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Author ORCID Identifier

https://orcid.org/0000-0002-5057-6708

AccessType

Open Access Dissertation

Document Type

dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemistry

Year Degree Awarded

2023

Month Degree Awarded

February

First Advisor

Craig Martin

Subject Categories

Biochemistry, Biophysics, and Structural Biology | Laboratory and Basic Science Research | Nucleic Acids, Nucleotides, and Nucleosides | Other Analytical, Diagnostic and Therapeutic Techniques and Equipment | Therapeutics

Abstract

RNA is poised to revolutionize medicine. By simply changing the sequence, one therapeutic can be converted into a wholly new one, with little or no change in manufacturing and formulation. While a single mRNA vaccine produced at massive scale can treat billions, the re-codability of RNA will also enable the widespread growth of personalized medicines. T7 RNA polymerase is highly efficient at the synthesis of therapeutic RNA, but is known to produce unintended RNA impurities during synthesis. These products arise from the encoded RNA rebinding the enzyme such that its 3’ end serves as a primer for extension. This leads to double stranded RNA that can trigger the inflammatory innate immune response. We have established two approaches to dramatically reduce this secondary reaction during synthesis.

Rebinding of RNA can be inhibited by elevated salt, but promoter binding is also weakened. To restore promoter binding, we first introduce simple and novel modifications in the DNA to strengthen promoter binding. For example, the enzymatic introduction of a targeted gap in the DNA strengthens promoter binding. In this system, high salt transcription of RNA produces higher yield and purity. The approach can readily be applied to the synthesis of long mRNA.

Secondly, we strengthen promoter binding by covalently tethering chloroalkyl-modified promoter DNA to HaloTag® T7 RNA polymerase. The tethered system products. Tethering this binary catalyst to a solid support allows many rounds of synthesis, increasing yields per DNA, and will serve as a basis for continuous flow reactors.

Finally, we assess RNA structural features that modulate primed extension. Very short hairpins effectively prime extension, while hairpins too large to fit into the RNA-DNA binding cleft do not. We discuss both results in a structural and functional context, but in any case, the results will provide design guidance for those who aim to reduce primer extension by appropriate sequence re-design of RNA.

Together these approaches will be instrumental in enabling the production of higher yields of higher purity RNA for a diverse range of therapeutic applications.


DOI

https://doi.org/10.7275/31544120

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