Eukaryotic mRNA's 5' cap (m7GpppG) protects transcripts and aids translation; its fluorescent labeling enables visualizing biological dynamics, similar to how 3,5-dinitropyridin-2-yl groups detect metabolites.
Creative Biolabs' Fluorescent Cap Service uses proprietary synthesis, fluorophores (e.g., Ant, Mant), and high-purity Cap 1 modification to quantify mRNA localization/expression kinetics. It provides real-time data to solve long development cycles, delivery/translation issues, cut trial-and-error, and accelerate preclinical programs.
The Fluorescent Cap is a synthetic analog of the natural 5' cap, m7GpppNp, where the methylguanosine moiety is covalently linked to a small-molecule fluorophore, such as Anthraniloyl (Ant) or N-methylanthraniloyl (Mant). This low molecular weight label is chosen specifically to avoid steric interference with the eukaryotic initiation factor 4E (eIF4E) protein, which recognizes the cap to initiate translation.
The attached fluorophore allows the mRNA molecule to be visualized using standard fluorescence microscopy or FRET. By tracking the fluorescent signal, researchers gain direct, real-time insights into the location, movement, and eventual degradation of the mRNA therapeutic within a cellular or tissue context.
Fig.1 Schematic diagram of the FA probe structure of common cap-binding proteins.1
The complexity of synthesizing high-purity, functional, and fluorescently-capped mRNA requires a rigorous, multi-stage process to ensure optimal results suitable for preclinical studies.
| Stage | Activity |
|---|---|
| Project Consultation & Design | Required Starting Materials: Target sequence/plasmid design, desired fluorophore (e.g., Anthraniloyl, Mant), and intended delivery vehicle specifications (LNP/Polyplex). |
| Template Synthesis & IVT | Manufacture and linearize the DNA template. Execute In Vitro Transcription (IVT) using high-quality enzymes and modified nucleotides (e.g., Pseudouridine, 5-Methylcytidine) to enhance stability. |
| Fluorescent Capping & Purification | Perform Post-transcriptional Enzymatic Capping to ensure near 100% capping efficiency, or Co-transcriptional integration. Follow with stringent purification steps, typically HPLC, to remove unconverted templates and truncated transcripts. |
| Comprehensive QC & Characterization | Analysis of Cap Efficiency (e.g., using a Cap-dependent translation assay), structural integrity (denaturing gel), purity (A260/280), and residual contaminants (endotoxin, dsRNA). |
| Final Delivery & Technical Support | Lyophilization and packaging of the final product. Shipment with all quality control documentation and a detailed project report. |
| Estimated Timeframe: | The typical timeframe for this service ranges from 4 to 8 weeks, depending on the complexity of the template and the scale of the required synthesis (up to gram scales). |
Final Deliverables:
As an expert in biology, you demand precision and scalability. The Creative Biolabs' Fluorescent Cap Service is not just a modification; it is a meticulously engineered solution backed by a quality system built for both research discovery and therapeutic development. We provide a fully integrated pipeline that guarantees superior product quality and a streamlined path to your next milestone.
Custom-Tailored Capping Strategy
Dedicated optimization of the capping methodology (enzymatic or co-transcriptional) to perfectly match your gene, scale, and intended application (e.g., in vivo imaging vs. ex vivo cell sorting).
GMP-Focused Quality System
Creative Biolabs implements Quality-by-Design (QbD) principles and utilizes Process Analytical Techniques (PAT) throughout the entire synthesis and purification workflow, setting the foundation for future clinical transitions.
High-Fidelity Upstream/Downstream Processing
Efficient upstream design, including optimization of codon usage to maximize translational output, is coupled with stringent downstream HPLC purification to guarantee high purity of the final capped construct.
Comprehensive Quality Control (QC)
High-standard QC tools are employed to quantify and evaluate the quality of the final product, including assays for Cap efficiency, RNA integrity, and residual enzyme/DNA removal.
Guaranteed Functional Stability
We take precautions and follow the Hazard Analysis Critical Control Point (HACCP) approach, ensuring the stability of the final fluorescently capped mRNA construct is verified and guaranteed throughout the manufacturing and storage process.
A: The Fluorescent Cap tracks the mRNA molecule itself, providing instantaneous data on delivery and degradation kinetics. A reporter protein tracks the translated protein, which involves a significant delay and often requires stable protein accumulation. Our Cap Service provides data on the before and during phases, which is critical for vector optimization.
A: Absolutely. Our high-purity, large-scale synthesis capabilities are specifically designed to support robust in vivo animal studies. The low molecular weight fluorophores we use are suitable for tracking localization within organs and tissues using advanced imaging techniques.
A: We use proprietary, low molecular weight fluorophores (like Mant and Ant) known to minimize steric interference at the eIF4E binding site. While any modification can theoretically have an effect, our rigorous QC and functional assays are designed to detect and mitigate any significant impact. This commitment to functional integrity is a key reason to inquire with us before starting your synthesis.
A: Yes, customization is a core feature of our service. We offer a selection of different fluorophores with varied excitation/emission spectra to seamlessly integrate with your existing microscopy, flow cytometry, or FRET instruments.
Creative Biolabs' Fluorescent Cap Service provides an essential, high-resolution tool for every stage of mRNA therapeutic development. By delivering traceable, high-purity mRNA with verified Cap efficiency and diverse fluorophore options, we empower clients to accelerate delivery vector optimization, validate translational integrity, and obtain the rigorous data necessary for regulatory submissions.
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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