Oligonucleotides have shown efficient therapeutic effects in various fields, indicating their potential for disease treatment. However, due to poor in vivo stability, ineffective cellular uptake, and off-target effects including immune stimulation, the translation from laboratory to clinical application faces significant challenges.
As a result, there has been an awareness that chemical modifications to natural DNA and RNA structures are necessary to make them suitable as therapeutics. Introducing chemical modifications enhances the stability of oligonucleotides, increases their affinity for targets, facilitates cellular uptake, and improves their bioavailability in vivo. Chemical modifications of nucleic acids can be categorized into three types: (1)modified internucleotide, (2) sugar rings, and (3) base modifications. In addition to direct nucleic acid modifications, conjugation is another highly effective method for improving intracellular delivery or pharmacokinetics and biodistribution of oligonucleotides. Here, we discuss modifications frequently found in oligonucleotide drugs.
Raw Materials for Oligonucleotides Synthesis
- Nucleosides
- Phosphoramidites for oligonucleotide synthesis
- Nucleotides applied on Proteins/Enzymes
- Nucleotides
- Nucleotides applied on DNA/RNA
- Unmodified Nucleotides
- Biotinylated Nucleotides
- Cyclic Nucleotides
- Phosphate modified Nucleotides
Internucleotide bond modifications
In oligonucleotide synthesis, nucleotide monomers are linked by phosphodiester bonds to form oligonucleotides, where the phosphodiester bonds between nucleotides are negatively charged and easily cleaved by nucleases and exonucleases in serum and mammalian cells at physiological pH. To enhance the stability of oligonucleotides, chemical modifications to these bonds have been extensively studied. Some modification strategies have been proven to be highly successful in improving nuclease resistance and are compatible with RNase H-mediated mRNA cleavage mechanisms, while others provide oligonucleotides with additional beneficial properties.
Phosphorothioate modification
One of the most common backbone modifications is the phosphorothioate modification, where the oxygen in the phosphodiester bond is replaced by sulfur. Compared to phosphoester bonds, thiophosphoryl ester bonds exhibit stronger nuclease resistance and promote in vivo cellular uptake and bioavailability. This is because they have increased binding affinity to serum proteins (such as albumin), which helps them evade blood clearance long enough to reach their target tissues.
phosphorothioate modification(Roberts, Thomas C., et al., 2020)
Peptide nucleic acid (PNA)
The second modification is peptide nucleic acid (PNA), which is a completely different class of oligonucleotide analogs where the sugar-phosphate backbone is replaced by a peptide backbone, yet it still retains the ability to base-pair with complementary RNA and DNA. PNA has a neutral charge backbone composed of N-(2-aminoethyl)glycine, showing significant nuclease and protein resistance, along with higher affinity. PNA does not activate RNase H and can act through a translation inhibition mechanism, also displaying splice-switching activity. However, pharmacokinetic and potency challenges remain for PNA oligonucleotide drugs, and there are concerns about whether this modification may be too stable in tissues.
Peptide nucleic acid(Roberts, Thomas C., et al., 2020)
Phosphorodiamidate Morpholino Oligomers (PMOs)
The third modification is PMOs (Phosphorodiamidate Morpholino Oligomers), which feature a neutral backbone where the furanose sugar ring is replaced by a morpholine ring, and the diamidophosphonate bond connects the morpholine nitrogen atom to the 5-hydroxyl group. Due to the diamidophosphonate bond, PMO oligonucleotides are neutral, stable against nucleases, and exhibit higher binding affinity for the target strand. They can be used as translation inhibitors and anti-miRNA agents. PMOs are resistant to nuclease degradation and show promise for splice modulation and translation arrest.
Phosphorodiamidate Morpholino Oligomers(Roberts, Thomas C., et al., 2020)
Ribose Modifications
Modifications to the ribose significantly affect the sugar conformation in nucleotides, which determines the oligonucleotide’s binding affinity to its complementary strand and its double-stranded structure.
