Controlled sulfur-based engineering confers mouldability to phosphorothioate antisense oligonucleotides

Area of Science:

• Oligonucleotide chemistry and biophysics
• Therapeutic oligonucleotide design
• Molecular mechanisms of gene silencing

Background:

• Phosphorothioate (PS) modifications enhance nuclease resistance and bioavailability of therapeutic oligonucleotides like antisense oligonucleotides (ASOs).
• Despite widespread use in ASO therapies for cancer and neurodegenerative diseases, the structural impact of PS substitutions, particularly chirality, on DNA·RNA hybrids is poorly understood.
• Existing knowledge on the role of phosphorothioate chirality in modulating ASO properties is scarce and controversial.

Purpose of the Study:

• To investigate the impact of phosphorothioate chirality on the structural properties of DNA-based antisense oligonucleotides.
• To elucidate how different phosphorothioate diastereomers affect DNA topology, stability, and flexibility in DNA·RNA hybrids.
• To determine the roles of pro-Sp S and pro-Rp S configurations in interactions with DNA Exonuclease and Human Ribonuclease H, key enzymes affecting ASO efficacy.

Main Methods:

• Comprehensive computational investigations (e.g., molecular dynamics simulations).
• Experimental measurements (e.g., biophysical assays).
• Analysis of DNA·RNA hybrid structures and stability.

Main Results:

• Detailed, full-atom insights into structural aberrations induced by PS substitutions in DNA·RNA hybrids.
• Mechanistic understanding of how PS chirality influences DNA topology, stability, and flexibility.
• Identification of specific roles for pro-Sp S and pro-Rp S phosphorothioate linkages concerning DNA Exonuclease and Human Ribonuclease H activity.

Conclusions:

• PS substitutions significantly alter DNA·RNA hybrid structures, with chirality playing a critical role.
• The findings explain the origin of nuclease resistance conferred by PS linkages.
• This knowledge is crucial for optimizing the design and efficacy of current and future antisense oligonucleotide-based therapies.