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The Molecular Origins of Life, Munich conference addresses one of the most fundamental questions of science: How could life originate? With 24 talks by renowned scientists accompanied by Q&A, panel discussions and poster sessions, this international conference brings together scientists from a wide range of disciplines, namely: astronomy, bio-/chemistry, bio-/physics, geosciences and theoretical chemistry and physics. Only the combined effort from various disciplines can be successful in retracing the origins of life under experimental conditions and pave the way towards answering some of the most pertinent questions: What were the conditions on early Earth? Which chemicals could serve as precursors for the synthesis of living systems on Earth and/or on other planets? How did the very first genetic material in life forms develop? How could Darwinian evolution emerge? What were the first metabolic pathways? The conference's aim is to represent and to discuss the state of the art in the Origin of Life field.
The Molecular Origins of Life, Munich 2022 is sponsored by DFG funded Collaborative Research Center 235 Emergence of Life and the attendance to the event is free of charge.
Registration is mandatory to be able to attend the event both online and on site.
Spatial organization is a defining characteristic of all living organisms but has been challenging to engineer from the bottom up. By colocalizing specific reactions and separating others, synthetic compartments have the potential to improve the biochemical capabilities of artificial cells and cell-free systems. How can we encode spatial organization in a nucleotide sequence? To answer this question, my lab uses cell-free synthesis to produce molecules that have the ability to self-assemble or self-organize into compartments and patterns. I will present examples of our recent work on engineering spatial organization in biomimetic systems. In an effort to generate and engineer self-organized patterns in communities artificial cells, we have developed polymeric cell mimics that communicate diffusively with their neighbors. Cell mimics contain DNA-hydrogel compartments that code for diffusive protein and RNA signals, which support the formation of gene expression gradients in cell mimic communities. Furthermore, in addition to microfluidically produced cell mimics, we explore self-assembled and programmable coacervate systems to compartmentalize cell-free reactions. Our results show that a combination of biological and synthetic materials facilitates the engineering of spatial organization in biomimetic systems and will help integrate increasingly complex functions in artificial cells and cell-free systems.
Molecular self-assembly is when molecules combine into superstructures held together through non-covalent interactions. Over the last decades, supramolecular chemists have perfected this art, and we can now create Gigadalton structures in which each atom is placed with angstrom precision. More importantly, the unique properties of the emerging assemblies have found their way into everyday life, like, for example, the liquid crystals in our displays. Nevertheless, we are entirely overshadowed by biology when it comes to assembly with molecular building blocks. Indeed, the biological cell has the same molecular toolbox for creating structures; it also uses non-covalent interactions to hold molecules together. Biology uses another trick. Biological structures are governed not only by non-covalent interactions but also by reactions forming covalent ones. Arguably, molecular self-assembly offers the structures; chemical reactions govern the dynamics and functions of these structures. Biological structures are sustained and regulated in the non-equilibrium regime through chemical reaction cycles that convert energy. The implications, rules, and mechanisms there are poorly understood.
In this lecture, I will discuss my team’s effort to elucidate the rules of non-equilibrium self-assembly fueled by chemical reactions. Next, I will describe a simple yet versatile chemical reaction cycle that can be coupled to self-assembly to create chemically fueled assemblies. Finally, I will highlight chemically fueled droplets that can be created and our pathway towards the synthesis of life. We aim to understand the fundamental mechanisms of the properties we typically associate with life with these droplets. Mechanisms like compartmentalization, division, and evolution.
The most profound transition in the biochemistry of life is thought to have been the one from a primordial RNA-peptide world to the modern DNA-protein world. The reasons for this transition are a matter of speculation, but may have included changes in planetary conditions that disfavored long RNA molecules. Whatever the reasons, the original combination of both catalysis and replication in one class of molecules (RNA) gave way to the tripartite division of labor we still observe today, with proteins providing the catalytic activity, DNA serving as the information repository, and RNA mediating between them.
