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The power of vitro transcribed (IVT) mRNA

The power of IVT mRNA technology became aware again with the latest development of covid-19 vaccines. Messenger RNA (mRNA) vaccine is a type of vaccine that uses mRNA molecules, which encode disease-specific antigens to trigger an immune response that will provide protection against future infections. Unlike traditional vaccines, mRNA vaccines are quickly designed and produced at a faster rate in a cell-independent process and are non-infectious and therefore safer.

In addition to the pharmaceutical industry’s growing interest in mRNA for vaccine development, RNA molecules have proven to be versatile tools for various applications in academic and industrial research. Such applications include biochemical and molecular characterization of RNA for RNA-protein interactions and structural analyses, generation of RNA aptamers, synthesis of functional mRNAs for expression, and generation of small RNAs for alteration of gene expression (e.g., guide RNAs, RNAi). Furthermore, the use of in vitro synthesized RNA has been instrumental for CRISPR/Cas9 genome editing tools, generation of pluripotent stem cells, screening of RNA inhibitors, as well as development of RNA amplification-based diagnostics.

Synthesis of high-quality RNA in vitro requires template generation and transcription via RNA polymerases, NTPs and optimized buffers. In addition, production of mature mRNA requires 5’ capping and 3’ tailing. Most eukaryotic mRNAs require a 7-methyl guanosine (m7G) cap structure at the 5′ end and a poly(A) tail at the 3′ end that will increase mRNA stability, translation efficiency and decrease the immune response.

New England Biolabs (NEB) offers six in vitro RNA synthesis kits for convenient in vitro transcription (IVT) workflows. All kits have been optimized to generate reproducible yields of quality RNA and depending on the requirements, individual components are available from the NEB portfolio for template generation (e.g. Q5 polymerases and master mixes), in vitro transcription (IVT Kits, Capping and tailing reagents) and RNA purification (Monarch RNA Clean Up Kits).

HiScribe T7 mRNA Kit with CleanCap Reagent AG (#E2080) is the latest product developed by NEB for in vitro RNA transcription. Cap-1 mRNA synthesized from this kit is suitable for many applications including transfection, microinjection, in vitro translation, preclinical mRNA therapeutic studies, as well as RNA structure and function analysis. The natural Cap-1 structure of the synthesized mRNA results in maximal translatability and minimizes the cellular innate immune response of synthetic mRNA. In addition, the kit stands out for its streamlined workflow with single-step co-transcriptional capping that results in high capping efficiency (> 95%) and optimized high yields.

Depending on the scale of mRNA synthesis required and number of templates to be transcribed, NEB offers RNA capping options that include (1) post-transcriptional capping by standard synthesis with enzyme-based capping following the transcription reaction, (2) co-transcriptional capping by incorporation of a dinucleotide cap analog during transcription and (3) CleanCap co-transcriptional capping by using a cap analog such as CleanCap during in vitro transcription. Post-transcriptional capping offers the advantage of the capability of 100% capping on large quantity of in vitro transcripts. On the other hand, co-transcriptional capping using cap analog allows for a single-step workflow and the flexibility to incorporate non-canonical cap structures to the RNA.

A summary of all NEB’s in vitro RNA synthesis kits is presented below. For information regarding suitable applications for the HiScribe kits, capping options and strategy, as well as companion products for template generation, transcription, capping, tailing, and RNA purification please contact us at BioNordika.

NEBs in vitro RNA transcription kits

  • HiScribe T7 mRNA Kit with CleanCap® Reagent AG (includes capping) (#E2080)
  • HiScribe™ T7 ARCA mRNA Synthesis Kit (includes capping and tailing) (#E2060)
  • HiScribe T7 ARCA mRNA Synthesis Kit (includes capping) (#E2065)
  • HiScribe™ T7 High Yield RNA Synthesis Kit (#E2040)
  • HiScribe T7 Quick High Yield RNA Synthesis Kit (#2050)
  • HiScribe™ SP6 RNA Synthesis Kit (#E2070)