ORCID

https://orcid.org/0009-0002-0997-8980

Date of Award

Summer 2026

Language

English

Embargo Period

7-9-2026

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Chemistry

Program

Chemistry

First Advisor

Dr. Maksim Royzen

Committee Members

Dr. Mehmet Yigit; Dr. Jia Sheng; Dr. Qiang Zhang; Dr. Maksim Royzen.

Keywords

Nucleotide Synthesis, Nonchromatographic Purification, Organic Chemistry

Subject Categories

Organic Chemistry

Abstract

The scope of RNA-based therapeutics continues to expand as more siRNA, mRNA, and CRISPR-based treatments are approved for various medical applications. Purification of synthetic RNA drugs is inherently challenging and requires considerable time and resources. New cost-effective and efficient purification strategies will expedite drug development process and will lower the drug costs. In this thesis I describe two generations of photolabile linkers for the non-chromatographic purification of therapeutic RNAs. In addition, the photolabile linker was utilized for regulation CRISPR-Cas9 gene editing. In the first part of the thesis, I describe the first generation of the photolabile coumarin-based linker for non-chromatographic purification of synthetic RNAs. After the solid phase RNA synthesis, cleavage from solid support is achieved by irradiation with 456 nm blue light for click-affinity isolation of the successful sequences. We report the effective purification of several model 20-nt RNA sequences, as well as purification of single guide RNA (sgRNA) for the CRISPR-Cas9 gene editing experiments. Purified RNAs were analyzed by HPLC and against control samples. Model RNAs containing phosphorothioate backbone were synthesized and tested. The purification protocol was then applied to sgRNA for CRISPR-Cas9 editing, targeting the pBR322 plasmid. The biological activity of the experimentally purified sgRNA was compared against traditional gel electrophoresis purified sgRNA. This formed the basis for the second work of this thesis, to synthesize a second generation RNA-anchoring photolabile linker for nonchromatographic purification. In the second part of this thesis, I developed a second generation of photolabile linker to anchor a uridine nucleotide in place of the first generation’s thymidine nucleotide. This second generation of photolabile linker was utilized to synthesize sgRNA for the CRISPR-Cas12a gene editing system, targeting eGFP plasmid. The purified sgRNA was compared against the sequence synthesized with the first-generation photolabile linker for isolated yield and purity. These RNAs were applied to in vitro CRISPR-Cas12a gene editing for activity comparison. Lastly, the experimentally purified sgRNA was applied to live 8 Human Epithelial Kidney (HEK293) cells which express GFP. GFP expression was analyzed by flow cytometry, to compare the activity of gel-purified sgRNA against the secondgeneration purified sgRNA. In the third part of this thesis, I developed a photolabile linker phosphoramidite which can be incorporated into sgRNA using solid phase synthesis. The synthesized sgRNA can be cleaved readily with irradiation of 456 nm light, to afford a temporal control method for rapid On-Off regulation of the CRISPR-Cas9 system. After synthesizing the 103-nt guide RNA for CRISPR-Cas9 containing the photolabile linker, the cleavage efficiency was established in vitro. Next, sgRNA samples with and without the photolabile linker were applied to CRISPR-Cas9 experimentation in live HEK293 cells to compare their activities. The control of the activity correlated to the time in which the sgRNA was irradiated. GFP expression was analyzed by flow cytometry for in cell application. This method for On-Off regulation via photocleavage has potential to lower the off-target effects during CRISPR-Cas9 experiments.

License

Creative Commons Attribution 4.0 International License
This work is licensed under a Creative Commons Attribution 4.0 International License.

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