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  • Post-transcriptional capping
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Home › Learning centers › mRNA and cDNA synthesis › mRNA synthesis › 5-prime capping of mRNA › Post-transcriptional capping

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Tech Note

Post-transcriptional capping

Post-transcriptional capping is when enzymatic modification at the 5’ end of mRNA is performed as a separate step after the in vitro transcription reaction.

Because enzymatic capping is patent-free, inexpensive, and has a high capping efficiency of 80–100% (Grudzien et al., 2004), many scientists use this approach for capping in vitro-transcribed mRNAs.

Reagents Procedure

Reagents  

One of the major capping enzymes, Vaccinia Capping Enzyme (VCE, Cat. # 2460), adds 7-methylguanylate cap structures (m7G or cap-0) to the 5’ end of in vitro-transcribed, uncapped mRNA. All reagents for setting up the capping reaction—10X Capping Buffer, GTP, and the methyl donor, S-adenosylmethionine (SAM)—are included with our VCE. The process takes less than one hour. 

Components Concentration Size*
Vaccinia Capping Enzyme 10 U/µl 500 U
10X Capping Buffer 10X 100 µl
S-adenosylmethionine (SAM) 32 mM 100 µl
GTP 10 mM 50 µl

*The 500 U size (Cat. # 2460A) supports 50 x 20 µl reactions. The 2,000 U size (Cat. # 2460B) is also available. 

mRNA Cap 2'-O-Methyltransferase (2’-O-MTase, Cat. # 2470) then uses SAM as a methyl donor to add a methyl group at the 2’-O position of the first nucleotide (+1) at the 5’ end of the mRNAs, resulting in the cap-1 structure.

Components Concentration Size*
mRNA Cap 2'-O-Methyltransferase 50 U/µl 2500 U
10X Capping Buffer 10X 100 µl
S-adenosylmethionine (SAM) 32 mM 100 µl

*The 2500 U size (Cat. # 2470A) supports 50 x 20 µl reactions. The 10,000 U size (Cat. # 2470B) is also available.

Procedure  

VCE and 2'-O-MTase: one-step vs. two-step protocol

VCE and 2’-O-MTase work sequentially. That is, mRNA requires the cap-0 structure created by VCE (or ARCA) before it can be a substrate for 2’-O-MTase. This scenario is the 2-step reaction shown in Figure 1. However, for the 1-step reaction, transfer of the methyl group by 2’-O-MTase can occur simultaneously with the VCE’s capping reaction (Figure 1). Details of the 1-step and 2-step protocols are included in the Data Sheet found on the product page.

Figure 1. Post-transcriptional capping procedure. The reactions performed by Vaccinia Capping Enzyme (VCE, Cat. # 2460) and mRNA Cap 2'-O-Methyltransferase (2'-O-MTase, Cat. # 2470) can be a 1-step or 2-step process. A full protocol is available in the Data Sheet.

Explore our comprehensive selection of in-vitro transcription products and learn more about their features.

Related Products

Cat. # Product Size License Quantity Details
6141 Takara IVTpro™ mRNA Synthesis System 1 Set USD $398.00

Takara IVTpro mRNA Synthesis System includes all components necessary from DNA template preparation to high-yield in vitro transcription reaction. Easily scale up to 10-fold (200 μl reaction volume) without affecting the expected mRNA yield. This kit includes the Cloning Kit for mRNA Template (Cat. # 6143) and Takara IVTpro mRNA Synthesis Kit (Cat. # 6144), which are also sold separately.

Notice to purchaser

This product is for in vitro diagnostic use. Takara products may not be resold or transferred, modified for resale or transfer, or used to manufacture commercial products without written approval from Takara Bio Inc. If you require licenses for other use, please contact us by phone at +81 77 .565. 6976 or from our website at https://www.takara-bio.co.jp/inquiry/ivd/. Your use of this product is also subject to compliance with any applicable licensing requirements described on the product web page. It is your responsibility to review, understand and adhere to any restrictions imposed by such statements. All trademarks are the property of their respective owners. Certain trademarks may not be registered in all jurisdictions.

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Affect of CleanCap Reagent AG (3’ OMe) on RNA yield and the expression of FLuc mRNA

Affect of CleanCap Reagent AG (3’ OMe) on RNA yield and the expression of FLuc mRNA

Addition of the CleanCap Reagent AG (3’ OMe) in IVT reaction has no significant effect on mRNA yield (Panel A). HEK293T cells transfected with mRNA containing a cap structure show significatly higher protein expression compared to cells transfected with mRNA without a cap (Panel B).

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Increasing IVT reaction volume has no effect on RNA yield or quality.

Increasing IVT reaction volume has no effect on RNA yield or quality.

The mRNA yield was proportional to the reaction volume up to 200 µl with no significant change in mRNA concentration (Panel A). Scaling up of the IVT reaction volume does not affect the quality of RNA product (Panel B).

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Effect of different RNA purification methods on RNA yield and protein expression.

Effect of different RNA purification methods on RNA yield and protein expression.

Purification of RNA product using LiCl was comparable to purification with NucleoSpin column when two elutions were performed (Panel A). HEK293T cells transfected with mRNA purified using LiCl shows protein expression comparable to mRNA purified using NucleoSpin columns (Panel B).

