Macromolecular crowding in cells influences processes such as folding, association and diffusion of proteins and polynucleic acids. Direct spatiotemporal readout of crowding would be a powerful approach for unraveling the structure of the cytoplasm and determining the impact of excluded volume on protein function in living cells. Here, we introduce a genetically encodable fluorescence resonance energy transfer (FRET) sensor for quantifying macromolecular crowding and discuss our application of the sensor in bacterial and mammalian cells.

This study shows that the role of DNA in the DNA-based enantioselective Diels-Alder reaction of azachalcone with cyclopentadiene is not limited to that of a chiral scaffold. DNA in combination with the copper complex of 4,4'-dimethyl-2,2'-bipyridine (Cu-L1) gives rise to a rate acceleration of up to 2 orders of magnitude compared to Cu-L1 catalysis alone. Furthermore, both the enantioselectivity and the rate enhancement prove to be dependent on the DNA-sequence. These features are the main reasons for the efficient and enantioselective catalysis observed with salmon testes DNA/Cu-L1 in the Diels-Alder reaction. The fact that absolute levels of stereocontrol can be achieved with a simple and weak DNA-binding complex like Cu-L1 is a clear demonstration of the power of the supramolecular approach to hybrid catalysis.

The unique chiral structure of DNA has been a source of inspiration for the development of a new class of bio-inspired catalysts. The novel concept of DNA-based asymmetric catalysis, which was introduced only five years ago, has been applied successfully in a variety of catalytic enantioselective reactions. In this tutorial review, the ideas behind this novel concept will be introduced, an overview of the catalytic chemistry available to date will be given and the role of DNA in catalysis will be discussed. Finally, an overview of new developments of potential interest for DNA-based asymmetric catalysis will be provided.

Macromolecular crowding affects the mobility of biomolecules, protein folding and stability, and the association of macromolecules with each other. Local differences in crowding that arise as a result of subcellular components and supramolecular assemblies contribute to the structural organization of the cytoplasm. In this Opinion article we discuss how macromolecular crowding affects the physicochemistry of the cytoplasm and how this, in turn, affects microbial physiology. We propose that cells maintain the overall concentration of macromolecules within a narrow range and discuss possible mechanisms for achieving crowding homeostasis. In addition, we propose that the term 'homeocrowding' is used to describe the process by which cells maintain relatively constant levels of macromolecules.

The enantioselective addition of water to olefins in an aqueous environment is a common transformation in biological systems, but was beyond the ability of synthetic chemists. Here, we present the first examples of a non-enzymatic catalytic enantioselective hydration of enones, for which we used a catalyst that comprises a copper complex, based on an achiral ligand, non-covalently bound to (deoxy)ribonucleic acid, which is the only source of chirality present under the reaction conditions. The chiral β-hydroxy ketone product was obtained in up to 82% enantiomeric excess. Deuterium-labelling studies demonstrated that the reaction is diastereospecific, with only the syn hydration product formed. So far, this diastereospecific and enantioselective reaction had no equivalent in conventional homogeneous catalysis.

A new approach to DNA-based asymmetric catalysis is presented, which gives rise to very high enantioselectivities (up to 99% ee) in the copper catalyzed Diels-Alder reaction in water.

The cell is highly crowded with biomacromolecules, and the excluded volume influences processes such as diffusion, folding, conformation, and aggregation or association of proteins and polynucleic acids. In Escherichia coli, the values reported for the total macromolecular content range from 200 to 400 mg/mL. Knowledge of the macromolecular crowding is needed to understand behavior and especially interactions of biomolecules in vivo, be it for drug development, fundamental knowledge, or to support computational efforts to model the living cell. Direct spatiotemporal readout of the crowding would be a powerful asset to unravel the structure of the cytoplasm and the impact of excluded volume on protein function in living cells. Here, we introduce a Förster resonance energy transfer (FRET) sensor for quantification of the macromolecular crowding and apply the sensor in living cells.

Cells are highly crowded with proteins and polynucleotides. Any reaction that depends on the available volume can be affected by macromolecular crowding, but the effects of crowding in cells are complex and difficult to track. Here, we present a set of Fö rster resonance energy transfer (FRET)-based crowding-sensitive probes and investigate the role of the linker design. We investigate the sensors in vitro and in vivo and by molecular dynamics simulations. We find that in vitro all the probes can be compressed by crowding, with a magnitude that increases with the probe size, the crowder concentration, and the crowder size. We capture the role of the linker in a heuristic scaling model, and we find that compression is a function of size of the probe and volume fraction of the crowder. The FRET changes observed in Escherichia coli are more complicated, where FRETincreases and scaling behavior are observed solely with probes that contain the helices in the linker. The probe with the highest sensitivity to crowding in vivo yields the same macromolecular volume fractions as previously obtained from cell dry weight. The collection of new probes provides more detailed readouts on the macromolecular crowding than a single sensor.