The change from RNA to DNA as the main repository of biological information seems chemically unchallenging, but there is a major obstacle on the path from peptides to proteins, the so-called protein folding problem. Chemical activity requires structure and whereas the peptides of the RNA-peptide world could use nucleic acids as scaffolds, proteins needed to start folding autonomously after most RNA scaffolds went extinct. Since folding is an exceedingly rare property in random polypeptides, an origin of folding by random concatenation of amino acids does not seem attractive. Instead, a few simple mechanisms have been substantiated that can lead from unstructured precursors to folded proteins. These mechanisms are a long-standing focus of our research.
Here we discuss an exploration of these mechanisms using fragments of ribosomal proteins as a starting point. The ribosome is the main survivor of the transition to the DNA-protein world, due to its irreplaceable function in the synthesis of new proteins, and its proteins provide the earliest examples of polypeptides on their way to folding autonomously. Our results suggest that repetition of building blocks was the most straight-forward path to structure, whereas the recombination of preoptimized building blocks was largely unsuccessful.
The replication of genetic information is closely linked to the origin of life. The molecular basis of replication is genetic copying, a reaction in which the sequence of a template strand is transmitted to a copy. In cells, genetic copying is catalyzed by polymerases, which, in turn, are encoded in genes, making the origin of replication a difficult evolutionary problem. One proposal how this problem may have been solved posits that RNA was the biocatalyst for its own replication. This proposal is part of the 'RNA world' hypothesis . Again, there is a dilemma, as the known RNA-based polymerases  are too large to have a reasonable probability of being formed spontaneously from nucleotides oligomerizing into random sequences. A simpler scenario relies on base pairing and chemical reactivity alone. In this scenario, nucleotides oligomerize to oligonucleotides, and template-directed chain extension reactions account for the transmission of genetic information. Experimental studies on enzyme-free genetic copying were pioneered by the Orgel group , and have been performed extensively by Szostak  and others. We have studied the factors limiting the yield of copying with activated ribonucleotides , and we then established in situ activation combined with organocatalysis to reduce inhibition . Recently, we reported dinucleotides as building blocks for copying . Building on this work and the results from the first enzyme-free replication of DNA sequences , we have now identified reaction conditions that lead to the copying of up to 12 nucleotides in an RNA template strand .
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Some of the most interesting open questions about the origins of life and molecular sciences center on chemical evolution and the spontaneous generation of new complex and functional chemical species. The spectacular polymers that underlay biology demonstrate an untapped, by modern science, creative potential. We hypothesized that prebiotic chemical evolutionary processes leading to biopolymers were not idiosyncratic one-off events. We have developed an experimental platform that accomplishes chemical evolution in the laboratory. We have observed empirical outcomes, some of which were not foreseen. We have constructed experimental chemical systems that: (i) undergo continuous recursive change with transitions to new chemical spaces while not converging, (ii) demonstrate stringent chemical selection, during which combinatorial explosion is avoided, (iii) maintain synchronicity of molecular sub-populations, and (iv) harvest environmental energy that is invested in chemical reactions. These results suggest that chemical evolution can be adapted to produce a broad array of molecules with novel structures and functions.
Over the past few centuries, biologists have revealed an exhaustive list of the molecular components of life and their interactions. So, why can’t we synthesize life from scratch? A critical challenge in understanding the synthesis and origin of life is to understand how bioactivity could be regulated in prebiotic environments. We are studying how Lipid membranes can serve as organizational platforms for riboregulation in prebiotically plausible synthetic systems. RNA can serve as both catalyst and information carrier, giving it central importance in the origin and synthesis of life. We recently demonstrated that RNA can interact directly with lipids in a sequence-dependent manner, and that this changes ribozyme activity (Czerniak and Saenz, 2022). Our observations demonstrate a functional interaction between RNA and lipids and gives a simple answer to a fundamental question surrounding the origin of life – how could primordial RNA molecules be regulated?
