Phosphorothioate Modification Service

Introduction

Phosphorothioate (PS) modification, replacing a non-bridging oxygen in the phosphate backbone of oligonucleotide with sulfur, enhances therapeutic oligonucleotides' enzymatic resistance, boosting bioavailability, extending half-life, and enabling gene silencing/editing by addressing instability and off-target issues. Our service leverages this to enhance nucleic acid drug stability and efficacy, improving pharmacokinetics, broadening therapeutic windows, and delivering products with preserved activity, reducing dosing frequency, and improving outcomes via rigorous characterization.

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Phosphorothioate Modification

Fig.1 The monomer structures of thiophosphates and dithiophosphates obtained by replacing one or two non-bridging oxygen atoms with sulfur atoms.¹

Phosphorothioate modification, one of the most commonly used oligonucleotide chemical modifications, involves substituting a non-bridging oxygen in the natural oligonucleotide's phosphodiester bond (-O-P-O-) with a sulfur atom to form a phosphorothioate bond (-O-P-S-). This seemingly minor structural adjustment significantly improves the oligonucleotide's biological properties. While retaining the negative charge of natural nucleotides (making them the closest analogues), it confers distinct advantages, leading to wide applications in research, diagnostics, and therapeutic fields.

Key Advantages

• Enhanced Nuclease Resistance: Sulfur-induced steric hindrance and electronic changes boost resistance to exo/endonucleases, preserving structure in biological environments, extending half-life, and sustaining activity.
• Improved Pharmacokinetic Profile: Increased plasma protein binding, reduced renal clearance, and enhanced bioavailability; stability enables less frequent dosing, aiding compliance.
• Preserved Biological Activity: Maintains specific base-pairing with target nucleic acids, ensuring efficacy in gene silencing, targeted binding, etc.
• Immunogenicity Regulation: Moderate modification reduces immune overactivation; some promote cellular uptake via binding to specific proteins (e.g., cell surface receptors).

Considerations for Optimal Design

• Optimization of Modification Degree and Mode: Adjust modification range (full/partial) by application: excess reduces targeting; insufficiency limits stability. Design modes (e.g., 3'/5' end modification, intermittent phosphorothioate insertion) to balance stability and targeting.
• Control of Non-Specific Interactions: Modification may bind non-specifically to endogenous components (heparin-like proteins, etc.) via enhanced charge/altered backbone, raising off-target risk. Use sequence optimization and site screening to reduce such interactions, ensuring efficacy and specificity.
• Balance Between Immunogenicity and Toxicity: Moderate modification reduces immunogenicity; high proportions may activate innate immunity, causing inflammation/toxicity. Determine optimal ratio via gradient experiments to avoid overactive immune pathways.
• Synthesis and Purity Control: Phosphorothioate bonds increase synthesis difficulty, with diastereoisomer impurities affecting homogeneity/activity. Use high-precision synthesis and strict purification for accurate modification and compliant impurities.

Workflow

1. Required Starting Materials

   • Target Gene/RNA Sequence: The specific genetic target (e.g., mRNA sequence for ASO/siRNA) you aim to modulate.
   • Desired Oligonucleotide Sequence: The proposed sequence of your therapeutic oligonucleotide.
   • Specific Therapeutic Application Details: Information regarding the disease area, intended delivery route, and desired in vivo stability.

2. Consultation & Design Optimization

   Experts collaborate to understand goals, design optimal phosphorothioate strategies (considering placement, density, off-target risks), tailoring for stability, efficacy, and minimal side effects.

3. High-Quality Oligonucleotide Synthesis

   State-of-the-art automated platforms precisely synthesize custom oligonucleotides with phosphorothioate linkages, ensuring high purity and yield via robust protocols.

4. Rigorous Quality Control & Purification

   Stringent QC (MS, HPLC) verifies sequence integrity, purity, and modification incorporation; advanced purification removes impurities for top-quality products.

5. Comprehensive Biophysical & Biochemical Characterization

   In-depth studies (nuclease stability, T_m analysis, protein binding) evaluate PS modification impacts, providing insights into in vivo behavior.

6. Final Deliverables

   • Comprehensive Synthesis Report: Detailed documentation of the synthesis process, including reaction parameters and raw data.
   • Analytical Data Package: Full characterization data (e.g., MS, HPLC chromatograms, purity analysis, nuclease stability profiles).
   • Optimized Oligonucleotide Product: The high-purity, PS-modified oligonucleotide is ready for your research or preclinical studies.

7. Estimated Timeframe Our Phosphorothioate Modification service typically takes 6-12 weeks, depending on sequence complexity, synthesis scale, and characterization depth. Expedited options may be available.

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FAQs

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References

1. Novikova, Daria, et al. "A visual compendium of principal modifications within the nucleic acid sugar phosphate backbone." Molecules 29.13 (2024): 3025. DOI: 10.1038/s41467-022-31636-2. DOI: 10.3390/molecules29133025. Distributed under Open Access license CC BY 4.0, A part of the picture was cropped.