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Superior System Supporting Large-scale Production

High Yield High Purity High Capping Rate

Hi-Yield T7 In Vitro Transcription Reagent

Co-Capping T7 In Vitro Transcription Reagent

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 Cap1-structured mRNA.

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.

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.

Product Overview

Advantages

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.

Six independent NTPs for free substitution of modified nucleotides

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.

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

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.

T7 RNA polymerase Mix

10 X Transcription Buffer

ATP (100 mM)

CTP (100 mM)

GTP (100 mM)

UTP (100 mM)

N1-Me-pUTP (100 mM)

pUTP (100 mM)

CAP GAG(3’OMe)

(100 mM)

CAP GAG (100 mM)

Control DNA Template

(500 ng/μL)

company

198.2

145.3 75.60% 195.8 88.70% 176.8 81.10% 207.6 76.30% 201.2 76.00% 217.49 83.70%

81.10% 230.9 91.20% 203.8 87.20% 202.8 85.90% 198.9 84.50% 220.47 89.70%

228.5 74.50% 241.4 87.30% 240.1 83.20% 214.4 76.20% 211.5 78.40% 220.61 83.20%

160.9 76.00% 177.8 84.40% 166.7 76.10% 162.2 77.20% 156.4 80.50% 163.03 85.60%

216.3 76.40% 243.6 88.70% 230.7 82.90% 213.3 80.50% 204.1 80.10% 219.72 84.70%

Components HBP001505 HBP001506 HBP001507 HBP001508 HBP001509 HBP001510

Product Components & Selection Guide

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.

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.

EL(1000nt~) Product Luc (2000nt~) BK(4000nt~) SP(4000nt~)GGG (5000nt~) SP(4000nt~)AGG

Yield (µg) Purity% Yield (µg) Purity% Yield (µg) Purity% Yield (µg) Purity% Yield (µg) Purity% Yield (µg) Purity%

HZYMES

Performance

Z company

V company

J company

K company

UTP

pUTP

N1-Me-pUTP

UTP

pUTP

N1-Me-pUTP

UTP

pUTP

N1-Me-pUTP

UTP

pUTP

N1-Me-pUTP

UTP

pUTP

N1-Me-pUTP

90.10%

90.00%

89.60%

88.60%

87.30%

86.10%

87.20%

83.70%

82.10%

85.50%

87.60%

88.90%

87.10%

88.00%

87.70%

194.05

186.28

187.1

197.36

188.32

168.87

203.45

191.9

194.21

155.91

145.63

145.42

203.66

198.3

191.97

88.00%

86.70%

86.80%

73.60%

72.30%

68.10%

76.40%

74.90%

75.40%

79.90%

79.20%

77.70%

80.80%

79.50%

81.80%

231.89

210.14

215.62

197.64

176.22

153.61

231.21

212.05

218.05

165.2

131.57

152.22

232.16

211.04

213.75

Product Modified Nucleotides Luc (2000nt~) SP(4000nt~)GGG

Cap AG

Cap 3'OMe

pUTP

N1-Me-pUTP

pUTP

N1-Me-pUTP

100%

96.48%

96.16%

179

178

185

182.5

Modified

Nucleotides Cap1 β-Gal

Yield Purity% Capping Rate

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%.

Yield (µg) Purity% Yield (µg) Purity%

HZYMES

Ordering Information

Products Cat. No. Specification Storage

Temperature

Hi-Yield T7 In Vitro Transcription Reagent

(N1-Me-pUTP)

Hi-Yield T7 In Vitro Transcription Reagent

(pUTP)

Co-Capping T7 In Vitro Transcription Reagent

(pUTP, CAP GAG(3’OMe) )

Co-Capping T7 In Vitro Transcription Reagent

(pUTP, CAP GAG)

Co-Capping T7 In Vitro Transcription Reagent

(N1-Me-pUTP, CAP GAG(3’OMe))

Co-Capping T7 In Vitro Transcription Reagent

(N1-Me-pUTP, CAP GAG)

HBP001505

HBP001506

HBP001507

HBP001508

HBP001509

HBP001510

50T

-25℃ ~ -15℃

Marketing & Sales Center：Hzymes Building, Shanghai, China

Production Center: Precision Medical Industry Base, Wuhan, China

+86 021-60879177 | info@hzymes.com

www.hzymesbiotech.com

HZYMES BIOTECHNOLOGY CO.,LTD

"CAP GAG(3’OMe)"for making modified RNA molecules:

CAP: This likely refers to the 5' cap structure found on eukaryotic mRNA molecules. The cap structure consists of a modified nucleotide (7-methylguanosine) attached in a reverse orientation to the first transcribed nucleotide. This cap plays crucial roles in mRNA stability, nuclear export, and translation initiation.

GAG: This could refer to a specific sequence within an RNA molecule. GAG is a sequence of three nucleotides, which could encode for an amino acid (glycine-alanine-glycine, for instance) or might be a part of a larger sequence.

(3’OMe): This indicates a modification at the 3' end of the RNA molecule. "OMe" often refers to a methyl group attached to the 3' end of the RNA molecule. Such modifications can alter RNA stability, function, or interactions with other molecules.

Putting it together, "CAP GAG(3’OMe)" might describe an RNA molecule with a 5' cap structure, containing a specific sequence "GAG," and having a methyl group modification at the 3' end. This molecule could be used in research or therapeutics, where such modifications might be employed to enhance stability or modulate function.

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