An in Vitro Cytomimetic of In-Cell RNA Folding

Abstract

To discover the cytomimetic that accounts for cytoplasmic crowding and sticking on RNA stability, we conducted a two-dimensional scan of mixtures of artificial crowding and sticking agents, PEG10k and M-PER^TM . As our model RNA, we investigate the fourU RNA thermometer motif of Salmonella, a hairpin-structured RNA that regulates translation by unfolding and exposing its ribosome binding site (RBS) in response to temperature perturbations. We found that the addition of artificial crowding and sticking agents leads to a stabilization and destabilization of RNA folding, respectively, through the excluded volume effect and surface interactions. FRET-labels were added to the fourU RNA and Fast Relaxation Imaging (FReI), fluorescence microscopy coupled to temperature-jump spectroscopy, probed differences between folding stability of RNA inside single living cells and in vitro. Our results suggest that the cytoplasmic environment affecting RNA folding is comparable to a combination of 20 % v/v M-PER^TM and 150 g/L PEG10k.

Keywords: RNA folding; fast relaxation imaging (FReI); fluorescence resonance energy transfer (FRET); fourU RNA (4U RNA); in-cell RNA; thermal stability.