Product Overview

In vitro transcription (IVT) is the process of rapidly synthesizing large amounts of RNA under cell-free conditions using RNA polymerase, DNA as template, and NTP as substrate. The biological activity of mRNA in vivo can be enhanced by further structural modifications. For most mature eukaryotes, mRNAs must have a 5'-end cap structure (m7G) and a 3'-end Poly(A) structure, both of which are crucial for mRNA stability, in vivo translation efficiency, and immunogenicity.

Hzymes offers our brand-new series of T7 in vitro transcription kits. These kits feature a genetically engineered T7 RNA polymerase, offering an optimized high-yield RNA synthesis system. Addition ally, our kits include CAP GAG analogs and provide five independently packaged NTPs, allowing for the flexible substitution of modified nucleotide substrates. These kits are designed for one-step, rapid production of a substantial quantity of Cap1-structured mRNA.

Hi-Yield T7 In Vitro Transcription Reagent

Two Hi-Yield T7 In Vitro Transcription Reagents developed by Hzymes are systematically optimized for in vitro transcription reaction systems. This series of kits utilizes genetically engineered T7 RNA polymerase and linear double-stranded DNA templates containing T7 promoter sequences, along with NTPs and modified nucleotides as substrates, to transcribe downstream DNA sequences. These kits efficiently synthesize RNA transcripts, with 1 µg of template input producing 180-200 µg of mRNA.

Co-Capping T7 In Vitro Transcription Reagent

Hzymes has developed four T7 co-transcription kits optimized for one-step co-transcription reactions. This series of kits utilizes genetically engineered T7 RNA polymerase and linear double-stranded DNA templates containing T7 promoter sequences, along with NTPs, modified nucleotides, and CAP GAG analog as substrates, for transcription of downstream DNA sequences. These kits synthesize RNA transcripts in a co-transcriptional manner, efficiently incorporating modified nucleotide substrates and using cap analogs as primers for mRNA synthesis. Typically, 1 µg of template input can produce over 180 µg of Cap 1 mRNA.

Six independent NTPs for free substitution of modified nucleotides

The high immunogenicity of mRNAs synthesized in vitro from natural NTPs has been a major bottle neck limiting the clinical application of mRNA therapy. A great deal of research has been conducted by the scientific community over the past decade to address this issue. It has been found that the strategy of replacing natural nucleotides with modified nucleotides can effectively reduce the immunogenicity of mRNAs without affecting their translational properties, and this discovery has enabled the large-scale clinical application of mRNA therapy. Hzymes T7 In Vitro Transcription Reagent series provide individually packaged four natural nucleotides, A/G/C/U, as well as two modified nucleotides -- pUTP and N1-Me-pUTP, which can reduce the immunogenicity of mRNA and increase the efficiency of protein expression.

Two CAP GAG analogs available for one-step realization of Cap 1 structured mRNA

Hzymes provides two cap analogs, Cap GAG and Cap GAG (3'OMe), which can be freely selected by customers according to the process route, providing high activity and low toxicity Cap1 structured mRNA synthesis system. The Cap GAG analog has the structure m7G(5') ppp(5') (2'OMeA) pG and is suitable for co-transcription capping reactions. It exhibits higher capping efficiency compared to ARCA, enabling the efficient production of natural Cap 1 structured mRNAs. As a result, it helps reduce the immunogenicity of mRNA in vivo. The Cap GAG (3'OMe) analog has the structure m7(3'OMeG) (5') ppp(5') (2'OMeA) pG. Compared to CAP GAG, this cap analog has anti-reverse transcription properties and demonstrates better biological performance in some DNA templates or specific sequences. Cap GAG (3'OMe) typically generates >90% of the natural Cap 1 structure. It is compatible with Hzymes T7 RNA polymerase. The final capping efficiency depends on the cap analog reagent, DNA template, and the ultimate mRNA sequence. Secondary structures formed by RNA length and base composition may also influence the final capping efficiency.

Advantages

• Convenient Operation: The reaction system has been carefully optimized for efficient Cap1 mRNA synthesis in just one step, suitable for rapid setup of the reaction system.

• High Capping Rate: One-step co-capping process, capping rate can reach 95-100%.

• High Yield: 1 µg of template input can produce more than 180 µg of Cap 1 mRNA.

• High Purity: The series uses engineered T7 RNA polymerase with high specific activity, hence lower enzyme dosage. The purity of the mRNA product is stabilized at above 85%.

• High Expression Efficiency: Cap 1 mRNA generated has higher expression efficiency at the cellular level.

Product Components & Selection Guide

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Performance

Optimized Hi-Yield T7 In Vitro Transcription Reagents are compatible with templates of varying lengths, offering superior performance in terms of both yield and purity.

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As shown in the figure above, through optimization, the mRNA yield and purity are at the top level using Hzymes Hi-Yield T7 In Vitro Transcription Reagents compared with several competitors under 6 different length templates. Templates with GGG and AGG as starting sequences both exhibit higher yields and purity using Hzymes Hi-Yield T7 In Vitro Transcription Reagents.

Performance

Hzymes Hi-Yield T7 In Vitro Transcription Reagents are adapted to different modified nucleotides, and the products exhibit better yield and purity compared to those of competitors.

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Using LC-MS platform for detecting the capping efficiency of the Co-Capping T7 In Vitro Transcription Reagents, the capping efficiency exceeds 95%.

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Ordering Information

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