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6141: Takara IVTpro mRNA Synthesis System

6141: Takara IVTpro mRNA Synthesis System
6143 Cloning Kit for mRNA Template 10 Rxns USD $179.00

Cloning Kit for mRNA Template enables easy cloning of a template DNA into a pre-linearized plasmid (included in the kit) for downstream in vitro transcription reactions. The pre-linearized plasmid contains the T7 promoter, transcription initiation sequence (AGG), 5'- and 3'-UTRs (untranslated region), and poly(A) sequence. The kit also includes an In-Fusion Snap Assembly Master Mix for seamless cloning of the target DNA into the pre-linearized vector and a FLuc Control Fragment, consisting of 15 bp 3' and 5' In-Fusion sequences and Fluc CDS (1,683 bp).

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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6143: Cloning Kit for mRNA Template

6143: Cloning Kit for mRNA Template
6144 Takara IVTpro™ T7 mRNA Synthesis Kit 20 Rxns USD $248.00

Takara IVTpro mRNA Synthesis Kit enables the production of large amounts of mRNA through in vitro transcription using an optimized, highly efficient T7 RNA polymerase. The kit includes separate NTPs for easy optimization or replacement with modified nucleosides, such as pseudouridine. The kit synthesizes up to 200 μg or more of mRNA per 20 μl reaction, includes DNase I to digest the template DNA after the in vitro transcription reaction and a lithium chloride (LiCl) solution to purify the synthesized mRNA for downstream applications. 

The kit also includes a Positive Control Template (FLuc) plasmid containing the T7 promoter, transcription initiation sequence (AGG), 5'- and 3'-UTRs, FLuc CDS, and poly(A) sequence.

Easily scale up to 10-fold (200 μl reaction volume) without affecting the expected mRNA yield.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components Image Data

Back

Affect of CleanCap Reagent AG (3’ OMe) on RNA yield and the expression of FLuc mRNA

Affect of CleanCap Reagent AG (3’ OMe) on RNA yield and the expression of FLuc mRNA

Addition of the CleanCap Reagent AG (3’ OMe) in IVT reaction has no significant effect on mRNA yield (Panel A). HEK293T cells transfected with mRNA containing a cap structure show significatly higher protein expression compared to cells transfected with mRNA without a cap (Panel B).

Back

Increasing IVT reaction volume has no effect on RNA yield or quality.

Increasing IVT reaction volume has no effect on RNA yield or quality.

The mRNA yield was proportional to the reaction volume up to 200 µl with no significant change in mRNA concentration (Panel A). Scaling up of the IVT reaction volume does not affect the quality of RNA product (Panel B).

Back

Effect of different RNA purification methods on RNA yield and protein expression.

Effect of different RNA purification methods on RNA yield and protein expression.

Purification of RNA product using LiCl was comparable to purification with NucleoSpin column when two elutions were performed (Panel A). HEK293T cells transfected with mRNA purified using LiCl shows protein expression comparable to mRNA purified using NucleoSpin columns (Panel B).

Back

6144: Takara IVTpro T7 mRNA Synthesis Kit

6144: Takara IVTpro T7 mRNA Synthesis Kit
2460A Vaccinia Capping Enzyme 500 U USD $175.00

Vaccinia Capping Enzyme adds 7-methylguanylate cap structures (Cap-0) to the 5’ ends of uncapped RNA. Vaccinia Capping Enzyme (10 U/µl), 10X Capping Buffer, S-adenosylmethionine (SAM), and GTP are included.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components Image Data

Back

2460A: Vaccinia Capping Enzyme

2460A: Vaccinia Capping Enzyme
2460B Vaccinia Capping Enzyme 2,000 U USD $640.00

Vaccinia Capping Enzyme adds 7-methylguanylate cap structures (Cap-0) to the 5’ ends of uncapped RNA. Vaccinia Capping Enzyme (10 U/µl), 10X Capping Buffer, S-adenosylmethionine (SAM), and GTP are included.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components Image Data

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2460B: Vaccinia Capping Enzyme

2460B: Vaccinia Capping Enzyme
2470A mRNA Cap 2'-O-Methyltransferase 2500 U USD $72.50

The mRNA Cap 2'-O-Methyltransferase creates a Cap-1 structure, which promotes translation efficiency, increasing subsequent protein expression. mRNA Cap 2'-O-Methyltransferase (50 U/µl), 10X Capping Buffer, and S-adenosylmethionine (SAM) are included.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components Image Data

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2470A: mRNA Cap 2'-O-Methyltransferase

2470A: mRNA Cap 2'-O-Methyltransferase
2470B mRNA Cap 2'-O-Methyltransferase 10,000 U USD $250.00

The mRNA Cap 2'-O-Methyltransferase creates a Cap-1 structure, which promotes translation efficiency, increasing subsequent protein expression. mRNA Cap 2'-O-Methyltransferase (50 U/µl), 10X Capping Buffer, and S-adenosylmethionine (SAM) are included. 

Cat. # 2470B contains 4 of Cat. # 2470A. Please refer to Cat. # 2470A for complete product documentation and resources.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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2470B: mRNA Cap 2'-O-Methyltransferase

2470B: mRNA Cap 2'-O-Methyltransferase

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