? ? Today we would like to share a research paper published in The FEBS Journal titled Engineered T7 RNA polymerase reduces dsRNA formation by lowering terminal transferase and RNA-dependent RNA polymerase activities. The study used the Droplet Entrapping Microfluidic Cell-sorter (DREM cell) in conjunction with directed enzyme evolution to successfully modify T7 RNAP. The modified T7 RNAP can significantly reduce dsRNA production during transcription (to just 1.8% of the wild type) while markedly improving the overall efficiency and quality of synthesized mRNA. This breakthrough holds promise for revolutionary changes in gene therapy and vaccine development, driving further advancements in related technologies.

01 Research Background

Double-stranded RNA (dsRNA) is a common byproduct of in vitro transcription (IVT), which can activate the host cell's immune response (such as inhibiting translation through the PKR and OAS pathways), affecting the safety and efficacy of mRNA drugs. T7 RNA polymerase (T7 RNAP) is a core tool in IVT, but its terminal transferase activity and RNA-dependent RNA polymerase (RDRP) activity lead to dsRNA generation. Traditional methods rely on purification to remove dsRNA, but this study proposes modifying T7 RNAP itself to reduce dsRNA generation, thereby improving the quality and safety of mRNA products.

02 Method and technology platform

1. High-throughput screening technology (FADS)

Principle: Droplets are generated based on the DREM cell device droplet microfluidic method. The droplets contain lysing reagent, fluorescent substrate and E. coli cells expressing mutant target enzymes. The incubation process is used for cell lysis and RNA transcription, and then the droplets are introduced into the sorting chip for fluorescence detection and sorting.

Advantages: Ultra-high throughput (screening 10⁶-10⁸ droplets per day), low reagent consumption (pico-liter reaction volume).

Process: Build random mutation library → transcription in droplets → fluorescence detection → sorting of high fluorescence droplets (corresponding to low dsRNA mutants)

2. Molecular beacon and dual probe detection

Molecular beacon: Combined with the 3' end of RNA, the fluorescence intensity is positively correlated with the length of complementary region, which is used to distinguish complete RNA and dsRNA.

Dual probe system: 5 'end Cy5 probe detects RNA yield, 3' end FAM probe detects dsRNA content, and high yield and low by-product mutants are screened by fluorescence ratio.

3. Semi-rational design and structural analysis

Target selection: Targeted saturation mutations are performed on key regions of T7 RNAP conformational transition (such as helix C and H subdomains) by combining molecular dynamics simulation (GROMACS) and FoldX/PROSS software.

Key mutation sites: A70Q (stable extension conformation), F162S/A247T (reduction of RDRP activity), and K180E (reduction of template combination bias).

4. Function verification experiment

dsRNA detection: J2 antibody immunoblotting and ELISA quantification.

Immunogenicity assessment: Transfected RAW264.7 cells are used to detect IFN-β expression, and HEK293 cells are employed to assess EGFP translation efficiency.

Enzyme activity analysis: RDRP activity is detected by hairpin-structured substrate, and endomutase activity is evaluated by NGS analysis of RNA 3' end heterogeneity.

03 Main conclusions

1. Mutant screening and performance

Two million droplet libraries (Lib3 and Lib5) were generated using DREM cell, and key candidate mutants were successfully selected after two rounds of sorting

Single-point mutants: M1 (V214A), M7 (F162S/A247T), M11 (K180E), and M14 (A70Q) significantly reduced dsRNA production (3-7% of the wild type).

Composite mutant: M17 (A70Q/F162S/K180E), which showed the best performance, had only 1.8% dsRNA content compared with the wild type and only 0.007 ng/μg in the screening system.

This result has not only been verified in vitro experiments but also shown significant reduced immunogenicity and increased protein translation efficiency in cell experiments. After introducing the mRNA transcribed from the mutant into RAW264.7 cells, the expression levels of interferon β (IFN-β) mRNA and protein induced by the optimal mutant M11 product were only 9.7% and 12.93 pg/mL of those in the wild type, respectively. In HEK293 cells, the number of cells expressing EGFP from the mutant mRNA significantly increased, with stable fluorescence intensity. This indicates that reducing dsRNA can effectively relieve PKR-mediated translation inhibition, unlocking the therapeutic potential of mRNA.

2. Mechanism analysis

Terminal transferase activity: The wild-type T7 RNAP tends to add extra nucleotides (such as cytosine) to the 3' end of RNA, promoting complementary pairing; the mutant (such as M17) reduces this activity by more than 50%.

RDRP activity: The mutant has a reduced ability to bind to RNA templates, reducing the synthesis of complementary chains with RNA as the template.

Conformational stability: Mutations (such as A70Q) reduce the production of aborted RNA by stabilizing extended conformation, thus indirectly inhibiting dsRNA formation.

In order to understand the mechanism by which the mutant reduces dsRNA, we revealed through computer simulation and functional experiments that the mutant reduces dsRNA due to the reduced activity of RNA-dependent RNA polymerase (RDRP) and terminal transferase. This finding provides an important theoretical basis for further optimization of T7 RNAP in the future.

3. Application potential

The immunogenicity of mutant mRNA was significantly reduced (IFN-β expression decreased by 90%), and the translation efficiency was improved (the number of EGFP positive cells increased).

It provides a safer and more efficient T7 RNAP tool for mRNA vaccine/drug production.

Summary of research highlights

1. Random mutation library + DREM cell: A high-throughput sorting system based on molecular beacon fluorescent probe was constructed, and more than 107 droplets were screened by DREM cell, greatly improving the screening efficiency.

2. Micro-droplet reaction system: Cell lysis, transcription and fluorescent probe combination processes were carried out in the droplet to accurately capture low-yield dsRNA mutants, reducing reagent consumption by 100 times.

3. Breakthrough in enzyme engineering: In the T7 RNAP combinatorial mutant M17 (A70Q/F162S/K180E), dsRNA content was reduced to 1.8% of the wild-type level..

4. Cell experiment verification: M17 mutant significantly reduced the expression of immune factor IFN-β and improved the translation efficiency of EGFP protein.

5. Mechanism: The terminal transferase activity is the "culprit". The mutant reduces the activity of terminal transferase and RNA-dependent RNA polymerase (RDRP) to reduce the addition of non-template nucleotides at the 3' end, and block the complementary pairing of dsRNA.

Droplet Entrapping Microfluidic Cell-sorter (DREM cell)

DREM cell is a multifunctional, high-throughput, fully automated device that integrates droplet generation, droplet sorting, micro-injection of droplets, and droplet printing. It combines microfluidic droplet technology and electrophoretic sorting technology, comprising multipass fluorescence detection system, high-speed microscopic imaging system, auto-focus stage, micro-total analysis system, electrophoretic sorting system, image monitoring system, and powerful data processing system. It can be used for detecting, sorting, picking, and separating single cells, cell clusters, spheres, and organoid, providing accurate, efficient, convenient, and economical automated tools for research in disease detection, personalized therapy, vaccine development, monoclonal antibody preparation, and bioproduct manufacturing.