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Huntington's disease is a neurodegenerative disorder associated with the expansion of the polyglutamine tract in the exon-1 domain of the huntingtin protein (htte1). Above a threshold of 37 glutamine residues, htte1 starts to aggregate in a nucleation-dependent manner. A 17-residue N-terminal fragment of htte1 (N17) has been suggested to play a crucial role in modulating the aggregation propensity and toxicity of htte1. Here, we identify N17 as a potential target for novel therapeutic intervention using the molecular tweezer CLR01. A combination of biochemical experiments and computer simulations shows that binding of CLR01 induces structural rearrangements within the htte1 monomer and inhibits htte1 aggregation, underpinning the key role of N17 in modulating htte1 toxicity.

Changes of the extracellular milieu could affect cellular crowding. To prevent detrimental effects, cells use adaptation mechanisms to react to such conditions. Using fluorescent crowding sensors, we show that the initial response to osmotic stress is fast but imperfect, while the slow response renders cells more tolerant to stress, particularly in the presence of osmolytes.

The influence of the cellular milieu, a complex and crowded solvent, is often neglected when biomolecular structure and function are studied in vitro. To mimic the cellular environment, crowding effects are commonly induced in vitro using artificial crowding agents like Ficoll or dextran. However, it is unclear if such effects are also observed in cellulo. Diverging results on protein stability in living cells point out the need for new quantitative methods to investigate the contributions of excluded volume and nonspecific interactions to the cellular crowding effect. We show how new crowding sensitive probes may be utilized to directly investigate crowding effects in living cells. Moreover, we discuss processes where crowding effects could play a crucial role in molecular cell biology.

Precise secondary and tertiary structure formation is critically important for the cellular functionality of ribonucleic acids (RNAs). RNA folding studies were mainly conducted in vitro, without the possibility of validating these experiments inside cells. Here, we directly resolve the folding stability of a hairpin‐structured RNA inside live mammalian cells. We find that the stability inside the cell is comparable to that in dilute physiological buffer. On the contrary, the addition of in vitro artificial crowding agents, with the exception of high‐molecular‐weight PEG, leads to a destabilization of the hairpin structure through surface interactions and reduction in water activity. We further show that RNA stability is highly variable within cell populations as well as within subcellular regions of the cytosol and nucleus. We conclude that inside cells the RNA is subject to (localized) stabilizing and destabilizing effects that lead to an on average only marginal modulation compared to diluted buffer.