Background and Significance
The fidelity of T7 RNA polymerase is a critical quality attribute in mRNA drug development, as transcriptional errors may lead to altered protein expression, reduced efficacy, or potential safety risks. Ensuring low error rates during in vitro transcription (IVT) is therefore essential for producing high-quality therapeutic mRNA.
Experimental Design
To evaluate the transcriptional fidelity of different T7 RNA polymerases, comparative studies were conducted in two experimental groups:
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Group 1: Comparison between Hieff T7 RNA Polymerase (Low dsRNA) and NEB T7 RNA Polymerase
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Group 2: Comparative analysis including Yeasen Wild-type T7 RNA Polymerase
In both groups, eGFP and luciferase mRNAs were synthesized by in vitro transcription using N1-methyl-pseudouridine (m¹Ψ-UTP) to better reflect mRNA therapeutic production conditions.
Methods
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In vitro transcription (IVT) of eGFP and luciferase mRNA
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Reverse transcription of RNA into cDNA
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Second-strand cDNA synthesis
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Library construction using an enzymatic fragmentation–based NGS library preparation kit
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High-throughput sequencing and bioinformatic analysis of mutation profiles
Mutation rates were evaluated across three major error types:
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Base mismatches
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Insertions
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Deletions
Results
|
Exp 1
|
T7 RNA Pol and Catalog No. |
Mismatch Rate (x10-6) |
Insertion Rate error/base (x10-6) |
Deletion Rate error/base (x10-6) |
RNA |
|
NEB T7 Pol (M0460T) |
338.85 |
12.16 |
38.00 |
eGFP |
|
|
Hieff T7 Pol - Low dsRNA (10628ES) |
328.66 |
12.61 |
42.55 |
||
|
314.46 |
12.08 |
43.93 |
|||
|
319.96 |
12.24 |
41.09 |
|||
|
NEB T7 Pol (M0460T) |
279.75 |
24.43 |
36.77 |
Luciferase |
|
|
Hieff T7 Pol - Low dsRNA (10628ES) |
279.00 |
26.41 |
19.03 |
||
|
287.03 |
28.44 |
21.03 |
|||
|
264.45 |
26.86 |
22.15 |
|||
|
Exp 2
|
T7 RNA polymerase and Catalog No. |
Mismatch Rate (x10-6) |
Insertion Rate error/base (x10-6) |
Deletion Rate error/base (x10-6) |
RNA |
|
NEB T7 Pol (M0460T) |
347.68 |
8.24 |
46.97 |
eGFP |
|
|
YEASEN Wild Type T7 Pol (10625ES) |
380.46 |
8.86 |
52.26 |
||
|
376 |
8.02 |
50.06 |
|||
|
378.61 |
8.09 |
51.07 |
|||
|
NEB T7 Pol (M0460T) |
301.10 |
8.09 |
17.63 |
Luciferase |
|
|
YEASEN Wild Type T7 Pol (10625ES) |
320.81 |
9.97 |
14.83 |
||
|
326.35 |
10.49 |
15.48 |
|||
|
321.59 |
10.10 |
16.06 |
NGS analysis demonstrated that:
-
NEB T7 RNA Polymerase,
-
Yeasen Wild-type T7 RNA Polymerase
-
Hieff T7 RNA Polymerase (Low dsRNA mutant)
exhibited comparable mutation rates across all three mutation categories (mismatch, insertion, and deletion).
No significant differences in transcriptional fidelity were observed among the tested enzymes under the experimental conditions.
Conclusion
These results indicate that Yeasen T7 RNA polymerase variants, including both the wild-type and Low dsRNA mutant, maintain an equivalent level of transcriptional fidelity. This high fidelity supports their suitability for mRNA therapeutic and vaccine development, where sequence accuracy is essential for product safety, consistency, and clinical performance.