A distinctive feature found in eukaryotic mRNA is the presence of a cap structure at the 5' terminus, which is an important modification and specifically interacts with numerous cellular proteins to involve in pre-mRNA splicing, RNA export, translation initiation and RNA turnover. With access to advanced instruments, fit-for-purpose laboratories, and professional knowledge, Creative Biolabs offers a comprehensive range of mRNA services, including but not limited to mRNA synthesis, modification, delivery, stability test, and mRNA-based cell reprogramming. Amongst, mRNA 5' fluorescent cap is an available and delicate service item in our mRNA modification portfolio.
Synthetic analogs of the mRNA cap have been proven to be valuable tools in the study of cap-associated processes without interfering with their biological functions, such as radioactive, fluorescent, and other spectroscopic labels. Fluorescence is a potent technique to obtain visualized information about the behavior and distribution of molecules within biological systems, usually aided by the fluorescence microscopy or Förster resonance energy transfer (FRET). Fluorescence mRNA also proves invaluable for mRNA dynamics, interactions and other biophysical studies. The most commonly used fluorophores in mRNA capping are low molecular weight groups that can not interfere with eIF4E binding interactions, such as anthraniloyl (Ant), N-methylanthraniloyl (Mant) and trinitrophenyl (TNP). These dyes are environmentally sensitive fluorophores that emit a stronger signal upon binding to a target mRNA. Nucleotides labeled with these tags can closely mimic naturally occurring nucleotides in their interactions with molecular targets.
Creative Biolabs offers a variety of strategies for the covalent linkage of fluorophores to RNA, mostly focusing on cotranscriptional or posttranscriptional enzymatic labeling approaches.
In vitro transcription (IVT) RNA is subjected to a dedicated enzymatic capping reaction. The capping enzymes are produced recombinantly in E. coli and developed from capping apparatuses of different eukaryotic organisms or DNA viruses (such as vaccinia virus). Usually, this method offers the advantage of the capability of 100% capping on a large quantity of IVT.
Fig.1 Schematic representation of post-transcriptional capping. 1
In this way, cap analogs are added directly to the IVT at the 5' end by RNA polymerases with relaxed substrate specificity (e.g. T3, T7 or SP6 RNA polymerases). Therefore, this route allows for a single-step workflow and permits more flexibility to incorporate non-canonical cap structures (besides m7GpppG) because RNA polymerases also accept several modified or alternative cap analogs.
Fig.2 Schematic representation of co-transcriptional capping with different cap analogs.1
Inquire About Our ServicesA: A fluorescent cap is a synthetic analog added to mRNA that includes a fluorescent label for visualization and tracking in biological systems.
A: Fluorescent caps enhance the ability to study mRNA dynamics, interactions, and localization using fluorescence microscopy or FRET.
A: Common fluorophores include anthraniloyl (Ant), N-methylanthraniloyl (Mant), and trinitrophenyl (TNP).
A: The cap can be added post-transcriptionally through enzymatic reactions or co-transcriptionally by incorporating cap analogs during in vitro transcription (IVT).
A: Yes, we offer custom-tailored capping strategies to meet specific research needs and applications.
A: Yes, fluorescent caps are suitable for in vivo applications to study mRNA behavior in living organisms.
A: The turnaround time depends on the complexity and customization but is typically a few weeks.
The article describes the development of five dinucleotide fluorescent cap analogues labeled with anthraniloyl (Ant) or N-methylanthraniloyl (Mant). These analogues are designed for mRNA labeling via in vitro transcription. Two of the analogues include methylene modifications in the triphosphate bridge, providing resistance against decapping enzymes Dcp2 and DcpS. The study evaluates the biological and spectroscopic properties of these compounds, showing acceptable fluorescence quantum yields, pH independence, and photostability. The cap analogues can be efficiently incorporated into mRNA transcripts and translated in vitro, making them useful for studying cap-related cellular processes and interactions with cap-binding proteins.
Fig.3 Sensitivity of Mant –labeled cap analog 2 to changes in local polarity caused by protein binding.2
| 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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