Czerniak T, Saenz JP. 2022. Lipid membranes modulate the activity of RNA through sequence-dependent interactions. P Natl Acad Sci Usa 119:e2119235119. doi:10.1073/pnas.2119235119
Several classes of biological reactions that are mediated by an enzyme-cofactor tandem can also occur without the enzyme and even without the cofactor under catalysis by metal ions – albeit significantly slower . These observations support the idea that precursors to some core metabolic pathways emerged under inorganic catalysis and were later refined by organocatalysis from cofactors and, eventually, by enzymes. Notably, transamination, the biological process by which ammonia is transferred between amino acids and α-keto acids, has been shown to be catalyzed efficiently by metal ions such as Cu2+, Ni2+, Co2+, and V5+, and its mechanism studied in detail . However, in biology this reaction is co-catalyzed by the cofactor pyridoxal phosphate (PLP) and an enzyme, without help from metal ions. To understand how and why the transition from metal catalysis to cofactor catalysis might have taken place, we systematically studied the influence of PLP on a transamination reaction catalyzed by 18 different metals ions. Although PLP was found to substantially accelerate the rate of the Cu2+ and V5+ catalyzed transamination, the largest rate increases came for the two most abundant metals in Earth’s crust, Al3+ and Fe3+, with rate accelerations of 90- and 225-fold, respectively. In the presence of PLP, the reactions co-catalyzed by Al3+ and Fe3+ become competitive with those catalyzed solely by much rarer metal ions such as Cu2+ and V5+. Kinetic and DFT studies performed to probe the mechanism of co-catalysis support a ping-pong mechanism of the reaction. Our results suggest that one of the main reasons for the emergence of PLP in metabolism might have been to allow highly Earth-abundant metals to take over those catalytic functions that were once carried out by less common metals. This trend is not so different from that currently seen in the field of chemical catalysis engineered by humans.
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All evolutionary biological processes lead to a change in heritable traits over successive generations. The responsible genetic information encoded in DNA is altered, selected, and inherited by mutation of the base sequence.
While this is well known at the biological level, an evolutionary change at the molecular level of small organic molecules is unknown but represents an important prerequisite for the emergence of life.
Here, I present a class of prebiotic imidazolidine-4-thione organocatalysts able to dynamically change their constitution and potentially capable to form an evolutionary system. These catalysts functionalize their own building blocks and dynamically adapt to their (self-modified) environment by mutation of their own structure.
Depending on the surrounding conditions, they show pronounced and opposing selectivity in their formation. Remarkably, the preferentially formed species can be associated with different catalytic properties, which enable multiple pathways to the formation of nucleotides, oligomers and lipids for the transition from abiotic matter to functional biomolecules.
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Prebiotic amphiphiles, like decanol and decanoic acid, are well-known to self-assemble into membranes and are proposed as precursors to lipid membranes for the origins of life. Their formation conditions rely heavily on the composition of amphiphiles and the environmental parameters of temperature, pH, ionic strength, solute composition. This talk will focus on the synthesis of these amphiphiles, their membrane formation conditions, and their function with respect to photochemistry.
A way to study the emergence of life is to create a physico-chemical system that is capable of open ended evolution. The aim is to search for most minimal requirements to maximize the probability to find it outside the lab. Starting life with three molecules in a one-pot geological non-equilibrium without human intervention would be a favorable scenario.
We revisited polymerization and templated ligation of RNA from nucleotides with 2’,3’ cyclic phosphates. Simple alkaline conditions at pH 9-11 without catalysts or added salts oligomerized the nucleotides up to 10mers over across 25-80°C within a day, both in the ‘dry’ state or in the wet-dry cycling at a heated air-water interface . The polymerization was dominated by G, but cold and dry conditions, achieved in the planet simulator of McMaster University yielded random sequences of GC or GCAU according to mass spectrometry.
