PhosphorothioatBackbones

Phosphorothioate backbones refer to a modification of the backbone of nucleic acids in which a non-bridging oxygen of the phosphate group is replaced by sulfur. This substitution creates phosphorothioate (PS) linkages throughout the oligonucleotide. PS backbones are widely used in synthetic oligonucleotides for research and therapeutic applications because they markedly increase resistance to nucleases and extend plasma circulation time.

Each phosphorothioate linkage is chiral at phosphorus, producing Rp and Sp diastereomers. In standard synthesis, mixtures of both configurations are generated, though newer methods can yield stereopure PS oligos. The sulfur substitution also influences biophysical properties: PS substitutions generally reduce duplex thermal stability relative to unmodified phosphodiesters and increase binding to serum proteins, which can alter distribution and half-life.

Biological activity and applications are closely tied to these properties. PS backbones are common in antisense oligonucleotides, small interfering RNA, and other therapeutic oligos. In antisense designs, a central DNA region (gapmer) can recruit RNase H to degrade the target RNA, while flanking regions are PS-modified to improve stability. Fully PS-modified RNAs may have different activity profiles and safety considerations. Synthesis is achieved by solid-phase methods using phosphorothioate phosphoramidites, with the sulfur substitution introducing stereochemistry at phosphorus.

Safety and considerations include non-specific protein interactions that can affect distribution and potential immune or toxic responses, underscoring the importance of careful design and testing. Phosphorothioate backbones remain a foundational tool in modern oligonucleotide therapeutics and research.