The motivation.
Our lab is interested in one of science’s greatest questions: how life as we know it could have started from a mix of simple molecules. It’s tricky because the building blocks of life, like RNA and proteins, are fragile and tend to break down. To understand how it began or to build (de) novo life from scratch, we need ways to select and keep these unstable molecules from a pool of simple molecules (Fig. 1).
Using man-made molecules that were never alive, the group aims to solve the fundamental challenge of stabilizing molecules. Besides, our group aims to create an entity that is considered alive by all definitions: a compartment that can sustain itself, move around, self-replicate, and evolve to adapt to changes in its environment. 1-9 With our research, we dissolve the boundaries between chemistry and biology.8,9 While learning and understanding ways to convert chemical systems into biological ones, we can come closer to understanding what life is and how it could have emerged.
Figure 1. Template-based copying controls selection in chemical-fueled libraries and alters the coacervate’s physical properties when happening inside of coacervates. Fuel oligomerizes monomers, and spontaneous hydrolysis deoligomerizes the oligoanhydrides in solution. The interaction of monomers and oligoanhydrides with a template accelerates oligomerization by selectively concentrating longer oligomers. It simultaneously decelerates deoligomerization through a protection mechanism. Template-assisted formation of oligomers within coacervate-based protocells (green droplets) changes coacervate’s physical properties, like their ability to fuse. Green pictograms represent the DNA or RNA template, and red pictograms depict the oligomers.
The origin of the project.
The origin of this project dates back to a research proposal to Volkswagen Stiftung in 2018. That proposal asked how life began as a symbiosis between two chemical subsystems, one that stored information and another that generated energy. To address this puzzle, theoreticians and experimentalists linked their expertise. Boekhoven and Gerland, experts in exploring metabolic reaction cycles, joined forces with Holliger, an expert in the evolution of genetic material.
The first experiments.
While nature had hundreds of millions of years to develop by chance through chemical reactions between water and the bases, the first information containing sequences, like RNA, we do not have this time. So, to simulate and study this process in the laboratory more quickly, we had to design a model system of RNA bases, which were repeatedly joined and then separated again and again by so-called hydrolysis on a “fast” time scale (Fig. 2a). This challenge triggered Christine Kriebisch from the Boekhoven group. Inspired by the daily plastic poly terephthalate, the idea was to use terephthalic acid or its derivative isophthalic acid to build the backbone of the transient synthetic polymer, to which nucleobases could be attached. With the group’s expertise in chemical-fueled reaction cycles8-15, the first experiments ran within one hour. It quickly became clear that isophthalic acid was the ideal candidate. We found that a mixture of oligomers was built by combining analytic high-performance liquid chromatography and mass spectrometry (Fig. 2b). Importantly, those oligomers degraded within minutes. The start was made, and in the follow-up weeks, we obtained a complete kinetic characterization of the library and a quantitative understanding of the kinetics that dominate the evolution of the library by writing a simple kinetic model that predicts the concentrations of all reactants and products. With this simple model, we already found feedback mechanisms that delayed the decay of labile molecules.16 Our first findings with our newly developed model, “chemical-fueled dynamic combinatorial libraries”, were published in JACS16, but it was clear that was just the beginning of our journey. A journey that aims to discover mechanisms by which labile molecules stabilize far from equilibrium. With our chemical-fueled dynamic combinatorial libraries, we had a great model at hand to study this fundamental challenge.
Figure 2. Description of the chemically fueled library. a) Schematic representation of the concept of the chemical-fueled dynamic combinatorial library. Molecular structures of monomer and oligomer backbone are shown. b) HPLC chromatogram of the library.
Our curiosity kept us thrilled.