Interestingly, the same conditions triggered (i) with Trimetaphosphate the specific cyclic phosphorylation and (ii) the templated ligation of oligonucleotides, the latter also under ‘dry’ conditions. Therefore, we envisage a dry RNA world where exponential replication is driven by long enough polymers, templated ligation and hydrolytic recombination using wet-dry cycles to separate the strands. Short, replicated RNA sequences could enhance their own ligation and start the first cycle of functional RNA evolution.
That CO2-water interfaces can drive the replication towards sequence lengths of up to 1300mers was demonstrated recently . The accumulation by capillary flow overcame the tyranny of the shortest. The long strands were shown to separate under the pH cycling provided by the Hadean atmosphere of CO2. While the replication was still implemented by a polymerase to enhance kinetics, the findings indicate that a similar strand separation will be possible for RNA based replication even under intermediate Mg2+ concentrations.
 Dass AV, Wunnava S, Langlais J, von der Esch B, Krusche M, Ufer L, et al. RNA auto-polymerisation from 2’,3’-cyclic nucleotides at air-water interfaces. ChemRxiv. Cambridge: Cambridge Open Engage (2022). https://doi.org/10.26434/chemrxiv-2022-zwh2t
 Ianeselli, A., Atienza, M., Kudella, P.W. et al. Water cycles in a Hadean CO2 atmosphere drive the evolution of long DNA. Nat. Phys. (2022). https://doi.org/10.1038/s41567-022-01516-z
Synthesis of RNA in early life forms required chemically activated nucleotides, perhaps in the same form of nucleoside 5′-triphosphates (NTPs) as in the contemporary biosphere. Here we show the development of a catalytic RNA (ribozyme) that generates the nucleoside triphosphate guanosine 5′-triphosphate (GTP) from the nucleoside guanosine and the prebiotically plausible cyclic trimetaphosphate (cTmp). Ribozymes were selected from about 1014 different randomized sequences by metabolically coupling the synthesis of 6-thio GTP to primer extension by an RNA polymerase ribozyme within 1016 emulsion droplets. Several functional RNAs were identified, one of which was characterized in more detail. Under optimized reaction conditions, this ribozyme produced GTP at a rate 18,000-fold higher than the uncatalyzed rate, with a turnover of 1.7-fold, and supported the incorporation of GTP into RNA oligomers in tandem with an RNA polymerase ribozyme. We are currently working on improving the low turnover of the GTP synthase ribozyme, with the long-term goal of establishing efficient synthesis of RNA polymers from nucleosides and cTmp. The results are discussed in the context of early life forms.
Our interest is in the evolution and adaptation of enzymes at a structural level to extreme environments. Understanding how temperature and pressure can affect where organism can survive may also provide a history of early life looked like. We are particularly interested if evolution towards or way extremes influences outcomes. Now, we are using Alpha Fold, a machine learning method, to expand the number of structures and molecular dynamics simulations to determine “material science” measures to quantitate P-T diagrams of how proteins vary with pressure and temperature.
The idea that life can act as a guide to its own origins has gained in strength in recent years with experimental work demonstrating that a biomimetic protometabolism starting from H2 and CO2 is indeed favoured in the absence of genes and enzymes. I will present some of our own recent work, both modelling and experimental, which shows that (i) CO2 fixation can be driven by a pH gradient across Fe(Ni)S barriers; (ii) protocells composed of simple amphiphiles can be formed readily under these conditions and are stable across a wide pH range; (iii) FeS clusters with redox potentials in the range needed to reduce CO2 can form spontaneously in the presence of monomeric cysteine or short peptides; and (iv) protometabolic pathways catalysed by metal ions are capable of synthesising amino acids and nucleobases including uracil. Taken together, these findings suggest that protocells could feasibly grow autotrophically by CO2 fixation via the acetyl CoA pathway and reverse incomplete Krebs cycle. Modelling work indicates that positive feedbacks in autotrophic protocells could amplify flux through the system, facilitating nucleotide synthesis. I will end with some thoughts on the emergence of genetic information in autotrophically growing protocells.
