How Do Antisense Oligonucleotides Work?

Antisense oligonucleotides (ASOs) are short, synthetic nucleic acid strands designed to bind specifically to RNA. They have potential for treating genetic disorders, viral infections, and certain cancers by selectively altering gene expression. Their ability to target RNA makes them a powerful tool for modulating protein production.

Understanding ASO function requires examining their structural components, binding mechanisms, enzymatic interactions, chemical modifications, and cellular uptake.

Key Structural Components

The composition of ASOs dictates their stability, specificity, and interaction with target RNA. Typically 12 to 25 nucleotides long, ASOs incorporate chemical modifications to enhance resistance to degradation and improve binding affinity. One key modification is the phosphorothioate linkage, which replaces a non-bridging oxygen in the phosphate group with sulfur, increasing nuclease resistance and extending half-life.

The sugar component also influences ASO stability and function. While natural nucleotides contain ribose or deoxyribose, ASOs often feature modifications like 2′-O-methyl (2′-OMe) or locked nucleic acids (LNAs). These enhance binding affinity by stabilizing duplex formation. LNAs, in particular, introduce a methylene bridge that locks the ribose in a rigid conformation, increasing thermal stability and reducing off-target effects.

Terminal modifications further refine ASO performance. Conjugates such as N-acetylgalactosamine (GalNAc) enhance targeted delivery, particularly to the liver, via receptor-mediated uptake. This strategy has been successfully employed in FDA-approved ASO therapies like inclisiran, which targets PCSK9 mRNA to lower cholesterol levels.

Mechanism Of Sequence Specific Binding

ASOs bind target RNA through Watson-Crick base pairing, ensuring sequence-specific hybridization. Designed to complement specific regions of messenger RNA (mRNA), they anneal with high precision. Factors such as nucleotide composition, sequence length, and chemical modifications influence binding affinity. Guanine-cytosine (GC) pairs contribute greater stability than adenine-uracil pairs, making sequence optimization crucial for strong, specific interactions.

Once an ASO forms a duplex with RNA, its structural design determines the biological outcome. Gapmer ASOs recruit RNase H1, an endogenous enzyme that selectively degrades the RNA strand in an RNA-DNA hybrid, preventing protein translation. In contrast, steric-blocking ASOs physically obstruct ribosome binding or spliceosome assembly, altering gene expression without degrading the RNA. The choice of mechanism depends on therapeutic goals—whether to downregulate a harmful protein or correct aberrant splicing.

Cellular conditions, such as ionic strength and protein interactions, affect ASO-RNA binding. Magnesium ions stabilize RNA secondary structures that may hinder ASO accessibility. To maximize effectiveness, ASOs are often designed to target single-stranded or loop regions of mRNA. Computational modeling and high-throughput screening help identify optimal binding sites, refining ASO efficacy before clinical application.

Role Of Enzymatic Degradation

RNA degradation is a key mechanism by which ASOs regulate gene expression. Once hybridized to target RNA, ASOs trigger endogenous enzymes to initiate degradation. RNase H1 plays a central role in this process when gapmer ASOs are used. It specifically recognizes RNA-DNA hybrids and cleaves the RNA strand while leaving the ASO intact, enabling multiple rounds of degradation and increasing potency.

RNase H1 activity depends on ASO sequence composition and the structural accessibility of target RNA. Highly structured mRNA regions or those bound by proteins can hinder enzyme recruitment, necessitating strategic target selection. Advances in bioinformatics and high-throughput screening help identify accessible RNA regions for effective ASO engagement. Additionally, RNase H1 concentration varies across cell types, influencing tissue-specific ASO efficacy.

Beyond RNase H1, alternative pathways contribute to RNA silencing. Some ASOs engage other nucleases, such as Tudor-SN, involved in small interfering RNA (siRNA) degradation. Others induce exon skipping or splicing alterations through steric hindrance rather than enzymatic cleavage. These diverse approaches allow ASOs to modulate gene expression based on therapeutic needs.

Types Of Chemical Modifications

Chemical modifications enhance ASO stability, affinity, and therapeutic potential. Phosphorothioate (PS) linkages, which replace a non-bridging oxygen atom with sulfur, increase nuclease resistance and prolong circulation half-life. This modification also enhances protein binding, aiding biodistribution but sometimes leading to off-target interactions. Researchers continue refining backbone modifications to balance these effects.

Sugar modifications further improve ASO performance. Locked nucleic acids (LNAs) rigidify the ribose conformation, increasing thermal stability and target specificity. Similarly, 2′-O-methyl (2′-OMe) and 2′-O-methoxyethyl (2′-MOE) modifications reduce immune activation and enhance duplex formation with target RNA, making them valuable in clinical applications. These modifications are particularly significant in splice-switching ASOs, where precise binding is necessary to modulate RNA processing.

Pathway For Cellular Uptake

For ASOs to function, they must enter cells and reach their target RNA in the cytoplasm or nucleus. Due to their size and negative charge, ASOs cannot passively diffuse across membranes and instead rely on endocytosis. Adsorptive and receptor-mediated endocytosis facilitate cellular entry, with scavenger receptors like stabilin-1 and stabilin-2 playing key roles, particularly in liver-targeted therapies.

Once internalized, ASOs must escape endosomal compartments to reach their site of action. A significant fraction remains trapped in endosomes and is eventually degraded in lysosomes, limiting bioavailability. Enhancing endosomal escape is a major challenge. Some chemical modifications, such as phosphorothioate backbones, promote interactions with membrane-disrupting proteins that aid escape. Additionally, conjugation to moieties like GalNAc increases receptor-specific uptake and influences intracellular trafficking. Advances in lipid nanoparticles and cell-penetrating peptides further improve ASO release from endosomes, maximizing therapeutic potential.