Kinetic competition between theophylline sensitive ribozyme cleavage and termination in living cells by 3D orbital tracking-fluorescent cross-correlation spectroscopy.

Abstract

Synthetic RNA-based regulatory tools such as riboswitches are promising candidates for the realization of complex engineered genetic circuits. However, previous efforts to implement such tools in synthetic biology have yielded inconsistent results. Often the quantification of performance is measured using protein expression, even though these devices act at the RNA level. To overcome this, we use in vivo RNA labeling of Hemoglobin B in Human Epithelial Kidney T-Rex Cells to determine two objectives. First, we will determine whether a theophylline-dependent hammerhead ribozyme riboswitch alters gene expression by inducing early termination of transcription or via post-transcriptional mRNA cleavage. The second objective will use a self-cleaving hammerhead ribozyme to separate transcription elongation from termination. This will allow for the determination of transcription termination kinetics. Quantification for both objectives will be completed using 3D Orbital Tracking-Fluorescent Cross-Correlation Spectroscopy (3DOT-FCCS). To determine the levels of post-transcription mRNA cleavage, we will use Raster Image Correlation Spectroscopy (RICS) to measure mRNA stability via mRNA lifetime. Preliminary data suggests that the theophylline aptamer stabilizes the ribozyme, correlating to an increased RNA dwell time and termination. However, when the aptamer is absent, there is a decrease. This indicates early termination and premature transcript release from the DNA template are occurring with the ribozyme. In the future, we will examine the transcribing order of Intron 1 and Intron 2, splicing behaviors, post-termination splicing using RICS, and RNA lifetime.