Poster abstracts

Poster number 34 submitted by Le Zhang

Monitoring RNA folding and degradation in living cells using the fluorogenic malachite green aptamer

Randall Reif (College of Pharmacy, University of Kentucky), Farzin Haque (College of Pharmacy, University of Kentucky), Le Zhang (College of Pharmacy, University of Kentucky), Peixuan Guo (College of Pharmacy, University of Kentucky)

Abstract:
The noncoding RNAs, including siRNA, miRNA, ribozyme, and riboswitch, have been found participating in the regulation of cell life cycle and their applications in medical and pharmaceutical areas have become routine practice. Although the folding, degradation, and intracellular half-life of RNA are interested in many studies, current methods to observe intracellular RNAs are extremely challenging. The common method to measure RNA half-life in vivo is the use of radioactive markers or fluorescence RNA labeling. The challenge is, after the degradation of RNA 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. Here we report a method to monitor RNA degradation in real time in living cells using fluorogenic RNA aptamer in combination with RNA nanotechnology. The RNA aptamer that binds Malachite Green (MG), the ribozyme that cleaves the hepatitis virus genome, and a siRNA for firefly luciferase were all integrated into a 3WJ derived from bacteriophage phi29 packaging RNA (pRNA) to generate RNA nanoparticles. MG is an organic dye which is not fluorogenic by itself, but it starts emitting fluorescent light when it binds to the RNA aptamer. The binding only happens when the RNA is folded in the correct conformation. Therefore, the MG aptamer fluorescence can be used as a measure of the degradation and folding of the RNA nanoparticles using epifluorescence microscopy and fluorescence spectroscopy method without lysing the cells. The half-life of the electroporated RNA nanoparticle containing MG aptamer was found to be 4.3 hours after electroporation into cells.

References:
1. Reif R, Haque F, Guo P. Fluorogenic RNA Nanoparticles for Monitoring RNA Folding and Degradation in Real Time in Living Cells. In press.
2. Shu D, Shu Y, Haque F, Abdelmawla S, Guo P. Thermodynamically stable RNA three-way junction as a platform for constructing multifunctional nanoparticles for delivery of therapeutics. Nature Nanotech. 2011; 6(10):658-67.
3. Haque F, Shu D, Shu Y, Shlyakhtenko LS, Rychahou PG, Evers BM, Guo P. Ultrastable synergistic tetravalent RNA nanoparticles for targeting to cancers. Nano Today. 2012. 7: 245—257
4. Guo P. The emerging field of RNA nanotechnology. Nature Nanotech. 2010; 5:833–842.

Keywords: RNA, fluorescence tag, aptamer