Publication Date

5-2023

Advisor(s) - Committee Chair

Moon-Soo Kim, Matthew Nee, Ajay Srivastava

Degree Program

Department of Chemistry

Degree Type

Master of Science

Abstract

As antibiotic resistance is one of the major health concerns in infectious diseases because of reducing the efficacy of antibiotics, rapid and sensitive detection of antibiotic resistance genes is critical for the effective treatment of infectious diseases. Polymerase chain reaction (PCR) is widely used to detect nucleic acids, but it requires additional steps involved in DNA denaturation and subsequent hybridization. A new class of DNA-binding domains called transcriptional activator-like effectors (TALEs) provide a novel scaffold for designing versatile DNA-binding proteins due to their modularity and predictability. Here, we developed a simple, rapid, and sensitive system for detecting antibiotic resistance genes utilizing novel TALEs as a new diagnostic probe along with 2D nanosheet graphene oxide. TALEs were engineered to recognize the specific double-stranded (ds) DNA sequences present in the tetracycline resistance gene (tetM), avoiding the need for DNA denaturation and renaturation. Quantum dots (QDs)-labeled TALEs are adsorbed on the GO surface, which will bring QDs in close proximity to GO. Due to the fluorescence quenching property of GO, QDs are expected to be quenched by GO via fluorescence resonance energy transfer (FRET). When target DNA is added, QD-labeled TALEs recognize and bind to the target dsDNA, leading to the conformational change, which would result in dissociation from the GO surface. Thus, the signal will be restored as FRET would no longer occur. Our sensing system was able to detect low concentrations of dsDNA sequences in the tetM gene after only 10- minute incubation, providing a limit of detection as low as 1 fM. This study demonstrated that our approach of using TALEs as a new diagnostic probe along with GO as a sensing platform can provide a highly sensitive and rapid method for direct detection of the specific dsDNA present in antibiotic resistance genes without requiring DNA amplification or labeling.

Disciplines

Biotechnology | Immunology and Infectious Disease | Life Sciences | Pharmacology, Toxicology and Environmental Health

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