What is mRNA synthesis?
- Custom mRNA Synthesis
baseclick provides customised in vitro transcription (IVT) services to produce high-quality custom-designed mRNA for use in research and clinical applications. Our IVT High-Performance T7 RNA Synthesis Kit provides full-length mRNA using GMP-grade T7 RNA polymerase and NTPs. The process is optimised for both standard and modified nucleotides, ensuring compatibility with a variety of downstream applications:
- Therapeutics: to express therapeutic proteins, gene editing, or cancer immunotherapies.
- Vaccines: mRNA encoding viral antigens for immune response stimulation.
- Research: Functional studies of gene expression, RNA stability, and cellular delivery.
The service supports the synthesis of mRNA with specific modifications, including 5’-capping and poly-A tailing, to enhance stability and translational efficiency.
- mRNA Modification
Key modification services include:
- 5’-end modification: baseclick offers a wide range of cap structures and cap analogues for modifying the 5' end of mRNA. These enhance translational efficiency, enable real-time tracking and improve cell-specific targeting.
Azide-modified cap analogues, for example, are reacted with alkyne-bearing molecules to create functional cap structures.
- 3’end (Poly-A Tail) Modification: The introduction of modified NTPs at the 3′ end or to the poly-A tails can significantly improve the stability of mRNA and the efficiency of translation.
baseclick’s approach involves incorporating alkyne- or azide -modified nucleotides (e.g., EATP, 2’N3-2’-ATP) into the poly-A tail, which can then be conjugated with azide- or alkyne-containing molecules, such as fluorescent dyes or targeting ligands, without affecting mRNA functionality.
- Internal Nucleotide Modification: Alkyne-modified nucleotides (5-ethynyluridine-EU) can be incorporated into the mRNA sequence during IVT, enabling specific conjugation of functional moieties like biotin, fluorescent dyes, or cell-targeting ligands (e.g., sugars).
At the same time, this approach reduces immunogenicity in mRNA applications. The incorporation of EU into RNA, mimics natural modified nucleotides, suppressing immune activation by evading recognition by pattern recognition receptors (e.g., TLR7, RIG-I).
This minimizes inflammatory responses, such as type I interferon production, enhancing mRNA stability and translational efficiency for therapeutic applications like vaccines and protein replacement therapies. (1)
- Click Conjugation for Functionalization
baseclick’s core innovation lies in its use of copper-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC) for precise, site-specific mRNA labeling. These bioorthogonal click chemistry techniques allow for the attachment of functional molecules to mRNA without compromising its integrity, offering significant advantages over traditional chemical or enzymatic conjugation methods.
baseclick’s click chemistry platform facilitates the modular attachment of a wide range of functional molecules to mRNA, enhancing its utility in therapeutic and diagnostic applications. Their conjugation services include:
- Fluorescent Labeling: mRNA can be labeled with fluorescent dyes (e.g., Eterneon Red 645, Dye 488, Dye 647) for imaging and tracking in vivo or in vitro. This is particularly useful for studying mRNA biodistribution and cellular uptake, with applications in fluorescence-activated cell sorting (FACS) and microscopy.
- Targeting Ligands: Click chemistry enables the conjugation of cell- or tissue-specific ligands, such as sugars (e.g., Tri-GalNAc), peptides, or aptamers, to enhance targeted delivery to specific cell types, including cancer cells or immune cells. This improves the specificity and efficacy of mRNA therapeutics.
- Applications in mRNA-Based Technologies
baseclick’s mRNA modification and conjugation services support a broad range of applications:
- Therapeutics and Vaccines: Modified mRNAs with enhanced stability, improved translation, and targeted delivery are used in cancer immunotherapy, infectious disease vaccines, and protein replacement therapies. For example, click-modified mRNA can encode specific antigens or therapeutic proteins while incorporating targeting ligands for precise delivery.
- Diagnostics: Synthetic RNA serves as a standard or probe in quantitative reverse transcription PCR (RT-qPCR) and molecular beacon assays for detecting and quantifying RNA biomarkers.
- Biodistribution and Imaging: Click-labeled mRNA with fluorescent enables real-time tracking of mRNA in vivo, critical for understanding pharmacokinetics and optimizing therapeutic delivery.
- Research Tools: Modified mRNAs are used to study RNA stability, and cellular uptake mechanisms, with applications in cell proliferation assays and fluorescence in situ hybridization (FISH).
- Advantages of baseclick’s Click Chemistry Approach
- High Specificity and Efficiency: CuAAC and SPAAC reactions ensure precise, high-yield conjugations with minimal off-target effects, outperforming traditional methods like NHS ester conjugation.
- Modularity: The bioorthogonal nature of click chemistry allows for flexible, sequential attachment of multiple functional groups (e.g., click-click or click-click-click conjugations), enabling complex mRNA designs.
- Stability: The 1,2,3-triazole linkage formed during click reactions is highly stable against hydrolysis, oxidation, and reduction, ensuring the integrity of modified mRNA under physiological conditions.
- Compatibility: click chemistry reactions are compatible with ex vivo and in vivo delivery strategies, including LNPs, and support applications in personalized immunotherapies and vaccines.
- Custom Services and Collaboration
baseclick offers end-to-end support, from early-stage consulting to custom synthesis and conjugation, tailored to specific project needs.
baseclick also welcomes inquiries for research collaborations, technology licensing, and clinical partnerships to advance mRNA therapeutics.