2ʹ-Ribose substitutions(Roberts, Thomas C., et al., 2020)
2′-OMe modification
2′-OMe modification is one of the most widely used modifications in oligonucleotide therapeutics. The 2′-OMe modification increases the binding affinity for complementary RNA and enhances nuclease stability. It has been extensively applied in antisense oligonucleotide drugs, especially in “gapmer” structure antisense oligonucleotides. This modification is also highly suitable for siRNA, as it provides good nuclease resistance and reduces immune stimulation of siRNA.
2′-MOE (2-O-(2-methoxyethyl)) modification
2′-MOE (2-O-(2-methoxyethyl)) modification is another common modification. The 2′-MOE modification increases target binding affinity and improves nuclease stability.
2′-F modification
2′-F modification is another popular chemical modification, especially for siRNA. Like 2′-OMe, 2′-F is an RNA modification that increases binding affinity for target mRNA sequences and offers good nuclease resistance. 2′-F can be used for full sequence modifications of RNA, and this modification does not interfere with binding to RISC, providing more stable and effective duplexes.
Locked Nucleic Acid (LNA)
Locked Nucleic Acid (LNA) is an RNA modification in which a methylene bridge forms a covalent bond between the 2′-OH and 4′-C of the sugar. LNA increases binding to the target and exhibits the best duplex stability among all chemical modifications.
Cyclohexene Nucleic Acid (cEt)
Cyclohexene Nucleic Acid (cEt) has now been successfully applied in second-generation therapeutic antisense oligonucleotides. cEt and LNA show similar in vitro and in vivo activities. Compared to LNA, cEt also has the advantage of improving the toxicity profile of oligonucleotides.
(Roberts, Thomas C., et al., 2020)
Flexible Unlocked Nucleic Acid (UNA) modification
Flexible Unlocked Nucleic Acid (UNA) modification lacks the C2-C3 covalent bond in the ribose and is not constrained by conformation. UNA can be used to influence the flexibility of oligonucleotides, as detailed in Figure 6. UNA helps with the selection of the antisense strand for entry into the RISC complex and significantly reduces off-target effects.
Base Modifications
Chemically modified bases have also been applied in oligonucleotide drug development. By modifying the bases of nucleotides, such as the 5′-methyl modification of pyrimidines, higher affinity for the target mRNA can be achieved, and it also increases the thermal stability of the duplex formed between the oligonucleotide and its target mRNA. This greatly enhances activity during mRNA silencing. This is because when the oligonucleotide binds tightly to its target RNA, it can mask splice sites, prevent ribosome assembly, and inhibit translation. Additionally, base modifications can enhance the 3D structure of aptamers. However, it should be noted that while the enhanced binding affinity can improve efficacy, it may also increase the risk of off-target binding, thereby raising the potential for adverse reactions.
Nucleobase modifications(Roberts, Thomas C., et al., 2020)
Bioconjugation
In addition to modifying the internal chemical structure of oligonucleotides, oligonucleotides can also be chemically modified by conjugating other molecules. This can affect the targeting and uptake of oligonucleotides at the tissue and cellular levels, as well as alter their pharmacokinetics. When N-acetylgalactosamine (GalNAc) is conjugated to an oligonucleotide, it enhances the uptake and storage of the oligonucleotide within cellular compartments, thereby ensuring sustained efficacy in vivo. Using this approach, a single dose of GalNAc-conjugated siRNA can result in target gene silencing lasting for several weeks.
Bioconjugates can also include other molecules such as cholesterol, aptamers, antibodies, and peptides. Cell-penetrating peptides (CPPs) have been used as bioconjugates to enhance the activity of covalently linked oligonucleotides. CPPs can be designed to work by forming pores in the plasma membrane or destabilizing endosomes for cellular uptake.
Summary
The above briefly introduces some of the modifications of oligonucleotides and their corresponding effects. The discovery of these modifications has significantly enhanced the stability of oligonucleotides, increased their affinity for targets, facilitated cellular uptake, improved their bioavailability in vivo, reduced immunogenicity, and greatly increased the success rate of oligonucleotide drug development.