Single-Molecule Analysis of Synthetic RNA Devices in Living Cells By Fluorescence Cross-Correlation Spectroscopy

Synthetic regulatory elements that utilize RNA-aptamers offer the potential for programmable gene regulation. The nuclear environment of mammalian and other Eukaryotic cells offer numerous RNA processing events that could be exploited for regulation by synthetic elements. However, the temporal and spatial coordination of these events also presents challenges for design and evaluation. In addition, most devices are monitor at the protein level using reporter proteins, despite the fact that the devices are designed at the RNA level. Here, we present our approach to detect how synthetic regulatory elements alter the rates of several RNA processing events (transcription, splicing, cleavage, decay). To do this, we have embedded small molecule responsive RNA devices (theophylline aptazymes and splice-site aptamers) into two-color RNA-based reporters that track an intron and a terminal exon. The fluorescent signal from these reporters is monitored over time in live-cells at the point of transcription. The resulting time-series fluorescence data is deconvoluted for correlations that are then fit to a model to extract rates of transcription, splicing, cleavage at the poly-adenylation signal. The reporters can also be monitored away from the transcription site for half-life determination and localization studies. By monitoring these rates in the presence and absence of our effector molecule (theophylline), we can determine the actual point of action of these synthetic regulatory elements, which will guide future design and optimization.