CpG Oligonucleotide Synthesis Service
Overview
CD BioGlyco recognizes the profound scientific impact of unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs) in modern medicine and research. CpG ODNs are synthetic short single-stranded DNA molecules that mimic motifs found in bacterial and viral DNA, serving as potent immune adjuvants. These sequences are specifically recognized by toll-like receptor 9 (TLR9), a pattern recognition receptor localized within endosomes of key immune cells like B cells, plasmacytoid dendritic cells (pDCs), macrophages, and monocytes. Activation of TLR9 by CpG ODNs acts as an innate "danger signal," propelling a robust and targeted immune response characterized by the secretion of pro-inflammatory cytokines such as IL-6, IL-12, and IFN-γ, effectively linking innate and adaptive immunity for durable protection. CD BioGlyco leverages state-of-the-art chemical synthesis platforms to deliver high-purity, structurally customized CpG ODNs, ensuring your therapeutic and diagnostic projects are built upon a foundation of uncompromising quality and biological efficacy.
Core Technologies
The reliable and efficient production of high-quality CpG ODNs, especially those required for clinical and in vivo studies, relies on the gold-standard technology: solid-phase phosphoramidite chemistry. This automated method allows for the sequential and precise addition of nucleotide units in the 3' to 5' direction onto a solid support, typically controlled pore glass (CPG) or macroporous polystyrene (MPPS). Our advanced synthesis infrastructure is designed for maximum efficiency and flexibility:
- Automated, High-Efficiency Synthesis
We employ computer-controlled oligonucleotide synthesizers to execute thousands of sequential chemical steps with exceptional coupling efficiency (typically 98-99.5% per step), minimizing the generation of truncated "deletion sequences".
- Phosphorothioate (PS) Backbone Modification
Crucial for in vivo applications, the natural phosphodiester (PO) linkage is replaced with the nuclease-resistant phosphorothioate (PS) backbone during the synthesis cycle through a dedicated sulfurization step, dramatically enhancing the stability and half-life of the CpG ODNs in serum and complex biological matrices.
- Advanced Chemical Building Blocks
We utilize specialized phosphoramidite reagents for incorporating a vast library of non-standard modifications, including locked nucleic acids (LNA), 2'-O-methyl (2'-OMe) RNA analogs, and base modifications, which are essential for tailoring physicochemical properties, binding affinity, and overall biological activity of the final product.
CpG ODNs: Accelerating Next-Generation Immunotherapeutics
Our service offers unmatched breadth and depth to support every phase of drug development, from initial discovery to commercial production.
- Scale and Purity Customization
We offer synthesis scales from small research quantities (mg) for initial screening up to large-scale, multi-kilogram quantities for advanced preclinical trials and commercial manufacturing, with purity options ranging from 80% to ultra-pure 95% verified by HPLC or CE.
- Comprehensive Structure Classes
We provide reliable synthesis of all major immunostimulatory classes, including the B-class (strong B-cell activation, vaccine adjuvants), A-class (potent IFN-ɑ induction, pDC activation), C-class (combining B and A effects), and P-class ODNs (dimeric structures, enhanced NK stimulation).
- Custom Modification Integration
Our synthesis capabilities extend to over 200 chemical modifications, supporting complex designs for enhanced function, stability, and targeting, including diverse terminal and internal conjugations.
- Specialized Therapeutic Design
We possess expertise in synthesizing complex oligonucleotide architectures, such as ODNs containing advanced prodrug modifications (e.g., thermolytic protecting groups for tunable in vivo release) and specific fluorescent tags for cellular tracking and mechanistic studies.
Workflow
Sequence Design and Consultation
The process begins with our expert team advising on the optimal CpG ODN sequence, class selection (A, B, C, or P), and necessary chemical modifications (e.g., PS backbone, ligand conjugation) to match the client's specific biological requirements, such as targeting B cells versus pDCs.
