Fluorescence-Activated Cell Sorting Enables Rapid Screening of RNA Polymerase-Promoter Interaction in E. coli

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• RNA polymerases are crucial for synthesizing RNA for therapeutic mRNA transcripts.
• Current methods for characterizing RNA polymerase promoters are slow and laborious.
• RNA polymerase specificity is essential for preventing abortive transcripts and byproducts.

Purpose of the Study:

• To develop a high-throughput strategy for rapid identification and characterization of RNA polymerase promoters.
• To overcome limitations of current laborious promoter characterization techniques.
• To accelerate RNA polymerase applications in mRNA manufacturing.

Main Methods:

• A two-plasmid screening platform was established in *E. coli* for promoter identification.
• Fluorescence-activated cell sorting (FACS) was employed for analyzing and sorting cell populations.
• Sequence verification was performed after cell sorting to confirm promoter functionality.

Main Results:

• The screening platform successfully sorted cell populations based on high fluorescence, validating its functionality with T7 RNA polymerase and three promoters.
• The system was evaluated using two novel RNA polymerases and their cognate promoters.
• The proof-of-concept demonstrated efficient identification of both RNA polymerase and promoter pairs.

Conclusions:

• The developed high-throughput strategy significantly accelerates promoter identification and characterization.
• This approach supports the optimization of RNA polymerase specificity for improved mRNA manufacturing.
• The platform facilitates advancements in therapeutic mRNA production by streamlining enzyme-promoter pairing.