During the third reporting period of the CLASSY project, significant results have been achieved according to the general project goals (Figure 1). The main results include:
In the last project stage, the consortium has kept advancing various concepts that relate catalytic synthetic approaches to the way in which catalysis occurs in biology. That implies, for example, the efforts carried out to tune catalytic activity through self-assembly processes, making use of nucleic acid analogues and peptide (NA-pep) chimeras where the peptide is responsible for the catalytic activity and the NA sequence to control the self-assembled structure. Extending such an approach to NA-pep replicating systems, allows us to control two complementary orthogonal functions such as replication and catalysis (Figure 1B). Currently, we are optimising the dynamics of both processes so that they reinforce each oher. In all these studies it is very important to understand the systems behaviour of the involved assemblies.
Another concept of biology that affects catalysis and has been investigated within the CLASSY project is that of compartmentalisation. The compartmentalisation of catalytic reactions can be performed in different ways, and CLASSY has explored various of them, including the use of microfluidic flow reactors, different types of NA-pep assemblies (e.g. liquid droplets as a consequence of liquid-liquid phase separation), and photoswitchable NA-based hydrogels. Importantly, in studies with peptide replication networks, it has been demonstrated that compartmentalisation in microfluidic flow reactors can lead to chemical oscillations. For studies in flow (Figure 1A), two different microfluidic setups have been developed. The first involves a microfluidic platform of multiple reactors that are connected and can be filled with enzyme-loaded beads. As a second strategy, an alternative way to deal with reaction networks (e.g. enzymatic ones) has been explored intensively in the third reporting period, and is based on a prototype of a microfluidic cell-like molecular assembly line where the syringe pumps, the microfluidic reactor, an ultraviolet–visible (UV-vis) spectrophotometer and a trapped ion mobility spectrometry – time of flight (timsTOF) mass spectrometer are all connected.
Finally, various teams of the CLASSY consortium have concentrated on the demonstration of life-like enzymatic reaction sequences and networks, employing microfluidic flow setups to facilitate the experiments (Figure 1C). On one hand, aiming for cascades with separated sites of reaction and also the possibility to control the activity with light, a light-dependent decarboxylase has been successfully combined with the biocatalytic hydrolysis of triolein in flow. On the other hand, for developing a prototype of a cell-like molecular assembly line, a suitable microfluidic setup (see above) has allowed ten distinct species to be mixed directly in the reaction chamber, where a cascade reaction takes place. Advanced trapped ion mobility spectrometry – time of flight (timsTOF) instrumentation was then used to quantitively monitor species of interest online. As a proof of concept of the first cell-like molecular assembly line, the pentose phosphate pathway has been investigated using free enzymes in flow conditions.
