Carolin Holzhauser

RNA “Traffic Lights”: An Analytical Tool to Monitor siRNA Integrity

The combination of thiazole orange and thiazole red as an internal energy transfer-based fluorophore pair in oligonucleotides provides an outstanding analytical tool to follow DNA/RNA hybridization through a distinct fluorescence color change from red to green. Herein, we demonstrate that this concept can be applied to small interfering RNA (siRNA) to monitor RNA integrity in living cells in real time with a remarkable dynamic range and excellent contrast ratios in cellular media. Furthermore, we show that our siRNA-sensors still possess their gene silencing function toward the knockdown of enhanced green fluorescent protein in CHO-K1 cells.

Photochemically Active Fluorophore-DNA/RNA Conjugates for Cellular Imaging of Nucleic Acids by Readout in Electron Microscopy.

Imaging is a key research goal for the understanding of biological processes by proteins or nucleic acids inside the living cell.[1–3] By using classical confocal fluorescence microscopy it is possible to track those biomolecules that are labeled covalently with a suitable fluorophore[4, 5] or fluorescent protein.[6] Fluorescence microscopy as a noninvasive and nondestructive method (if the applied fluorophores are sufficiently photostable) offers the advantage to follow biological processes in real time over hours or even days. Over the last two decades, advanced fluorescence spectroscopy methods, such as stimulated emission depletion (STED),[7] photoactivated localization microscopy (PALM)[8] and others, have revitalized light-based microscopy for cell biology. Additionally, cell images obtained by electron microscopy allow the characterization of subcellular structures. The most complete picture would be drawn from imaging, if dynamic fluorescence and static electron microscopy were combined and correlated by applying a single label that gives readout in both types of microscopy. So far, this has been achieved to a certain extent for observing and localizing lipids and proteins inside cells but not for nucleic acids.