PMO Synthesis Service
Overview
Phosphorodiamidate morpholino oligomers (PMOs) are synthetic nucleic acid analogues recognized for their superior stability and efficacy as antisense oligonucleotides (ASOs). Unlike DNA or RNA, PMOs possess a unique backbone structure where the deoxyribose sugar ring is replaced by a morpholine ring, and the anionic phosphodiester linkage is replaced by a non-ionic phosphorodiamidate linkage. This modification grants PMOs exceptional nuclease resistance, ensuring long-term cellular stability and low non-specific toxicity. CD BioGlyco's specialized PMO synthesis service delivers custom, high-purity compounds essential for highly specific applications such as steric blocking, exon skipping, and targeted gene knockdown across diverse biological systems, from in vitro models to in vivo translational studies.
Core Technologies
We leverage proprietary, high-efficiency chemistry optimized specifically for the synthesis of PMOs. Our technology stack is built upon years of refinement in solid-phase oligonucleotide synthesis, ensuring the delivery of analogues with unmatched quality and reproducibility.
- Optimized Solid-Phase Synthesis Platform
We utilize a highly specialized solid-phase synthesis strategy, crucial for accurately assembling the PMO backbone. This technology enables rapid, scalable, and reproducible synthesis of sequences ranging from standard lengths to complex, longer oligomers, ensuring batch-to-batch consistency for both research and requirements.
- Proprietary Monomer Building Blocks
The integrity of the final PMO product relies entirely on the purity and quality of the starting materials. We employ highly characterized, specific PMO monomer building blocks, ensuring that each coupling step proceeds with maximum efficiency and minimal side-product formation, which is a critical factor in achieving high-yield, high-purity end products.
- Advanced Conjugation Chemistry
To address the challenge of systemic delivery, we offer advanced conjugation services. This includes linking PMOs to cell-penetrating peptides (CPPs), targeting ligands, or specialized fluorophores. Our expertise in complex conjugation chemistry guarantees that these modifications are seamlessly incorporated without compromising the PMO's structural or functional integrity.
- Rigorous Quality Control (QC)
Every synthesized PMO undergoes comprehensive analysis using high-end analytical instrumentation. This includes high-performance liquid chromatography (HPLC) for exceptional purity assessment and mass spectrometry (MS) for precise molecular weight and sequence confirmation. This stringent QC ensures that our clients receive products meeting the strictest purity specifications.
Integrated PMO Solutions: Unifying Strategy with Execution.
- Custom Sequence Synthesis
Production of any user-defined PMO sequence, with lengths optimized for specific gene targeting mechanisms (e.g., 18-30mers).
- Variety of Synthesis Scales
Ranging from milligram (mg) quantities for early-stage screening and basic research to gram (g) quantities for large-scale in vivo preclinical testing and scale-up feasibility assessments.
- Specialized Modifications
- Vivo-PMO Synthesis: Synthesis of PMOs conjugated to delivery moieties, such as CPPs, for superior systemic uptake, crucial for translational research.
- Reporter Tagging: Integration of fluorophores (e.g., FITC, Rhodamine) or biotin for visualization, tracking, and pull-down assays.
- Chimeric Oligomers: Synthesis of oligo structures that incorporate PMO sections alongside other chemistries for unique functional properties.
- Purity Grades
Offering guaranteed purity levels, typically 90% for standard research applications, and up to 95% or higher for demanding early-phase preclinical studies.
Workflow
Project Consultation & Design
The process begins with a detailed consultation with our scientific experts to define the PMO sequence, scale, and desired modifications (e.g., vvivo-PMOs, dye labels). We review targeting strategy (e.g., splice site, translation initiation) and provide constructive feedback to optimize the design for maximum biological activity.
High-Efficiency Solid-Phase Synthesis
Utilizing our state-of-the-art automated synthesizer, the PMO is constructed base-by-base on a solid support. This stage involves iterative coupling, capping, oxidation, and deprotection steps, employing optimized phosphorodiamidate chemistry specific to the PMO structure.
Custom Conjugation & Modification
For projects requiring enhanced delivery or detection, modifications such as attachment of CPPs (e.g., for targeted muscle delivery in DMD research) or fluorescent reporters are performed. We use validated methods to ensure high conjugation yield and stability.
Deprotection and Cleavage
The crude PMO is cleaved from the solid support and simultaneously deprotected. Unlike standard DNA/RNA synthesis, PMO deprotection is highly specific, and we use proprietary conditions optimized to maintain the integrity of the unique backbone.
Multi-Stage Purification (HPLC & Desalting)
The cleaved product undergoes comprehensive purification. Preparative HPLC is used to isolate the target full-length PMO sequence from truncated or incomplete side products, followed by desalting to remove residual salts and reagents, yielding the required purity grade.
Quality Control and Finalization
The purified PMO is subjected to our stringent QC panel (HPLC purity analysis and MS confirmation). A detailed certificate of analysis (CoA) is generated, confirming purity, concentration, and molecular identity, preparing the product for final lyophilization and shipment.
