Optimizing Transcription Efficiency through Mutations in the T7 RNA Polymerase

Authors

Claire N. Novak, University of Nebraska - LincolnFollow

First Advisor

Catherine Eichhorn

Second Advisor

Joseph Yesselman

Date of this Version

Spring 3-31-2025

Document Type

Thesis

Citation

Novak, C. 2025. Optimizing Transcription Efficiency through Mutations in the T7 RNA Polymerase. Undergraduate Honors Thesis. University of Nebraska-Lincoln.

Comments

Copyright Claire Novak 2025.

Abstract

The production of RNA is vital for many commercial and research purposes, with one of the more notable reasons being vaccine production. The RNA polymerase, derived from T7 bacteriophage, is commonly used for RNA production due to its ability to produce full-length RNA transcripts. However, researchers have faced limitations in optimizing this polymerase. For example, the polymerase generates heterogeneous RNA products that require additional purification steps during RNA production. Here, we selected three amino acid point substitutions based on a recent publication from Moderna that showed increased RNA purity compared to the wild type T7 RNA polymerase sequence. We created plasmid constructs with individual or multiple mutations, recombinantly expressed them in E. coli, and then purified the mutant polymerase from the rest of the proteins using affinity chromatography. We evaluated these constructs' purity and RNA synthesis activity compared to the wild type sequence. After testing the activity of a combination of mutations, we found that some T7 polymerase constructs showed increased purity of RNA products, and others showed decreased products and activities. After transcribing a 330-nucleotide RNA construct, we saw evidence that specific mutations could display increased activity and purity in an RNA sequence-dependent manner compared to the wild type. Identification of optimized T7 polymerase constructs that vastly increase RNA purity and yield can help to streamline the RNA production process by making it less costly and time-consuming to produce large quantities of RNA.