In vitro transcription of tRNASec and its effects on human cells

Author

• Safi Eddine M. Khiati, University at Albany, State University of New YorkFollow

ORCID

Safi Eddine Khiati (0009-0002-7294-7829) - My ORCID

Date of Award

Summer 2026

Language

English

Embargo Period

7-29-2028

Document Type

Master's Thesis

Degree Name

Master of Science (MS)

College/School/Department

Department of Biological Sciences

Program

Biology

First Advisor

Thomas J. Begley

Committee Members

Gabriele Fuchs, Shenglong Zhang, Andres J. Melendez

Keywords

tRNA, tRNASec, tRNASelenocysteine, Selenocysteine, Selenoproteins, MLC-seq, T7 termination, oxidative stress, ROS, reactive oxygene species, antioxidant

Subject Categories

Molecular Biology

Abstract

Reactive oxygen species (ROS) are produced during metabolism and can cause DNA, protein and lipid damage. ROS levels can be mitigated by cellular antioxidant systems that include catalase, superoxide dismutase (SOD), and the selenocysteine-containing glutathione peroxidases (GPxs) and thioredoxin reductases (TXNRDs). There are 25 selenocysteine containing proteins with 18 dedicated to ROS mitigation and maintaining redox balance, with their translation requiring specialized cis (internal stop codon, SECIS element) and trans factors (tRNA^Sec and translation factors). We used T7 RNA polymerase to in vitro transcribe (IVT) tRNA^Sec (unlabeled and fluorescently labeled), purified it and transfected it into human embryonic kidney cells (HEK293G). We have shown successful in vitro transcription and purification of tRNA^Sec and the control tRNA^Leu(CAG). T7-mediated in vitro transcription commonly produces heterogenous N+1 products. We developed chemically modified DNA templates that should force transcription termination with single-nucleotide accuracy, with mixed success. We have used fluorescently labeled tRNA^Sec to demonstrate successful transfection of the IVT-tRNA^Sec into human cells. Further studies will be used to force precise T7 transcription termination and assess the effects of IVT-tRNA^Sec in transfected HEK293G cells under normal and ROS-stress conditions. Our work is important because it is developing a novel agent with the potential to mitigate ROS damage as a cellular antioxidant.

License

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

Recommended Citation

Khiati, Safi Eddine M., "In vitro transcription of tRNASec and its effects on human cells" (2026). Electronic Theses & Dissertations (2024 - present). 526.
https://scholarsarchive.library.albany.edu/etd/526