After 70 years of experimentation, we still have not reconstructed the origin of life – but in the meantime many interesting experiments have been conducted, some more promising than others. One of the constraints may be that the topic is approached from too much of an organic chemistry point of view and not enough from the environmental side. Happily, there is budding awareness of the importance of environmental conditions during the Hadean, when life emerged on Earth. But the danger lies in the desire of chemists to constrain their experiments to their own idea of Hadean conditions – because their experiments work better if there is some control.
Life emerged as a natural evolution of chemistry into biology more than 4 billion years ago on a world that was totally different to the Earth today. Origins of life researchers suggest life emerging in submarine hydrothermal vents and their environments, in pumice rafts, in subaerial lakes fuelled by radioactive energy sources, in rivers or ponds, or in subaerial hydrothermal systems. The difficulty is establishing the local-scale conditions that fomented prebiotic chemistry 4 billion years ago. Much can be learned from the ancient rock record but there are limitations. The oldest, well-preserved rocks formed 1 billion years after the consolidation of the Earth. Rare older rocks are highly altered. Piecing together the local environments of Hadean Earth from the rock record is challenging.
It is certain that the different environments evoked for the emergence of life generally have compelling attributes (pumice rafts, however, are not very plausible, nor is the idea of uranium-rich placer sands, since U only became significantly mobile after the Great Oxygenation Event at ~2.4 Ga). It is likely that some degree of molecular evolution took place in most of them. The question is, to what extent? Simply the formation of complex molecules or did living cells emerge? Did life emerge in more than one environment? Would it have been possible for a variety of interesting molecules formed in subaerial to subaqueous environments to mix in the “middle ground, i.e. at the edges of shallow marine basins with hydrothermal activity?
Another question concerns the emergence of extraterrestrial life. If life emerged in one environment on Earth, let us say on emerged landmasses, could it have emerged in a purely subaqueous environment on another planetary body, such as the icy moons Europa and Enceladus? We need open minds to consider dirty chemistry in realistic, and possibly different, geological environments.
The complexity of solution compositions in natural OOL scenarios is in stark contrast to the often specifc initial requirements of reactions in prebiotic chemistry or molecular evolution. Failure to meet these conditions, as is often the case in natural environments, leads quite often to a multitude of undesired side products with low or vanishing yields of the target products. Heat fluxes through water-filled thin cracks in geomaterials offer an interesting approach to this problem for nature: the ubiquitous heat fluxes lead to a strong concentration of prebiotically relevant substances in the thin fluid layers. We show experimentally that the strength of this effect differs significantly even for extremely similar chemicals such as linear or cyclic phosphorylated nucleotides or the canonical bases, but also for most canonical amino acids. If the said rock cracks form a interconnected system, the selective effect is enhanced even at small temperature differences leading to relative concentration differences of several orders of magnitude between different chemicals such as 2-aminooxazoles versus 2-aminoimidazoles. Locally, a variety of solution compositions can thus be implemented and a wide range of reaction conditions can be provided. We demonstrate this using the example of the enrichment of magnesium over sodium ions from basalt leachates, in which the high sodium concentrations actually inhibit the function of ligase ribozymes. Only heat flux-driven selective enrichment of magnesium ions establishes the necessary reaction conditions and enables ligation. Selective concentration also leads to the separation of calcium from phosphate, providing a pathway for the release of phosphate from otherwise difficult-to-access apatite mineral. Even in simple salt solutions without further buffering, separation of ion pairs can produce pH differences of up to 4 units within a few centimeters. Heat fluxes through such plausible geomicrofluidics thus couple to a variety of chemically relevant gradients, and their wide availability provides ideal conditions for enabling prebiotically relevant reactions.
