With the rapid development of biotechnology, mRNA therapy, as an emerging treatment method, has attracted much attention due to its strong programmability and fast response speed. In the fields of mRNA vaccines and gene therapy, efficiently synthesizing high-quality mRNA is a key step in achieving therapeutic goals. During this process, T7 RNA polymerase (T7 RNAP) plays an essential role, it can efficiently catalyze the in vitro synthesis of mRNA.
Although T7 RNAP plays a crucial role in mRNA synthesis, it often produces double-stranded RNA (dsRNA) as a byproduct in practice. dsRNA is one of the hallmarks of many viruses, and therefore it is easily recognized by intracellular dsRNA-binding proteins, triggering innate immune responses and inflammatory reactions. This means that if mRNA formulations contain a higher level of dsRNA, it may lead to unnecessary immune activation, which could affect therapeutic efficacy or cause serious side effects. Excessive dsRNA can also interfere with the normal function of mRNA, including translation efficiency and mRNA stability, indirectly affecting the effectiveness of mRNA therapy.
Figure 1: The impact of dsRNA byproducts and the optimized T7 RNAP reaction.
Therefore, developing and adopting effective methods to reduce the generation of dsRNA is a crucial part of mRNA technology development. Researchers have developed various strategies, including the use of improved T7 RNA polymerases, modification nucleotides, optimization of IVT transcription buffers, and downstream purification processes. Yeasen Biology utilizes the capabilities of its ZymeEditor directed evolution platform to continuously evolve the core enzyme raw material for mRNA in vitro synthesis—T7 RNA polymerase. They have developed a low dsRNA T7 RNA polymerase that suppresses the RDRP activity of T7 RNA polymerase, fundamentally reducing the formation of dsRNA, thereby significantly lowering the content of dsRNA.
Product Data
Yield: over 9 mg/mL
dsRNA Content: the content of dsRNA has been significantly reduced by at least 10 times
Capping Efficiency: over 99%
Low dsRNA T7 RNA Polymerase Screening Process:
Through method 1) the construction of a random library and FADS high-throughput screening method, a random mutation library of over 10^6 was screened. Additionally, method 2) semi-rational design and microplate technology were used to screen a site-saturated library. Both methods yielded low dsRNA T7 RNA polymerase mutants. Considering the dsRNA content while ensuring that other indicators are not reduced (such as integrity/capping efficiency/yield, etc.), a mutant was selected for commercial application.
Product Data
In different length application scenarios, low dsRNA T7 RNA Polymerase has shown a highly significant decrease compared to both WT T7 and competitive products, while ensuring that other indicators are not reduced.
|
Fragment Length |
T7 RNA pol |
Yield（mg/mL） |
Integrity（%） |
dsRNA Content (ng of dsRNA produced per 1ug of RNA) |
|
4K |
T7-WT |
12.4 |
88.3 |
0.4273 |
|
T7-low dsRNA |
12.1 |
89.9 |
0.0129 |
|
|
Company A |
11.0 |
87.8 |
0.0323 |
|
|
9K |
T7-WT |
9.5 |
81.9 |
2.9180 |
|
T7-low dsRNA |
9.0 |
82.0 |
0.0379 |
|
|
Company A |
7.2 |
78.7 |
0.1805 |
2.By reducing the amount of Cap analog input, even at a concentration of 2.5 mM, excellent capping efficiency can still be achieved across different fragment lengths compared to WT. Additionally, superior performance is also observed at the cellular level.
|
Cap analog input（mM） |
Capping Efficiency（%）-4K |
1K |
|
|
WT |
Low dsRNA |
WT |
|
|
10 |
100 |
100 |
100 |
|
5 |
100 |
100 |
100 |
|
2.5 |
100 |
100 |
99.7 |
Yeasen discusses an innovative study. Based on the results of this research, Yeasen has submitted patent applications to China and the United States.
Product Information
|
Product Name |
Catalog number |
Specification |
Volume |
|
CleascripTM T7 RNA Polymerase GMP-grade (low dsRNA, 250 U/μL) |
10629ES60 |
100 KU |
400 μL |
|
10629ES86 |
2500 KU |
10 mL |
|
|
10629ES96 |
25MU |
100 mL |
|
|
10629ES99 |
100MU |
400 mL |