during automated oligonucleotide synthesis. Replacing the anionic oxygen atom with an alkoxy group provides the phosphotriester linkage which is also neutral and labile under basic conditions used to deprotect oligonucleotides after synthesis.
Furanose Sugar Modification
The furanose ring of natural nucleic acids has been a site of extensive modification within antisense medicinal chemistry. Modification of the sugar moiety can modulate ASO RNA-binding affinity, nuclease stability, and functional activity. Unlike base modifications that can only be applied in the context of ASO sequence, sugar modifications can be applied at any site within an ASO. Furthermore, sugar modifications can also be combined in a modular manner with nucleobase and backbone modifications to create a large universe of nucleic acid analogs. Small structural changes to the sugar moiety can have profound consequences on the biophysical and biological properties of oligonucleotides.
Reference
- Wan, W. B.;et al. (2016). The medicinal chemistry of therapeutic oligonucleotides. Journal of medicinal chemistry. 59(21): 9645-9667.
