Summary of achievements and conclusions of WP4 and WP5

Several important milestones have been reached towards the realization of container-, and metabolism-coupled chemical replicators based on DNA or PNA chemistry. Furthermore, replicators for real time monitoring by NMR, or by fluorescence (FRET) (e.g., in a microfluidics environment) have been constructed. In addition, a novel way to address the integration of replication and containment was demonstrated by preparing nanometer sized containers from artificially branched genetic materials. Moreover, studies on the self-construction of the azoarcus ribozyme from shorter pieces of RNA revealed that genetic materials can express anabolic autocatalysis, viz not acting as a template to direct the synthesis of copies via ligation steps, but acting as a metabolic autocatalyst on its own synthesis.

  • The most important milestone - one of the main reasons behind the PACE project in the first place - was to demonstrate that replicating (and eventually evolvable) systems can indeed be designed, supported to function, and monitored within programmable microfluidic technology: 1) redox-based ligation and subsequent replication of DNA has been successfully achieved compatible with proper microfluidic environment, and 2) an integrated metabolic-genetic (DNA) network produced in microfluidic fan reactor. FRET and NMR data of replicators were also theoretically modelled to derive information on the systems dynamics.
  • In relation to container coupling, it has specifically been shown that PNA duplexes have very significant stability in the presence of organic solvents, and therefore eventually may be incorporated directly in replication in oil droplets or vesicles.
  • Also PNA-lipid conjugates which bind to lipid vesicles have been produced.
  • Furthermore, in efforts to couple the genetic material to metabolism, in casu, directly to the anabolism of new vesicle components, DNA catalipids, in which the leaving group of a DNA replication step is a imidazole containing lipid have been constructed. While a completion of kinetic studies could not be achieved within project lifetime, DNA replication will now simultaneously lead to the growth of lipids and their supramolecular assemblies (micelles/vesicles).
  • In an other approach, PNA-Ru-bipyridyl complexes that in a photocatalysis reaction can produce fatty acids has been synthesized. In terms of chemical replicators it has been discovered that NMR can be used as a powerful method to follow replication in real time, and a software tool (SimFit) has been developed to simulate replications.
  • Finally, a FRET based PNA ligation/replication system has been set up. Unfortunately, attempts to implement and optimize this on a microfluidics platform was so far unsuccessful, due to technical problems in terms of PNA aggregation and adsorption to the walls of the microfluidics cells.

In terms of objectives and milestones, objectives O4.1-5 have been achieved as have milestones M4.1-2. The remaining objectives and milestones were all dependent on having a robust PNA template ligation/replication system that can be monitored by fluorescence detection, preferable using FRET.

Therefore, all efforts were invested in developing such a system. This was achieved, but do to time constraints could not be integrated into vesicle or microfluidics formats. Experiments were performed to examine the behaviour of PNA oligomers in and their possible manipulation in microfluidics systems. Fluorophore labelled but otherwise non-modified PNA oligomers showed extensive and uncontrollable aggregation in and adsorption to the microfluidics cell, and attemts to alleviate this problem using polyanionic (phosphonate modified) or polycationic (trimethyl lysine conjugated) PNAs was not successful.

A similar outcome holds for the objectives and milestones of WP5. The experimental demonstration of a fully operational minimal chemoton will certainly need several further years to come, but on the way to this goal results were achieved leading to a significatly improved understanding of artificial replicators. Tools like “kinetic NMR titration/SimFit” are expected to find many more applications for the chemistry of complex systems, most recently demonstrated in the analysis of a complex autocatalytic network leading to the self-construction of a functional ribozyme. The development of disulphide based DNA replicators and of DNA dodecahedral containment can be seen as chemical offspring from the funding from the IST/FET environment. The whole new field of “systems chemistry” now supported by COST and ESF at the level of networking and meetings point to the future of a mutualistic interplay of information science and chemistry, where both directions, chem4ict and ict4chem become operative.