Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Development of multicellular organisms depends on spatial and temporal expression of certain genes. The spatio-temporal control is achieved by several factors involved in the key phases of gene expression. Understanding the dynamics of transcription is hence necessary to dissect these regulatory mechanisms. Several imaging techniques have been developed that provide insights into the transcriptional events but their applicability has been limited by factors like choice of reporter and target selection. We have developed an aptamer-based reporter system to detect changes in polymerase II (Pol II) dependent promoter activity in living cells and in real time. The RNA reporters known as IMAGEtags (Intracellular MultiAptamer GEnetic tags) consist of a series of tandem aptamers that bind small membrane-permeable ligands as reporters for promoter activity. If the ligands are labeled with fluorophores, such as Cy3 or Cy5, the FRET (Förster resonance energy transfer) interaction can be used to monitor aptamer presence in the cells. In this study IMAGEtags were expressed from several Pol II promoters in yeast cells that were incubated with the labeled ligands. Both inducible and constitutive promoter activities were detected with IMAGEtag reporters, which were used to image real time changes in transcription from an inducible GAL1 promoter in yeast. The FRET output from aptamer ligands bound to IMAGEtags reflected variations in gene expression due to changes in media composition. In another imaging approach, aptamers were tested in bacteria and yeast for their capability of increasing the intracellular concentration of a supplied ligand. The aptamers were shown to increase the total and free intracellular concentrations of their ligands. Thus, aptamers can be applied to imaging the spatial and temporal expression of genes in living cells that express them.
Ray, Judhajeet, "Aptamer sensors for live-cell imaging of Pol II promoter activity" (2014). Graduate Theses and Dissertations. 14283.