An in Vitro Cytomimetic of In‐Cell RNA Folding

• Published in: Chembiochem : a European journal of chemical biology 2022-10, Vol.23 (20), p.e202200406-n/a
• Main Authors: Yoo, Hyejin, Davis, Caitlin M.
• Format: Article
• Language: English
• Subjects: Binding sites; Destabilization; fast relaxation imaging (FReI); Fluorescence; Fluorescence microscopy; Fluorescence resonance energy transfer; fluorescence resonance energy transfer (FRET); Folding; fourU RNA (4U RNA); in-cell RNA; Perturbation; Reagents; Ribonucleic acid; RNA; RNA folding; Spectroscopy; Stability; thermal stability
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.202200406

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‐PERTM. 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‐PERTM and 150 g/L PEG10k. Cellular environments can be reproduced by a combination of crowding and sticking agents: PEG10k and M‐PERTM in buffer solution. We investigated folding dynamics of the fourU thermometer, an RNA hairpin, in vitro and in cells. The best mimic of the cytoplasmic matrix for RNA is a mixture of 150 g/L PEGK10k and 20 % M‐PERTM.