Now that we had a fundamental understanding16 and feeling about our oligomer library, we were specifically curious about understanding the template’s role in stabilizing labile molecules. Templation is vital to the RNA world hypothesis1,17 and in today’s biology. Thus, we updated the molecular design of isophthalic acid by attaching nucleobases. This construct mimics RNA monomers well, as the 6-atom backbone length matches the one of the ribose (Fig. 3a). We were happy to see that the complementary DNA template strongly influences oligomerization and deoligomerization kinetics (Fig. 4a). Some sequences are more resistant and, therefore, remain stable for longer. Excited, we pushed the boundaries, creating a diverse monomer-based library (Fig. 3). To do so, Christine Kriebisch joined forces with Dr. Oleksii Zozulia and Michele Stasi, who are highly skilled in synthesizing tricky molecules. Our effort was rewarded, as we learned that certain sequences of bases can influence the binding behavior of other bases. These are the templates that thus prove to be a very effective tool for taming the chemical chaos into a stable copying process. On top of that, we excitingly found that when these processes happen inside simple cell-like structures called protocells, protocell properties change, like their ability to merge. This interaction between molecule production and physical properties is a critical step in life.
Figure 3. Template-based copying in chemically fueled libraries. Schematic representation of the concept of the chemical-fueled dynamic combinatorial library and the role of the template. Molecular structures of monomers and oligomers are shown. The reactive molecule sites, recognition motifs (e.g., thymine or cytosine), or complete molecular structures are color-coded as follows: monomer acid (blue), thymine (red), cytosine (blue functional group), adenine (green), oligomer anhydride (red), fuel EDC (purple), and waste EDU (black). The pictograms for thymine oligomers are colored red, for cytosine oligomers blue, and the DNA template is colored green. The chemical reaction system converts a chemical fuel (EDC) into waste (EDU) while building up oligomers that are part of a transient dynamic combinatorial library. b) HPLC chromatogram at 31 min of T/C oligomers with (top) and without (bottom) 2 mM template (dA)10, or (dG)10. * T5 was not observed without template (dA)10.
Going beyond our limits
We set up a kinetic model to quantitatively understand how the template influences exactly oligomerization and deoligomerization kinetics (Fig. 4a, b). We joined forces with the experts in this field, Ulrich Gerland and his student Ludwig Burger. Remembering our first chats, our excitement about our experimental findings skipped over, and the first model was quickly set up. The power of the kinetic model became immediately clear. Our initial hypothesis was that templates amplify oligomers by accelerating oligomerization reaction. But it turned out we were wrong. Instead, templates simply invert the statistical oligomer distribution compared to the solution (Fig. 4b). This was an eye-opener for us, as we learned that template-assisted ligation is far more than increased oligomerization rates, it is about inverting statistical distributions. The discovery of this mechanism changed our biased view of how templates assisted oligomer ligation in the RNA world hypothesis.
Another boundary was to overcome that we have had so far indirect evidence from the kinetics that oligomers hybridize with a complementary DNA template, but we missed direct evidence. Specifically powerful in this context are tools of biochemistry, such as native gel electrophoresis18, which is used to identify the structure of DNA molecules, i.e., single, double, or triple-stranded. Double-stranded molecules migrate slower than single strands and are thus well separated.19 By then, we had no DNA lab and no experience in gel electrophoresis. Thus, Dieter Braun and his student Alexander Floroni, experts in the origin of life, biophysics, and biochemistry, were immediately on board to help. With their expertise and well-equipped DNA lab, we had our direct proof in hand after several optimization rounds (Fig. 4c).
Figure 4. Dynamic library composition in solution and on the template. a) The concentration of oligomers T3 as a function of time with and without 8 mM template (dA)10. A kinetic model was used to fit the data. b) The kinetic model calculated the fraction of library members in solution (left) and on the template (right) during the reaction cycle. (c) The gel of (dT)10 and T oligomers with (dA)35 in native gel electrophoresis. Dots on the gels are dust inclusions. The weaker but slow migrating band most likely originates from the self-association of (dA)35 at lower pH, high salt, and DNA concentrations.20
Conclusion
A team of experimentalists and theoreticians joined forces to understand ways to select and keep unstable molecules that form at the expense of energy from a pool of simple molecules. Our fundamental understanding is likely highly important to, in turn, understand how life began and helps scientists aiming to build (de) novo life from scratch. In the future, we will broaden the scope of our system by introducing replication, mutation, and, eventually, evolution in our system.
The details of this work are available from Nature Chemistry 2024
https://www.nature.com/articles/s41557-024-01570-5
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