Solid-Phase Assembly (Cycle Synthesis)
The ODN is assembled nucleotide-by-nucleotide on a solid support using the phosphoramidite method, involving four core chemical steps: detritylation (deprotection of the 5'-hydroxyl group), coupling (addition of the new phosphoramidite monomer), capping (acetylation of unreacted hydroxyl groups to prevent errors), and oxidation/thiolation (stabilization of the phosphite triester, or introduction of the PS backbone).
Cleavage and Deprotection
Once the desired sequence length is reached, the full-length oligonucleotide is chemically cleaved from the solid support and simultaneously treated to remove all remaining protecting groups from the nucleobases, ensuring a fully functional and reactive molecule.
Purification and Desalting
Crude ODNs are subjected to rigorous purification methods, primarily high-performance liquid chromatography (HPLC) or polyacrylamide gel electrophoresis (PAGE), to isolate the full-length target sequence and remove truncated species and synthetic impurities, followed by necessary desalting.
Quality Control and Validation (QC)
Every batch undergoes stringent analytical testing, including mass spectrometry (MALDI-TOF or ESI-MS) for molecular weight confirmation, capillary electrophoresis (CE) or HPLC for purity assessment, UV spectroscopy for accurate concentration, and optional endotoxin testing (LAL method) for in vivo readiness.
Publication Data
Journal: Scientific Reports
DOI: 10.1038/s41598-024-61666-3
Published: 2024
IF: 3.9
Results: In this study, the authors investigated the unintended activation of the innate immune system via toll-like receptor 9 (TLR9) by antisense oligonucleotides (ASOs) containing immunostimulatory CpG motifs. To systematically analyze this effect, they first established a potent 18-mer phosphorothioate oligodeoxynucleotide model, SY-ODN18, and demonstrated that an unmethylated CpG motif near its 5'-end was indispensable for TLR9 activation. The core of their work involved engineering a series of SY-ODN18 variants by incorporating various sugar-modified nucleotides—such as 2′-OMe, 2′-MOE, and LNA—in designs mimicking therapeutic ASOs, including gapmer, mixmer, and fully modified structures. Their key finding was that the introduction of these sugar-modified nucleotides significantly suppressed TLR9-stimulatory activity, even when the CpG motifs remained unmethylated. This inhibitory effect was consistent across different modification types and oligonucleotide designs. The research provides crucial insights for the design of safer ASO therapeutics by outlining a strategic approach to mitigate potential immune-related side effects through specific chemical modifications, thereby enhancing their drug-like properties.
Applications
Next-Generation Vaccine Adjuvants
CpG ODNs are widely recognized as one of the most effective immune stimulants available for vaccine development, significantly boosting the immunogenicity of co-delivered antigens by stimulating a Th1-biased adaptive immune response crucial for long-lasting immunity against viral and bacterial pathogens.
Cancer Immunotherapy and Nanovaccines
Utilizing custom-synthesized CpG ODNs as adjuvants in tumor nanovaccines is a major research focus; our products facilitate the co-delivery of neoantigens and adjuvants to antigen-presenting cells (APCs), inducing robust cytotoxic T cell responses for effective tumor suppression and eradication.
Infectious Disease Intervention
By mimicking pathogen DNA, CpG ODNs can be deployed to prime the host's immune system, offering a prophylactic or therapeutic strategy to protect against or mitigate the effects of various challenging infections, a potential application strongly supported by preclinical models.
Allergy and Asthma Mitigation Therapies
Certain classes of CpG ODNs can shift the immune response away from a Th2-driven allergic reaction toward a Th1-biased profile, representing a highly promising area for developing new therapeutics aimed at controlling and reducing the severity of allergic diseases.
Advantages
Unique Delivery System Integration
We specialize in conjugating targeting ligands, such as cholesterol, PEG, or GalNAc, directly to the CpG ODN, which is key to improving cellular uptake, enhancing lymphatic accumulation, and delivering the TLR9 agonist precisely to the targeted immune cell populations.
Expertise in Novel Prodrug and Visualization Technologies
Our specialists are proficient in synthesizing cutting-edge ODNs, including those featuring thermolytic protection groups for controlled release in specific biological environments, and fluorescent nucleobase analogues for real-time cellular visualization and mechanism-of-action studies.