T. Matreux, K. Le Vay, A. Schmid, P. Aikkila, L. Belohlavek, A. Z. Çalışkanoğlu, E. Salibi, A. Kühnlein, C. Springsklee, B. Scheu, D. B. Dingwell, D. Braun, H. Mutschler, C. B. Mast, Nature Chemistry doi.org/10.1038/s41557-021-00772-5 (2021)
Life as we know it could most likely not have emerged in absence of active geological processes and the environments shaped by them. The quest of the most probable environment(s) is amongst the most challenging and heavily debated topics on the emergence of life. A key challenge remains however to identify environments favorable for a plethora of reactions allowing to progress from the synthesis of small molecules forming the simple building block of life, to complex organic molecules such as e.g. sugars or lipids and finally to functional polymers with the ability to metabolism and replication. Common to all promising geological environments are physical and /or chemical disequilibria driving change, in form of fluid-rock interaction such as e.g. leaching, precipitation and remineralization – all dynamically shaping their micro- and macro- environment. Fluid flow through a permeable (fracture) network enables efficient fluid-mediated transport and cycling of reactants and organic products, and is key to establish and/ or sustain chemical, temperature and pH gradients.
The scarcity of rock records for the early Archean Eon marks these periods as the least constrained ones in Earth’s history and fosters speculations about the earliest Earth geology and thus likely environments to explore on their feasibility for prebiotic chemical reactions. Geomaterials - glasses, minerals and rocks –exhibit different reactivity in contact with a fluid. The interesting highly reactive geomaterials, such as for instance glasses of mafic composition, however are not preserved in the rock records, with even their products likely to be altered and thus may be easily overlooked.
The earliest form of RNA replication may have been non-enzymatic, without requiring polymerase ribozymes. Template-directed synthesis of complementary strands forms double strands that are unlikely to separate unless temperature cycling drives melting. If there are multiple copies of identical sequences, re-annealing of existing strands prevents subsequent cycles of copying. However, if there is a diverse mixture of sequences, partially matching sequences can reanneal in configurations that allow continued strand growth. Here we present simulations that incorporate melting, reannealing, primer extension, and ligation. Strand growth occurs over multiple heating/cooling cycles, producing strands over 200 nucleotides in length. However, there is no exact copying of sequences, even if single base additions are fully accurate (no mutational errors). It has been proposed that RNA systems may contain a virtual circular genome consisting of partially overlapping sequences that can be assembled into a circle. We show that this situation is unlikely to arise naturally and cannot maintain itself in the presence of mutational errors or inflow of random oligomers. We show that even a short functional sequence like a tRNA cannot be encoded on a virtual circle because it contains repeated tetramers; hence sequence information on a longer length scale is not maintained. In contrast, we argue that the most likely way for replication to begin in the RNA world involves real circular strands that use the rolling circle mechanism. Multiple copies are produced from a single circle via strand displacement without requiring temperature cycling.
Pools of RNA oligomers are believed to play a central role for the spontaneous emergence of living systems. In suitable non-equilibrium environments, the RNA strands in such pools are thought to hybridize and dehybridize, ligate and break, such that they generate longer RNA molecules, which fold and function as ribozymes, ultimately enabling molecular evolution. However, concrete scenarios and possible pathways remain unclear. I will describe our ongoing effort to explore the complex dynamics of RNA pools computationally. The aim is to illustrate and understand how emergent behaviors arise from the interplay of the underlying molecular processes, and how the non-equilibrium environment affects these behaviors.
Although clearly not prebiotically plausible, I will show that DNA origami presents an excellent model to understand how early forms of cell walls made of nucleic acids could have formed in order to achieve non-spherically shaped protocells. By making use of simple self-assembly reactions, DNA origami are able to form large assemblies and stabilizing lattice networks on lipid membranes, including protocell-like lipid vesicles [1,2]. Additional stabilization may be achieved through biomineralization of such structures . Lipid vesicles, compared to modern cells and organisms are physically and mechanically not particularly stable towards different external environmental factors such as osmotic shocks as they are lacking a proper cell wall or cytoskeleton. Additionally, although compartmentalization is considered a key element for the emergence of life, there seemed to be very early appearances of distinct non-spherical compartment geometries. Modern nanotechnological tools may help us to understand how such anisotropic cell shapes came to be and how they were potentially even stabilized by early assemblies of nucleic acids. Although for simplicity we are employing DNA, our studies present important examples of symbiotic and regulatory interactions between primitive vesicle compartments and primitive genetic materials, which could equally have occurred in the realm of the more prebiotically relevant RNA world.
