Oligonucleotides are short, synthetic strands of nucleic acids, typically composed of 15 to 30 nucleotides, designed to interact with specific genetic sequences within a cell. These molecules mimic segments of DNA or RNA and have evolved from simple laboratory tools into sophisticated therapeutic agents. The primary goal of oligonucleotide therapies is to transform these nucleic acid fragments into stable, effective drugs that can precisely modulate gene expression to treat diseases. This application relies on chemical engineering to overcome biological barriers.
Why Natural Oligonucleotides Require Enhancement
Native DNA and RNA structures, when introduced into the body, are rapidly recognized and dismantled by the body’s natural defense systems. The most significant challenge is their susceptibility to rapid enzymatic degradation by nucleases, which are ubiquitous enzymes found in the bloodstream and within cells. These enzymes quickly cleave the natural phosphodiester bonds that link the nucleotides, resulting in a half-life of only minutes for an unmodified oligonucleotide in circulation. This rapid breakdown prevents the molecule from reaching its target site at a therapeutically relevant concentration.
Another major limitation is poor pharmacokinetics, specifically the difficulty in getting the molecules into the correct cells. Oligonucleotides are large, highly negatively charged molecules at physiological pH. This negative charge prevents them from easily crossing the hydrophobic, fatty barrier of the cell membrane, which is necessary to reach their target—messenger RNA (mRNA) or pre-mRNA—inside the cell’s cytoplasm or nucleus. Without modifications, these molecules are largely confined to the extracellular space or are quickly cleared from the body, making them ineffective as systemic drugs.
Chemical Strategies for Stability and Function
Chemical modifications are the foundation of oligonucleotide therapeutics, addressing stability and affinity issues. These modifications are strategically applied to three main parts of the nucleotide structure: the backbone, the sugar, and the nucleobase.
Backbone Modifications
Backbone modifications are the first line of defense against nucleases. This involves replacing a non-bridging oxygen atom in the natural phosphodiester bond with a sulfur atom, creating a phosphorothioate (PS) linkage. This change significantly increases nuclease resistance and improves the oligonucleotide’s ability to bind to plasma proteins, which helps prolong its circulation time in the blood.
Sugar Modifications
Sugar modifications enhance both stability and binding affinity to the target nucleic acid. Common alterations occur at the 2′-position of the ribose sugar ring, such as the addition of 2′-O-methyl (2′-OMe) or 2′-O-methoxyethyl (2′-MOE) groups. These groups enhance nuclease resistance and increase the thermal stability of the duplex formed when binding to target RNA. Another advanced modification is the Locked Nucleic Acid (LNA), where the furanose ring is “locked” by an extra bridge connecting the 2′ oxygen and 4′ carbon atoms, which dramatically increases binding affinity.
Base Modifications and Design
Base modifications are less common but are sometimes used to fine-tune activity, such as the 5-methyl modification of pyrimidines to increase binding affinity for the target mRNA. Researchers often combine these alterations using a “gapmer” design. This design features nuclease-resistant modified nucleotides at the ends and a central segment of DNA-like nucleotides, creating a molecule stable in the bloodstream yet capable of activating cellular machinery to destroy the target RNA. The specific combination of backbone and sugar modifications dictates the overall drug properties.
Modifying Oligonucleotides for Cellular Delivery
While chemical modifications enhance stability, separate strategies are required to ensure the stabilized oligonucleotide reaches the target cell and crosses the cell membrane. Delivery is achieved through conjugation, where a targeting ligand is chemically attached, or through encapsulation in a protective carrier.
A highly successful example of conjugation is the use of \(N\)-acetylgalactosamine (GalNAc), a sugar molecule attached in a trivalent arrangement. This GalNAc cluster is recognized by the asialoglycoprotein receptor (ASGPR), which is highly expressed on the surface of liver cells (hepatocytes). Binding to ASGPR triggers receptor-mediated endocytosis, delivering the oligonucleotide directly into the hepatocyte. This targeted approach has dramatically increased the efficiency of liver delivery, achieving over 80% hepatic uptake compared to the unconjugated molecule.
For systemic delivery to a wider range of tissues or for larger payloads like small interfering RNA (siRNA), encapsulation in Lipid Nanoparticles (LNPs) is often employed. These LNPs are synthetic spheres made of lipids that protect the nucleic acid cargo from degradation and facilitate its entry into cells.
Applications in Targeted Gene Regulation
The modifications and delivery systems enable oligonucleotides to act as powerful tools for targeted gene regulation, primarily through two mechanisms: Antisense Oligonucleotides (ASOs) and Small Interfering RNA (siRNA).
Antisense Oligonucleotides (ASOs)
ASOs are typically single-stranded molecules designed to bind to a specific sequence of messenger RNA (mRNA) or pre-mRNA. Depending on the modification pattern, ASOs function in several ways. They can block the translation of mRNA into a protein (steric blocking), or they can recruit the enzyme RNase H to cleave and destroy the target mRNA. ASOs can also modulate pre-mRNA splicing, allowing for the correction of genetic errors or the production of functional proteins.
Small Interfering RNA (siRNA)
In contrast, siRNA molecules are short, double-stranded RNA structures that trigger the cell’s natural RNA interference (RNAi) pathway. Once delivered, the siRNA is incorporated into the RNA-induced Silencing Complex (RISC). RISC uses one strand of the siRNA as a guide to locate and cleave complementary mRNA, leading to gene silencing. Both ASOs and siRNAs leverage the highly specific nature of base-pairing to target a single gene.
Beyond therapeutics, modified oligonucleotides are also used in diagnostics as aptamers. Aptamers are short nucleic acid sequences that fold into unique three-dimensional shapes to bind to specific proteins with high affinity, similar to antibodies.