Data-Driven Design and Technical Consultation
Our Ph.D.-level scientific support team provides comprehensive consultation on sequence design, modification compatibility, and purification strategy, leveraging a deep understanding of published CpG ODN structure-activity relationships.
Unmatched Modification Versatility for Immune Modulation
Our proprietary phosphoramidite chemistry enables the seamless incorporation of non-standard bases, sugar modifications (like LNA or 2'-OMe), and advanced functional groups, allowing you to fine-tune the immunomodulatory profile and receptor affinity of your CpG ODN with unparalleled precision.
Frequently Asked Questions
Customer Review
The lot of class C CpG ODNs provided by CD BioGlyco for our latest in vivo cancer model was outstanding. The certificate of analysis showed a confirmed purity of 97%, and the endotoxin levels were negligible, which is crucial for our sensitive animal studies. The low variation between batches has significantly improved the reproducibility of our anti-tumor efficacy data. CD BioGlyco has become our trusted synthesis partner.
— Dr. Patel, Principal Investigator, Immunotherapy Department
The fluorescently labeled CpG ODN CD BioGlyco, synthesized for us, incorporating a specialized internal nucleobase analog, was instrumental in deciphering the intracellular delivery mechanism of our nanoparticle system. The label's stability through our formulation process was exceptional, allowing for crystal-clear visualization and quantification of TLR9 colocalization.
— Dr. Schmidt, Research Scientist, Nanomedicine Laboratory
When scaling up our anti-viral ODN from milligrams to multi-gram quantities, CD BioGlyco's process development team ensured a seamless transition. Their adherence to strict quality protocols, including comprehensive stability testing, gave us high confidence in the material used for our GLP toxicology studies. Their full-service support saved us months of internal development time.
— Project Lead Chen, Preclinical Division, Infectious Disease Research
Associated Services
To fully leverage the power of CpG ODNs and other nucleic acid technologies, we offer a complementary suite of oligonucleotide synthesis and modification services designed to meet every research and therapeutic need.
Essential for applications requiring the ODN to be ligated or involved in enzymatic reactions, such as cloning, gene construction, or preparing duplexes. 5'-phosphorylation also prevents polymerase chain extension.
The full-spectrum PS service is dedicated to enhancing nuclease resistance and cellular uptake, a foundation for almost all in vivo CpG ODN applications.
Incorporation of 2'-OMe, 2'-fluoro (2'-F), or LNA to increase binding affinity, improve stability, and modulate ODN specificity, particularly valuable when developing hybrid CpG ODNs or ASOs.
Custom synthesis incorporating modified nucleobases like 5'-methylcytosine, deoxyinosine, or complex fluorescent nucleobase analogues for research in epigenetics, degeneracy, or tracking studies.
Specialized service for adding non-nucleotide linkers, such as polyethylene glycol (PEG), or custom spacers for optimizing conjugation distance and flexibility, critical when coupling antigens or targeting ligands to the CpG ODN.
A dedicated service for labeling ODNs with a vast array of fluorophores (e.g., FAM, Cy3, Cy5, ATTO dyes) at various positions for flow cytometry, imaging, and advanced diagnostic probe development.
CD BioGlyco is your definitive partner for high-quality, customized CpG ODN synthesis. Our dedication to solid-phase phosphoramidite chemistry, coupled with our expertise in advanced modifications—from nuclease-resistant PS backbones to cutting-edge prodrug and visualization systems—ensures that the critical TLR9 agonist component of your therapeutic or diagnostic project is delivered with the highest purity and functional integrity. To initiate your project, receive a confidential quote, or discuss the scientific and technical details of your complex oligonucleotide design, please contact us.
Reference
- Yoshida, T.; et al. Introduction of sugar-modified nucleotides into CpG-containing antisense oligonucleotides inhibits TLR9 activation. Scientific Reports. 2024, 14(1): 11540. (Open Access)