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Author ORCID Identifier

https://orcid.org/0000-0001-9284-745X

AccessType

Open Access Dissertation

Document Type

dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemistry

Year Degree Awarded

2021

Month Degree Awarded

May

First Advisor

Craig T. Martin

Subject Categories

Biochemical and Biomolecular Engineering | Biochemistry | Biophysics | Biotechnology | Molecular Biology

Abstract

High yields of RNA (e.g., mRNA, gRNA, lncRNA) are routinely prepared following a two-step approach: high yield in vitro transcription using T7 RNA polymerase, followed by extensive purification using gel or chromatic methods. In high yield transcription reactions, as RNA accumulates in solution, T7 RNA polymerase rebinds and extends the encoded RNA (using the RNA as a template), resulting in a product pool contaminated with longer than desired, (partially) double stranded impurities. Current purification methods often fail to fully eliminate these impurities which, if present in therapeutics, can stimulate the innate immune response with potentially fatal consequences. This study establishes novel in vitro transcription and purification technologies for high yield synthesis and purification of only encoded RNA.

First, we demonstrate a simple and economical in vitro transcription method carried out under high salt with partially single stranded promoter DNA (pss[-5]). This inhibits all non-promoter specific activity, including RNA self-primed extension, and the system exclusively generates high yields of encoded RNA. Second, we establish a novel in vitro transcription system where promoter DNA and T7 RNA polymerase are co-tethered in proximity on beads to drive promoter binding and initiation, and high salt eliminates all RNA product rebinding. The system is robust and reusable up to at least three times generating more yield of encoded RNA than conventional methods. Third, we develop novel affinity purification methods for in vitro transcribed RNA. Immobilized capture DNA selectively purifies only the desired RNA from a heterogeneous pool of products. In a novel approach, we improve binding capacity by increasing the binding sites per DNA oligo bound on beads using rolling circle amplification. We also introduce a universal capture DNA purification system that can capture any sequence of RNA without changing initial system components. Finally, we present a novel RNA microfactory that establishes the first flow in vitro transcription technology. In this new approach, encoded RNA is generated and continuously removed from the reaction chamber. This prevents primer extension and thus dramatically reduces double stranded impurities. By dramatically reducing extension of the desired RNA product, we achieve a high yield of relatively monodisperse, correct RNA products.

DOI

https://doi.org/10.7275/22479893.0

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