Background

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. Additionally, 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 Cap I-structured mRNA.

Product Description 

Hi-yield T7 in vitro transcription reagent (Trace dsRNA) is designed for in vitro transcription of RNA with low dsRNA byproduct using T7 RNA polymerase mutant with optimizing transcription reaction system. The kit is capable of synthesizing mRNA using linear double-stranded DNA containing T7 promoter as template and NTPs as substrate. The kit is suitable for incorporation of modified nucleotides to obtain biotin labeled, dye labeled and radiolabeled RNA. The kit also can be applied in synthesis of co-transcriptionally capping mRNAs with cap analogs. The kit contains modified nucleotides N1-Me-pUTP as alternative.

Each standard reaction yields up to 150-200 μg of RNA from 1μg DNA template. Reaction size can be scaled up to yield milligram-level RNA as needed.

RNA synthesized from the kit is suitable for many applications including RNA structure and function studies, ribozyme biochemistry, RNase protection assays and hybridization-based blots, anti-sense RNA and RNAi experiment. RNA synthesized from the kit also is suitable for producing enzymatic production of capped RNA by vaccinia capping enzyme (HBP000606) and 2’-O-Methyltransferase (HBP000701) and being tailed up with Poly (A) polymerase (HBP000801) to improve the stability and translational competence of RNA used for in vitro translation, transfection, and microinjection.

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 bottleneck 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.

Key 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

Performance Data

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.

As shown in the figure above, through optimization, the mRNA yield and purity are at the top level using Haymes 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 Haymes Hi-Yield T7 In Vitro Transcription Reagents.

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.

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

Transportation and storage

Transportation under 0°C and storage at -25～ -15°C.

Ordering Information