The 5' cap (N7-methylguanosine via 5' to 5' triphosphate bridge) is key to mature eukaryotic mRNA and mRNA therapeutics—it protects against 5' to 3' exonuclease degradation for stability, and anchors ribosomes to enable efficient translation into target proteins (e.g., therapeutic antigens).
Creative Biolabs' Custom mRNA 5' Capping Services use advanced enzymatic/co-transcriptional techniques to boost IVT mRNA therapeutics' translational efficiency and stability. It provides robust solutions for candidates like cancer vaccines, delivering mRNA mimicking natural eukaryotes to minimize immune detection and extend in vivo lifespan.
Discover How We Can Help - Request a Consultation TodayCreative Biolabs provides specialized cap analogs designed to meet diverse research and therapeutic needs, including specific applications in tracking, labeling, and interaction studies.
Fig.1 The structure of the 5' terminal cap of eukaryotic mRNA and the separation of impurities by reversed-phase high-performance liquid chromatography.1
Anti-Reverse Cap Analogs (ARCAs) are critical for ensuring the translational competence of IVT mRNA. Standard cap analogs can be incorporated in both the correct and reverse orientation. Only the correct orientation (5' to 5') is recognized by the ribosome. ARCA prevents this incorrect orientation, ensuring that a significantly higher percentage of your transcripts are functionally ready for high-efficiency protein synthesis. This modification is standard for maximizing the expression of vaccine antigens.
Fluorescent cap analogs allow researchers to tag the mRNA transcript at the point of its most crucial functional element. These molecules are invaluable tools for real-time tracking and visualization of the mRNA fate in vitro and in vivo. Applications include studying the kinetics of capping/decapping reactions, monitoring cellular uptake, tracking the distribution of delivery vehicles (like LNPs), and conducting biophysical studies on translation dynamics.
Fluorophosphate nucleotide analogs introduce a chemical handle that can be used for sophisticated enzyme activity monitoring and protein interaction studies. These compounds are particularly useful for analyzing the activity of cap-binding proteins and enzymes involved in mRNA processing. They serve as valuable reporting ligands for binding studies, allowing researchers to accurately monitor the specificity and requirements of various capping and translation factor enzymes.
The 6-Thioguanosine-Containing Cap is a highly specialized modification that allows for subsequent cross-linking with interacting macromolecules. The thioguanine residue can be activated by light, forming covalent bonds with proximal proteins or nucleic acids. This feature is crucial for performing photo-crosslinking experiments, which are used to map the physical proximity and interaction sites of cap-binding proteins (e.g., eIF4E) and other elements of the translation initiation complex.
Isolate, purify starting plasmid DNA, then linearize with restriction enzymes. Outcome: High-purity linear DNA template ready for transcription.
Transcribe linearized DNA into mRNA via RNA polymerase; incorporate desired cap analog (e.g., ARCA) during the process for efficient, correctly oriented capping. Outcome: Raw capped mRNA transcript.
Add enzymatic/templated poly(A) tail to mRNA 3' end (boosts stability, enhances translation), then purify to remove residual DNA, enzymes, and unincorporated nucleotides.
Use HPLC or mass spectrometry to confirm precise 5' cap structure (e.g., Cap 1) and overall integrity, ensuring functional optimization (critical QC step).
Formulate highly purified capped mRNA for stability; conduct final QC (RNase-free testing, cell-based functional translation assays, endotoxin analysis) to prepare for downstream in vivo studies.
The typical timeframe for this custom service ranges from 3 to 6 weeks, depending on the complexity of the mRNA sequence and the required level of cap structural analysis.
Our Advantage:
Customized Capping Strategy
Offer full-spectrum customized capping solutions (enzymatic, co-transcriptional with ARCA) to match your mRNA design and therapeutic application needs.
Optimized Performance
Ensure high-fidelity capping to maximize ribosomal binding and protein translation efficiency, boosting target antigen/enzyme expression yields.
Enhanced Stability and Immune Evasion
Produce advanced structures (Cap 1, Cap 2) to improve in vivo transcript stability, help mRNA be recognized as "self," and reduce unwanted innate immune responses.
Scalable Production Expertise
Provide reliable, high-throughput service supporting seamless scale transitions (lab mg-scale to preclinical/industrial gram-scale) with consistent quality.
Quality-by-Design (QbD) QC
Apply advanced analytical techniques (HPLC, Mass Spectrometry) via a mature quality system for definitive 5' cap structural verification, ensuring product integrity and regulatory compliance.
Integrated End-to-End Service
Offer complete options integrating custom capping with upstream vector design, downstream purification, polyadenylation, and quality assurance—providing a seamless one-stop solution for your full mRNA program.
A: Cap 0 (m7GpppN) is the initial cap structure. Cap 1 includes an additional methylation at the 2'-O position of the first transcribed nucleotide. Cap 1 is strongly preferred because it better mimics mature eukaryotic mRNA, which is essential for evading innate immune sensors and achieving higher translation efficiency.
A: Our multi-pronged approach includes using proprietary optimization methods during IVT, employing high-purity ARCAs, and incorporating a robust poly(A) tail. Crucially, we use specialized purification techniques to eliminate contaminants like dsRNA, which can trigger immune degradation pathways.
A: While the fluorescent tag is essential for tracking, any modification at the 5' end can potentially influence ribosomal binding. Our specialized fluorescent cap analogs are designed to maintain maximum translational fidelity.
A: Co-transcriptional capping (using analogs like ARCA) is typically more efficient and cost-effective for large-scale synthesis as it occurs simultaneously with transcription. Creative Biolabs offers both methods, but we generally recommend optimized co-transcriptional capping for therapeutic scaling.
A: Transitioning is seamless. We start with a full analysis of your existing protocol and data. We then generate pilot batches using our optimized ARCA/Cap 1 protocols, providing side-by-side comparative data (yield, purity, function) to demonstrate consistency and improvement before full-scale production.
Creative Biolabs is dedicated to providing the enabling technologies that maximize the performance and stability of your novel mRNA therapeutics and research tools. Our Custom mRNA 5' Capping Services are the essential foundation for robust protein expression and enhanced in vivo efficacy.
Contact Our Team for More Information and to Discuss Your Project!| Cat. No | Product Name | Promoter |
|---|---|---|
| CAT#: GTVCR-WQ001MR | IVTScrip™ pT7-mRNA-EGFP Vector | T7 |
| CAT#: GTVCR-WQ002MR | IVTScrip™ pT7-VEE-mRNA-EGFP Vector | T7 |
| CAT#: GTVCR-WQ003MR | IVTScrip™ pT7-VEE-mRNA-FLuc Vector | T7 |
| CAT#: GTVCR-WQ87MR | IVTScrip™ pT7-VEE-mRNA-Anti-SELP, 42-89-glycoprotein Vector | T7 |
| Cat. No | Product Name | Type |
|---|---|---|
| CAT#: GTTS-WQ001MR) | IVTScrip™ mRNA-EGFP (Cap 1, 30 nt-poly(A)) | Reporter Gene |
| CAT#: GTTS-WK18036MR | IVTScrip™ mRNA-Human AIMP2, (Cap 1, Pseudo-UTP, 120 nt-poly(A)) | Enzyme mRNA |
| (CAT#: GTTS-WQ004MR) | IVTScrip™ mRNA-Fluc (Cap 1, 30 nt-poly(A)) | Reporter Gene |
| (CAT#: GTTS-WQ009MR) | IVTScrip™ mRNA-β gal (Cap 1, 30 nt-poly(A)) | Reporter Gene |
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