A phosphorothioate bond is a synthetic modification to the chemical backbone of nucleic acids like DNA and RNA. This alteration, which does not occur naturally, is introduced into laboratory-made nucleic acid strands. The modification involves a specific atomic substitution within the phosphate group that links the building blocks of the nucleic acid chain together.
The Phosphorothioate Chemical Structure
The backbone of a natural DNA or RNA strand consists of a repeating chain of sugar and phosphate groups, forming a phosphodiester linkage. In a phosphorothioate (PS) bond, this structure is altered when one of the non-bridging oxygen atoms in the phosphate group is replaced with a sulfur atom. This substitution is the defining feature of a PS bond and changes the chemical properties of the nucleic acid backbone.
This atomic swap is comparable to changing a single link in a long chain, where the overall chain remains intact but the properties of that link are different. The introduction of a sulfur atom, which is larger and has different electronic properties than oxygen, alters the character of the bond. This also introduces complexity to the molecule’s three-dimensional shape.
The substitution of sulfur for oxygen creates a chiral center at the phosphorus atom. Chirality means the structure is not superimposable on its mirror image, similar to a left and a right hand. This results in two different stereoisomers, ‘Rp’ and ‘Sp’, for each PS bond. These isomers have distinct spatial arrangements that can influence how they interact with other molecules.
Mechanism of Nuclease Resistance
In biological environments, enzymes called nucleases break down DNA and RNA by cleaving the phosphodiester bonds that form their backbone. This natural degradation process poses a challenge for nucleic acid-based drugs. An unmodified therapeutic strand of DNA or RNA introduced into the body would be rapidly destroyed by nucleases before it could perform its intended function.
The phosphorothioate bond provides a defense against this enzymatic degradation. Nuclease enzymes have a precisely shaped active site configured to bind to the natural phosphodiester linkage. The presence of the larger sulfur atom in place of oxygen alters the geometry and electronic charge distribution of the bond, making it a poor fit for the enzyme’s active site.
This structural mismatch shields the bond from being cleaved, so a nuclease struggles to break a phosphorothioate linkage. As a result, nucleic acids that incorporate PS bonds are more resistant to degradation. This increased stability extends their half-life in serum and within cells, a property necessary for their use as therapeutic agents.
Role in Nucleic Acid Therapeutics
The stability from phosphorothioate bonds is central to designing nucleic acid therapeutics. These drugs, which treat diseases by altering gene expression, require enhanced resistance to nuclease degradation to survive in the body. This modification is widely used in two classes of nucleic acid drugs: antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs).
ASOs are short, single-stranded chains of synthetic DNA or RNA designed to bind to a specific messenger RNA (mRNA) molecule. The goal is to prevent the translation of that mRNA into a disease-causing protein, effectively silencing the gene. Incorporating PS bonds into the ASO’s backbone prevents it from being destroyed by nucleases, giving it time to find its target.
Similarly, siRNAs are short, double-stranded RNA molecules that silence genes through a pathway known as RNA interference. Once an siRNA enters a cell, it is incorporated into a protein complex that uses one strand to identify and destroy a complementary mRNA. The inclusion of PS bonds, often at the ends of the strands, protects them from being broken down.
Without the protective effect of this modification, both ASOs and siRNAs would have extremely short lifespans in the body, rendering them useless as drugs. The replacement of oxygen with sulfur in the nucleic acid backbone has enabled the development of therapies for a range of genetic disorders and other diseases.
Biological Consequences of Modification
While the introduction of phosphorothioate bonds is beneficial for drug stability, it is not without biological trade-offs. One consequence is the increased potential for non-specific binding to proteins. The sulfur atom imparts a “stickier” quality to the nucleic acid backbone, causing it to attach to various cellular and plasma proteins. This can lead to off-target effects or toxicities if the drug interferes with the normal function of these proteins.
Another consequence relates to the drug’s primary function. The structural change from a phosphodiester to a phosphorothioate bond can reduce the binding affinity of the therapeutic strand to its target RNA sequence. Altering this geometry can weaken this interaction, potentially reducing the potency of the drug.
The body’s immune system can sometimes recognize nucleic acids with extensive phosphorothioate modifications as foreign entities, which can trigger an innate immune response. Drug developers must carefully balance the number and placement of PS bonds in a therapeutic oligonucleotide. The goal is to maximize nuclease resistance while minimizing these undesirable biological consequences.