Nucleic acid therapeutics use molecules like DNA and RNA to target diseases at their genetic roots. These therapeutic agents are inherently fragile and face an immediate threat from enzymes that rapidly break them down. To overcome this, scientists employ a chemical alteration known as a phosphorothioate (PS) modification. This technique serves as a molecular shield, involving a subtle change to the backbone of the nucleic acid. This reinforcement protects the therapeutic molecule from degradation, allowing it to survive long enough to perform its function.
The Chemical Structure and Its Significance
The backbone of natural DNA and RNA is a repeating chain of sugar and phosphate groups, linked by phosphodiester (PO) bonds. This structure provides the framework that holds the genetic code. The phosphorothioate modification is a direct alteration of this backbone, involving the replacement of a single non-bridging oxygen atom within the phosphate group with a sulfur atom. This substitution creates a protective phosphorothioate linkage.
While the change from an oxygen to a sulfur atom is small, it introduces the property of chirality. The phosphorus atom at the site of the modification becomes a chiral center, meaning it can exist in two distinct three-dimensional arrangements. These two non-superimposable mirror-image forms are known as stereoisomers, designated as Rp and Sp. A synthesis of these modified nucleic acids results in a random mixture of both isomers at each linkage.
This induced chirality has direct biological consequences. The spatial orientation of the sulfur atom affects how the nucleic acid interacts with its environment. The Rp and Sp isomers can exhibit different levels of stability, binding affinity to target molecules, and interactions with cellular proteins. For instance, the Sp isomer shows greater resistance to certain enzymes, while the Rp isomer may form more stable bonds with its target RNA. This difference in behavior is a consideration in the design of these therapeutic molecules.
Primary Function and Mechanism of Action
The primary reason for applying phosphorothioate modifications is to protect therapeutic nucleic acids from destruction by the body’s defense mechanisms. Our bodies are filled with enzymes called nucleases, which identify and break down foreign or cellular DNA and RNA. Unmodified nucleic acid drugs are highly susceptible to these enzymes and are often degraded within minutes of entering the bloodstream.
The phosphorothioate modification provides protection against this enzymatic assault. The substitution of a sulfur atom for an oxygen atom in the phosphate backbone makes the linkage less recognizable to the active site of most nucleases. These enzymes are tuned to bind to and cleave the natural phosphodiester bond, and the presence of sulfur disrupts this interaction. The bond becomes resistant to being cut by the nuclease.
This resistance to degradation extends the half-life of the therapeutic molecule within the body. Instead of being destroyed almost instantly, a phosphorothioate-modified drug can persist for hours or even days. This enhanced stability allows the drug to travel through the bloodstream, distribute into tissues, and reach its intended cellular target.
Applications in Nucleic Acid Therapeutics
The stability provided by phosphorothioate modifications has enabled the development of several classes of nucleic acid drugs. This chemical alteration is foundational to a class of therapeutics known as antisense oligonucleotides (ASOs). ASOs are short, synthetic strands of DNA or RNA designed to bind to specific messenger RNA (mRNA) molecules, preventing the production of disease-causing proteins. Without PS modifications, ASOs would be degraded long before acting upon their target mRNA.
A prominent example of an ASO therapy is nusinersen, marketed as Spinraza, which is used to treat spinal muscular atrophy (SMA). SMA is a genetic disorder caused by insufficient levels of a protein called Survival Motor Neuron (SMN). Nusinersen is an ASO whose entire backbone consists of phosphorothioate linkages. It works by binding to the precursor mRNA of the SMN2 gene, correcting a splicing error and enabling the production of functional SMN protein, which helps improve motor function.
Beyond ASOs, phosphorothioate modifications are also used in other nucleic acid-based platforms. Small interfering RNAs (siRNAs) are molecules that silence genes through RNA interference, and PS modifications are placed at the ends of siRNA strands to protect them from exonucleases. Another application is in aptamers, which are single-stranded oligonucleotides that fold into specific shapes to bind to targets like proteins. The stability from PS linkages is important for aptamers to maintain their structure and function.
Potential Toxicities and Biological Challenges
While the phosphorothioate modification is beneficial for stability, it also presents challenges. The replacement of oxygen with sulfur increases the “stickiness” of the oligonucleotide backbone, leading to a higher propensity for non-specific binding to proteins. This off-target binding is a primary driver of the toxicities associated with this class of drugs.
These unintended interactions can lead to several adverse effects. The accumulation of PS-modified oligonucleotides bound to proteins can cause stress to organs, particularly the liver and kidneys. This protein binding can also trigger the innate immune system, leading to inflammatory responses like elevated liver enzymes, proteinuria (protein in the urine), and thrombocytopenia (low platelet counts).
The chirality of the phosphorothioate linkage also plays a role in these toxic effects. Because these drugs are a mixture of Rp and Sp stereoisomers, their biological behavior can be unpredictable. One isomer might be more prone to off-target protein binding than the other, contributing disproportionately to the overall toxicity profile. This has prompted research into developing stereopure drugs, which contain only the single, desired isomer. The goal is to create safer therapeutics by eliminating the isomer responsible for the majority of unwanted side effects.