Messenger RNA (mRNA) synthesized via in vitro transcription (IVT) is crucial for a wide range of applications, with IVT mRNA capping methods playing an essential role in the translation of that mRNA. IVT mRNA has become integral in areas such as mRNA vaccines, gene therapy and protein therapy due to its ability to be precisely engineered and rapidly produced. This enables efficient and targeted delivery of genetic information to cells, revolutionizing medical treatments which is particularly highlighted by the success of mRNA vaccines in combating COVID-19.
The cap structure at the 5′ end of IVT mRNA is essential for its stability, efficient translation and protection from exonucleases, ensuring that the mRNA can effectively produce the desired protein within cells. This modification significantly enhances the overall efficacy and longevity of the mRNA in therapeutic and research applications. This article delves into the history of mRNA capping and explores mRNA capping methods.
A Brief History of mRNA capping
The discovery of mRNA capping occurred in the 1970s. Researchers found that eukaryotic mRNA molecules possess a unique structure at their 5′ end, which became known as the 5′ cap. This cap structure consists of a methylated guanine nucleotide attached via a 5′-5′ triphosphate linkage. There are several variations of 5′ caps, including Cap 0, Cap 1, and Cap 2, which differ in their methylation patterns. Cap 0, found in lower eukaryotic mRNAs, consists of an N7-methylguanosine linked to the first nucleotide. Cap 1, common in higher eukaryotes such as mammals, includes an additional methyl group at the 2′-O position of the first nucleotide. Cap 2, present in some viral and higher eukaryotic RNAs, has methyl groups at the 2′-O positions of both the first and second nucleotides. These cap structures play crucial roles in enhancing mRNA stability, translation efficiency and immune system evasion.
The understanding of mRNA capping, including cap structure, has evolved significantly since its discovery, leading to advancements in molecular biology and biotechnology. Today, mRNA capping is a fundamental step in the synthesis of IVT mRNA, crucial for applications such as mRNA vaccines and gene therapy. For a deeper dive into key milestones and discoveries, check out this detailed review on the history of IVT.
IVT mRNA capping methods
Two mRNA capping methods are used to cap IVT mRNA, each with its own set of advantages and limitations: 1) Enzymatic capping, or 2) co-transcriptional capping.
- Enzymatic capping of IVT mRNA involves the addition of a 5′ cap structure to the mRNA molecule using enzymes such as guanylyltransferase and methyltransferase. This method ensures high capping efficiency, involves two purification steps, and can create a cap 0 or cap 1 structure.
Figure 1. Workflow for enzymatic capping of IVT mRNA. It requires multiple purification steps and additional enzymes.
- Co-transcriptional capping of IVT mRNA involves the incorporation of cap analogs, such as dinucleotides (mCap or ARCA) or trinucleotides (CleanCap reagent AG), directly during the transcription process. This method uses a single purification step and, depending on the cap analog, ensures high capping efficiency and can create cap 0 or cap 1 structure.
Figure 2. Workflow for co-transcriptional capping of IVT mRNA. Unlike enzymatic capping, it requires only a single purification step and no additional enzymes.
IVT mRNA cap structures
Each of these mRNA cap analogs have unique features and cap structures:
- mCap (m7GpppG): This is a cap 0 analog where the cap structure includes a 7-methylguanosine (m7G) linked to the first nucleotide via a triphosphate bridge. However, it will be incorporated in both the correct and incorrect orientation during transcription, leading to some mRNAs (~50%) untranslatable. Additionally, the requirement for a high cap:GTP ratio (often around 4:1) gives capping efficiencies around 70% but reduces the overall yield of full-length mRNA transcripts. This cap analog is found in our mMESSAGE mMACHINE™ T7 Transcription Kits.
- ARCA (Anti-Reverse Cap Analog, m7(3’-O-Me)GpppG): ARCA is a cap 0 analog and is designed to be incorporated only in the correct orientation, preventing reverse incorporation. This ensures that all capped mRNAs are translatable. However, like mCap, it also requires a high cap:GTP ratio, which will significantly lower the yield of full-length mRNAs. This cap analog can be found in our mMESSAGE mMACHINE™ T7 ULTRA Transcription Kits.
- CleanCap Reagent AG: This cap 1 analog is a newer method that provides a highly efficient and precise co-transcriptional capping approach. It does not require a high cap:GTP ratio, thus maintaining a more optimal balance during transcription. This results in higher yields of correctly capped, functional mRNA with improved stability and translational performance compared to mCap and ARCA methods.
This cap analog is in our newest kit mMESSAGE mMACHINE™ T7 mRNA Kit with CleanCap™ Reagent AG that has been optimized to generate high yields of mRNA (>5 mg/mL) with over 95% capping efficiencies. It has superior performance compared to the mCap or ARCA cap analogs (Figure 3). Note that this cap analog requires an AG initiation sequence instead of the standard GG sequence. More information about this can be found in our application note, Generating high-quality mRNA for in vivo delivery.
Figure 3. Performance comparison of different capping strategies. This dataset shows that the CleanCap Reagent AG cap analog outperforms the ARCA cap analog in terms of mRNA yields, capping efficiency and cell function.
Concluding thoughts on mRNA capping
mRNA capping is a critical step in the synthesis of IVT mRNA, significantly impacting its stability, translational efficiency and overall efficacy in various applications. The evolution of capping methods, from enzymatic to co-transcriptional, has provided researchers and clinicians with tools to produce high-quality mRNA for therapeutic and research purposes.
Among these methods, the CleanCap™ technology stands out due to its highly efficient and precise co-transcriptional capping approach that gives high yields. The mMESSAGE mMACHINE™ T7 mRNA Kit with CleanCap™ Reagent AG utilizes this technology to generate high yields of mRNA (>5 mg/mL) with over 95% capping efficiencies using a simple workflow.
As the demand for mRNA-based therapies continues to grow, the mMESSAGE mMACHINE™ T7 mRNA Kit with CleanCap™ Reagent AG offers a superior solution for producing high-quality mRNA, ensuring that therapeutic and research applications can achieve maximum efficacy. The advancements enabled by CleanCap™ technology are poised to drive the next generation of mRNA-based treatments, meeting the evolving needs of modern medicine and biotechnology.
Discover all the solutions and resources we offer for mRNA research at thermofisher.com/mRNAresearch.
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