Publication Data
Journal: Scientific reports
DOI: 10.1038/s41598-023-38698-2
IF: 3.9
Published: 2023
Results: This study presents a novel convergent synthesis method for PMOs using an H-phosphonate approach. The authors developed an efficient solution-phase strategy that employs phosphonium-type condensing reagents, such as PyNTP and MNTP, to directly form H-phosphonamidate linkages between morpholino nucleoside monomers. This method significantly reduces coupling times compared to conventional approaches. A key innovation was the successful implementation of fragment condensation, enabling the synthesis of PMOs up to 8-mers containing all four nucleobases with remarkable efficiency. The researchers optimized a one-pot reaction sequence involving condensation followed by oxidative amination to convert unstable H-phosphonamidate intermediates into stable phosphorodiamidate linkages. This convergent approach prevents the formation of difficult-to-separate N-1 mer by-products, simplifying purification due to significant lipophilicity differences between the target oligomers and fragment precursors. The methodology represents the first reported convergent synthesis of PMOs, offering substantial improvements in synthetic efficiency that could facilitate larger-scale production of these promising antisense therapeutics.
Applications
Therapeutic Exon Skipping for Genetic Disorders
PMOs are centrally important in the development of therapies for muscular dystrophies and other genetic diseases caused by frame-shifting mutations. Our high-purity PMOs enable precise splice modulation, restoring functional protein expression and supporting the critical preclinical development phases.
Antisense Antimicrobial and Antiviral Strategies
PMOs can be rationally designed to target and block the expression of essential genes within bacterial or viral pathogens. This application is a promising area for developing novel agents against multi-drug resistant infections, leveraging PMO's low toxicity profile in mammalian cells.
Splice Site Mutation Correction
Beyond classic exon skipping, PMOs can be designed to correct aberrant splicing caused by deep intronic mutations or splice enhancer/silencer modifications, offering precision tools for researchers investigating novel approaches to treating inherited diseases.
Functional Genomics Screening
PMOs can be arrayed or used in medium-throughput studies to systematically interrogate gene function across entire gene families or pathways, offering a powerful, stable alternative to traditional RNAi approaches for complex genomic analysis.
Advantages
Therapeutic Exon Skipping for Genetic Disorders
PMOs are centrally important in the development of therapies for muscular dystrophies and other genetic diseases caused by frame-shifting mutations. Our high-purity PMOs enable precise splice modulation, restoring functional protein expression and supporting the critical preclinical and clinical development phases.
Antisense Antimicrobial and Antiviral Strategies
PMOs can be rationally designed to target and block the expression of essential genes within bacterial or viral pathogens. This application is a promising area for developing novel agents against multidrug-resistant infections, leveraging PMO's low toxicity profile in mammalian cells.
Target Validation and Drug Screening
In drug discovery, our PMOs serve as highly specific reagents to validate novel drug targets by transiently and precisely knocking down gene expression, allowing researchers to accurately mimic a therapeutic intervention and assess phenotypic outcomes before committing to small-molecule development.
Splice Site Mutation Correction
Beyond classic exon skipping, PMOs can be designed to correct aberrant splicing caused by deep intronic mutations or splice enhancer/silencer modifications, offering precision tools for researchers investigating novel approaches to treating inherited diseases.
Frequently Asked Questions
Customer Review
CD BioGlyco managed the scale-up of our therapeutic PMO candidate seamlessly. Their process yielded a product purity consistently above 95%, meeting our stringent internal quality metrics for preclinical testing. The detailed COA and their proactive communication during the manufacturing phase demonstrated their commitment to partnership and quality.
— Manager D. Patel, Project Lead, Translational Medicine
We rely on CD BioGlyco for synthesizing our custom PMO libraries for pathway screening. Their rapid turnaround and competitive pricing, combined with their superior expertise in complex sequences, allow our facility to support multiple research groups without workflow bottlenecks. They are our go-to for specialized oligonucleotide needs.
— Research Associate E. Lopez, Genomics Core Facility
We required a custom PMO with a challenging dual-dye modification for visualization and tracking. They delivered the complex molecule on time, and the QC data confirmed both high purity and successful conjugation. This level of technical capability in handling complex modifications distinguishes them as an industry leader.
— Senior Scientist J. Williams, Gene Therapy Development
Associated Services
We are committed to providing end-to-end support for your oligonucleotide therapeutic journey. In addition to our flagship PMO synthesis service, we highly recommend integrating the following complementary services to ensure your project achieves its goals rapidly and reliably.
RNA-Cell Binding Interaction Analysis Services
Precisely determine the affinity and specificity of your PMO to its target RNA sequence and assess non-specific cellular binding.
Off-Target Activity Assessment Service
Employ advanced transcriptomics and bioinformatic analysis to rigorously screen for unintended effects, minimizing regulatory risk and ensuring the high specificity that PMOs are known for.
Conjugate Potency Assay Service
For vivo-PMOs, this service directly measures the functional biological activity (e.g., exon skipping efficiency or gene knockdown) in relevant cell lines, validating the efficacy of your chosen delivery moiety.
ADME Profiling Service
Essential for preclinical development, this service provides critical data on the absorption, distribution, metabolism, and excretion of your PMO, guiding dosing and formulation strategies for future in vivo studies.
CD BioGlyco stands as your expert partner in oligonucleotide therapeutics, providing high-end PMO synthesis services underpinned by decades of scientific mastery and a relentless commitment to quality. We deliver PMOs of unmatched stability and purity, customized with cutting-edge conjugation chemistry to drive breakthroughs in genetic disease therapy, infectious disease control, and fundamental gene function research. Contact us to leverage our specialized synthesis and complementary biological evaluation services, and your projects will benefit from a complete, end-to-end solution designed for success.
Reference
- Tsurusaki, T.; et al. Convergent synthesis of phosphorodiamidate morpholino oligonucleotides (PMOs) by the H-phosphonate approach. Scientific reports. 2023, 13(1): 12576. (Open Access)