Hieff™ T7 RNA polymerase (low dsRNA,250 U/μL)

Description:

This is an engineered T7 RNA polymerase variant (low dsRNA) derived from the wild-type T7 RNA polymerase and produced in Escherichia coli. It significantly reduces the production of double-stranded RNA (dsRNA) while efficiently incorporating cap analogs, and exhibiting highly efficient in vitro transcription (IVT) comparable to the wild-type T7 RNA polymerase. It catalyzes the 5'→3' synthesis of RNA on double-stranded DNA from its T7 promoter sequence (5'-TAATACGACTCACTATAG*-3') and uses NTPs as substrates.

Note: G* is the first base of the RNA transcript.

Source	Recombinant E. coli with T7 RNA Polymerase gene
Optimum Temperature	37℃
Storage Buffer	50 mM Tris-HCl, 1 mM EDTA, 10 mM DTT, 100 mM NaCl, 0.1% Triton X-100，50% (v/v) glycerin，pH7.9 at 25℃
Unit Definition	The amount of enzyme required to incorporate 1 nmol of [3H] GMP into the acid-insoluble precipitate within 1 hour at 37°C and pH 8.0 is defined as 1 unit.
Recommended Mg2+	30mM Magnesium Acetate

Items	Specification/Standard
Enzyme activity	250-300U/μL
Protein Purity	≥95%
Endotoxin	<20 EU/mg
Protease	Negative
Exonuclease	Negative
Nickase	Negative
RNase	Negative
E.coli host protein	<50ppm
E.coli host DNA	<10 fg/U
Mycoplasma examination	Negative

1. 8K Sequence Validation – WT-T7 vs. T7 (Low dsRNA)

2. Capping Efficiency & Cellular Function Validation:

For both 4K and 1K fragments, capping efficiency was evaluated at different cap analog input amounts, showing that the input can be reduced to as low as 2.5 mM without significant differences in capping efficiency or cellular function at 48 h. No significant differences were observed between T7 mutant and wild-type (WT) enzymes.

Customer feedback:

E

Figure 1. Testing of T7 RNA Polymerase mutants. dsRNA Content: The dsRNA content reduced by more than 10-fold (A). mRNA Integrity: Maintained at over 90% (B). 2K Fragment Capping Efficiency: Exceeded 99% (C, D). High integrity (E)

Figure 2. Evaluation of the immunogenicity of the IVT products in murine RAW264.7 cells (Figures 2A and 2B). IFN-β mRNA and protein levels were reduced in RAW264.7 cells transfected with mRNA produced by mutants compared to wild-type, indicating that mRNA synthesized by the wild-type T7 RNAP elicited the strongest immune response, while mRNA from the mutants showed a significantly reduced response.

Figure 3. The dsRNA content in mRNA synthesized with Hieff™ T7 RNA polymerase is lower than that in mRNA synthesized with the wild-type enzyme after cellulose treatment.

Shipping and Storage

The products are shipped with dry ice and can be stored at -15℃ ~ -25℃ for one year.