Fluorogenic RNA Nanoparticles for Monitoring RNA Folding and Degradation in Real Time in Living Cells

Abstract

Due to the discovery of more and more roles of cellular noncoding RNAs, the approaches for introducing RNAs including small interfering RNA (siRNA), micro RNA (miRNA), ribozyme, and riboswitch into cells for regulating cell life cycle and for the treatment of diseases have become routine practice. The understanding of RNA folding, degradation, and intracellular half-life after entering the cell is an intriguing question in biology and pharmacology. Currently, methods to detect RNA folding, degradation, and half-life in real time within the cell is extremely challenging. The common assay method to measure RNA half-life and degradation in vivo is the use of radioactive markers or fluorescence RNA labeling. The challenge is, after RNA becomes degraded or misfolded, the isotope or the fluorescence is still present in the cell, thus the signals are not a true indication of the presence of the RNA in the cell. The alternate method commonly used to measure RNA life is to isolate RNA from cells and distinguish between intact and degraded RNA by gel, chromatography, or capillary electrophoresis. However, when a cell is breaking down, ribonucleases (RNases) will be released from cell compartments, and degradation of small RNA in cell lysates occurs immediately after cell lysis. Here we report a method to monitor RNA degradation in real time in living cells using fluorogenic RNA in combination with RNA nanotechnology (Guo, 2010; Guo et al., 2012). The RNA aptamer that binds malachite green (MG), the ribozyme that cleaves the hepatitis virus genome, and a siRNA for firefly luciferase were all fused to the bacteriophage phi29 packaging RNA (pRNA) 3-way junction (3WJ) motif to generate RNA nanoparticles. The MG aptamer, the hepatitis B virus ribozyme, and the luciferase siRNA all retained their function independently after fusion into the nanoparticles. When the RNA nanoparticle is degraded, denatured, or misfolded, the fluorescence disappears. MG, which is not fluorescent by itself, is capable of binding to its aptamer and emitting fluorescent light only if the RNA remains folded in the correct conformation. Therefore, the MG aptamer fluorescence (in the presence of MG dye) can be used as a measure of the degradation and folding of RNA nanoparticles, the siRNA, the aptamer, and the ribozyme in the cell in real time using epifluorescence microscopy and fluorescence spectroscopy without lysing the cells. We show that the half-life (t½) of the electroporated MG aptamer containing RNA nanoparticle was 4.3 hours after electroporation into cells.