Engineering Molecular Sensors for The Detection of SOX9, RUNX2, and PPARG Transcription Factors
Open Access
- Author:
- Pera Mendes, Joao Paulo
- Area of Honors:
- Biomedical Engineering
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Pak Kin Wong, Thesis Supervisor
Jian Yang, Thesis Honors Advisor
Daniel J Hayes, Faculty Reader - Keywords:
- Nanosensor
Transcription Factor
DNA
Probe
SOX9
RUNX2
PPARG
FRET
iPSC
Molecular
Sensor
GNR - Abstract:
- In recent years, with the discovery and development of induced Pluripotent Stem Cells (iPSC), transcription factors (TF) have become an important tool in the process of stem cell differentiation. As such, detection and regulation of these essential proteins are of utmost importance to ensure desired development of iPSCs. With that goal, this study aimed at developing three molecular sensors capable of TF detection both in-vitro and in-vivo environments for SOX9, RUNX2, and PPARG. Using Fluorescence Resonance Energy Transfer (FRET) and TFs natural affinity towards to its binding site, a double stranded fluorophore tagged probe was created with the goal of contemplating three main aspects. Sensitivity, Specificity, and Incubation time dependency. Results have shown that the probe is capable of sensing TF with a range between 3-200 nM, as well as displaying specific behavior toward TFs. Additionally, probes have shown to operate after at least 30 minutes of incubation. A secondary detection model was proposed with complimentary single-stranded and double-stranded DNA probes. Results indicate a possible detection method for mRNA with ssDNA probes, while dsDNA probes showed non-discriminatory tendency towards multiple TFs. Finally, a brief study of Gold Nanorod (GNR) transfection and viability into cell’s cytoplasm was conducted. Results indicate good transfection rates, with decreased viability at higher GNR concentrations. Overall, our study revealed a sensitive, specific, and time dependent probe, capable of TF detection in a homogeneous, in-vitro solution.