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Fluorescent Cap

Background Fluorescent Cap Services Highlights FAQs Published Data

Background

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.

  • Importance of mRNA Cap
    Almost all eukaryotic mRNAs have a cap at the 5' terminus, which is a unique residue composed of a 7-methylguanosine moiety connected to the mRNA via a triphosphate linkage, m7GpppGpNp(Np)n. This specific methylated modification plays important roles in all stages of mRNA metabolism: synthesis, splicing, nucleocytosolic transport, intracellular localization, translation, and turnover.
    • mRNA cap protects the transcript from degradation.
    • mRNA cap helps the ribosome attach to the mRNA through recruiting translation factors.
    • mRNA cap functions as a protective group from 5' to 3' exonuclease cleavage and a unique identifier allows for a regulated degradation mechanism, enhances pre-RNA splicing and directs nuclear export.
  • Fluorescent Cap of mRNA

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.

Fluorescent Cap Services

Creative Biolabs offers a variety of strategies for the covalent linkage of fluorophores to RNA, mostly focusing on cotranscriptional or posttranscriptional enzymatic labeling approaches.

  • Post-transcriptional capping

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.

Diagram of the enzymatic capping reaction for IVT RNA.

Fig.1 Schematic representation of post-transcriptional capping. 1

  • Co-transcriptional capping

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.

Diagram of co-transcriptional capping with multiple cap analogues.

Fig.2 Schematic representation of co-transcriptional capping with different cap analogs.1

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Highlights

  • Custom-tailored capping strategy to meet specific application and program needs
  • Wide variety of capping moieties (different fluorophores) and mRNA purification options
  • Affordable custom synthesis up to gram scales of mRNA and long RNA

FAQ

Q: What is a fluorescent cap in mRNA?

A: A fluorescent cap is a synthetic analog added to mRNA that includes a fluorescent label for visualization and tracking in biological systems.

Q: What are the benefits of using a fluorescent cap?

A: Fluorescent caps enhance the ability to study mRNA dynamics, interactions, and localization using fluorescence microscopy or FRET.

Q: What types of fluorescent labels are available?

A: Common fluorophores include anthraniloyl (Ant), N-methylanthraniloyl (Mant), and trinitrophenyl (TNP).

Q: How is the fluorescent cap added to mRNA?

A: The cap can be added post-transcriptionally through enzymatic reactions or co-transcriptionally by incorporating cap analogs during in vitro transcription (IVT).

Q: Can Creative Biolabs customize the fluorescent cap?

A: Yes, we offer custom-tailored capping strategies to meet specific research needs and applications.

Q: Can fluorescent caps be used in living organisms?

A: Yes, fluorescent caps are suitable for in vivo applications to study mRNA behavior in living organisms.

Q: What is the turnaround time for producing fluorescently capped mRNA?

A: The turnaround time depends on the complexity and customization but is typically a few weeks.

Published Data

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

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Hot IVT Vectors

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

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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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References

  1. Muttach, F.; et al. Synthetic mRNA capping. Beilstein journal of organic chemistry. 2017, 13(1): 2819-2832.
  2. Ziemniak, Marcin, et al. "Synthesis and evaluation of fluorescent cap analogues for mRNA labelling." RSC advances 3.43 (2013): 20943-20958.
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