ORCID

https://orcid.org/0009-0004-8865-2210

Date of Award

Spring 2026

Language

English

Embargo Period

5-1-2026

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Chemistry

Program

Chemistry

First Advisor

Jia Sheng

Committee Members

Ting Wang, Ken Halvorsen, Mehmet Yigit

Keywords

Light-controlled RNA, Photoswitch, RNA structure& function, Azobenzene, RNA modification

Subject Categories

Analytical Chemistry | Biochemistry | Biophysics | Nucleic Acids, Nucleotides, and Nucleosides | Organic Chemistry

Abstract

RNA molecules perform many important biological functions by forming complex three-dimensional structures stabilized by base pairing, tertiary interactions, and metal ion coordination. Because RNA function depends strongly on its structure, methods that allow reversible control of RNA conformation and activity are important for understanding RNA behavior and for potential therapeutic applications. However, achieving reversible and site-specific control of RNA structure, especially at the single-nucleotide level, remains challenging. In this dissertation, I developed and applied azobenzene-modified nucleotides as a chemical tool to enable optical and spatial control of RNA structure and function.

The first part of this dissertation focuses on establishing a photoswitchable nucleotide platform for light-controlled RNA regulation. An azobenzene-modified cytidine (Azo-C) was designed, synthesized, converted into a phosphoramidite building block, and incorporated into RNA oligonucleotides through solid-phase synthesis. Upon light irradiation, the azobenzene modification undergoes reversible trans–cis photoisomerization, changing the local geometry of the RNA. This switching behavior was observed in both nucleoside and oligonucleotide forms. The trans and cis states showed different effects on RNA duplex stability and reverse transcriptase-mediated primer extension, indicating that a single photoswitchable nucleotide can be used to reversibly control RNA-related enzymatic activity with light.

The second part of this dissertation extends this approach to more structured RNA systems. By introducing Azo-C into an FMN riboswitch aptamer and hammerhead ribozyme systems, I found that light-triggered structural perturbation produces different functional outcomes depending on the position of the modification. In the FMN aptamer, photoisomerization at a site near the ligand-binding region caused a clear light-dependent change in ligand affinity, whereas modifications at other positions had much smaller effects. In hammerhead ribozyme systems, perturbations at different positions led to distinct changes in catalytic activity. These results indicate that the effect of optical control depends strongly on local RNA structure and the position of the modification.

The final part of this dissertation shows that the effect of local geometric perturbation extends beyond simple on/off control. In a hammerhead ribozyme targeting the RHO transcript, incorporation of azobenzene at different single-nucleotide positions generated distinct catalytic behaviors, including faster cleavage, slower but sustained cleavage, and strongly suppressed activity. Additional substitution studies using smaller chemical groups showed that these effects depend on both the position and the structural properties of the modification. These observations suggest that local chemical perturbation at a single nucleotide can reshape RNA catalytic behavior and may provide useful design principles for tuning ribozyme activity in disease-relevant systems.

Overall, this dissertation establishes azobenzene-modified nucleotides as a platform for optical and spatial control of RNA structure and function. It further shows that RNA activity can be reversibly regulated by light and tuned through position-dependent chemical perturbation.

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This work is licensed under the University at Albany Standard Author Agreement.

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