Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Chemistry

Language

English (en)

Date of Award

1-1-2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

John-Stephen Taylor

Abstract

This dissertation focuses on the study of imaging mRNA in living cells. To achieve this research objective, three approaches have been utilized:: 1) Imaging of a transgenic mRNA tagged by multiple repeats of malachite green: MG) binding aptamer.: 2) Imaging of inducible nitric oxide synthase: iNOS) mRNA by strand-displacement activated Peptide Nucleic Acid: PNA) probes.: 3) Imaging of iNOS mRNA by binary fluorescently labeled PNA probes. The first approach was based on the work of our former lab member Dr. Huafeng Fang, who had constructed a multiple MG binding aptamer tagged transgene: Flag-mβ2AR-GFP-MGVI), which could also express a green fluorescence protein associated with an adrenergic receptor protein. It has been reported that the tagged aptamer sequence can increase the fluorescence of MG up to 2000 fold by binding to MG. Total RNA extract of the transfected MDCK cells has shown up to 22 times increase of fluorescence in the presence of MG. Confocal fluorescence imaging study has shown that in the presence of MG, cells expressing the transgene showed both the fluorescence of GFP and enhanced fluorescence of MG. A flow cytometry study detected that in the presence of MG and transfected cells showed 1.3 fold increase of fluorescence compared to the wild type MDCK cells. The next approach was to use strand-displacement activated PNA probes to detect the iNOS mRNA in living RAW 264.7 mouse macrophage cells. A probe constitutes of an antisense 23-mer fluorescein: FAM) labeled antisense PNA and a 17-mer Dabcylplus labeled complementary DNA was used. The fluorescence of the FAM was quenched when the two strands hybridized to each other. In the presence of target mRNA, the shorter strand was displaced by the mRNA, which has more base pairs complementary to the PNA. The fluorescence of FAM was restored and thus could be used to detect the mRNA. The probe has been shown to be able to detect the target DNA and in vitro transcribed mRNA in solution. Fluorescence in situ hybridization: FISH) showed that the probes showed 3.6: ± 1.8)-fold increase of fluorescence between stimulated cells expressing a high level of iNOS mRNA and non-stimulated cells. Cationic Shell-crosslinked Knedel-like: cSCK) nanoparticles were employed to deliver probes into living cells and the fluorescence of the stimulated cells observed by confocal microscopy increased 16.6: ± 7.9)-fold. RT-PCR was conducted to determine the absolute copy number of the iNOS mRNA in cells. The detected increase of iNOS mRNA after 18 hours of stimulation was around 100 times, and the actual copy number of the mRNA per cell was around 70000. These results reveal that the under our current systems, strand-displacement probes are not sufficient to report quantitatively on the mRNA copy numbers in living cells, but they can be used to achieve mRNA imaging in living cells. The third approach was to use fluorescent labeled binary PNA probes to image the iNOS mRNA in living RAW 264.7 cells. PNAs bearing FAM and Cy5 and targeting the adjacent sites of the iNOS mRNA were synthesized and had shown FRET signal upon binding to the DNA target and in vitro transcribed iNOS mRNA in solution. The probes were delivered into living cells by hybridizing to their partially complementary DNAs and forming complexes with cSCK nanoparticles. Fluorescent images were taken by confocal microscope. The matched probes showed FRET image for stimulated cells while control probes showed almost no FRET signal and non-stimulated cells treated with matched probes showed weak signal. The average FRET intensity detected in stimulated cells was 3.8: ± 0.9) times higher than in non-stimulated cells.

Comments

Permanent URL: http://dx.doi.org/10.7936/K73J3B0Z

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