1. Czogalla, A., Franquelim, H.G., Schwille, P. (2016) “DNA Nanostructures on Membrane sas Tools for Synthetic Biology.” Biophys. J., 110(8), 1698 – 1707.
2. Franquelim, H.G., Khmelinskaia, A., Sobczak, J.P., Dietz, H., Schwille, P. (2018) “Membrane sculpting by curved DNA origami scaffolds.” Nat. Commun., 9(1), 1-10.
3. Nguyen, L., Döblinger, M., Liedl, T., Heuer-Jungemann, A. (2019) „DNA Origami-Templated Silica Growth by Sol Gel Chemistry” Angew. Chem. Int. Ed. 58(3), 912-916.
Early in the evolution of life the replication of genomes and the transcription of functional genes would have been of vital importance. RNA catalysts or ribozymes appear likely to have played a role in such activities. In extant biology, DNA replication is initiated by binding events at an origin of replication that assemble two topologically clamped replication forks capable of sustained polymerization. Similarly, transcription initiates by binding a polymerase to a DNA promoter, followed by a rearrangement into a topologically clamped RNA transcriptional elongation complex. The requirement for initiation of polymerization to be followed by a structural rearrangement into a topologically trapped elongation complex appears quite fundamental to both replication and transcription and we wondered if an RNA polymerase ribozyme could be selected with such properties.
We have selected an RNA polymerase ribozyme that can, just like a DNA dependent RNA polymerase, use a sigma-like specificity primer to locate a promoter sequence. Once found this RNA enzyme rearranges into a topologically clamped form able to stay associated with a single-stranded RNA template. The clamped polymerase stays associated with circular templates but falls off short linear templates indicating that it can move around the template during polymerization. This polymerase deals with randomly selected templates much better than previous ribozymes but still suffers in that its active site does not stay in register with the extending primer found on the template. Future selections may be able to establish this register and enable sustained polymerization.
The polymerase consists of three domains. The first domain was selected for its ability to ligate itself to an RNA primer by phosphodiester chemistry. The second domain was selected to enable the core catalytic core to allow NTP polymerization on a primed RNA template. The third, and most recently selected domain, confers promoter recognition and topological clamping. I will compare the evolution of this artificial ribozyme to that of other naturally existing ribozymes and suggest that such modular evolution was likely to have been common early in the evolution of life. Future in vitro selection may result in a fourth domain that could lead to the emergence of an RNA replicase ribozyme in the laboratory.
Since Louis Pasteur, it was beyond imagination that life could thrive at temperatures above the boiling point of water as bacteria could be safely killed when incubated at 100°C. The discovery of Archaea as third domain of life did not only revolutionize the tree of life, but led to the identification of heat-loving organisms that thrive at temperatures up to 122°C. Many of these hyperthermophilic Archaea were isolated from black smokers and hydrothermal vents, which are speculated to be the site for the emergence of early life forms. Considering that these organisms are fully adapted to optimally grow at extremely high temperatures, we might be able to learn what is required to maintain (or form) a functional cell in habitats with extreme temperatures and steep temperature gradients. At the Archaea Centre Regensburg, a wide range of hyperthermophilic Archaea were isolated and characterized for the first time allowing us to study how hyperthermophilic organisms adapt to extreme environmental conditions. In this talk, I will introduce you to some of the most fascinating and captivating hyperthermophilic Archaea. Moreover, I will highlight molecular strategies utilized by hyperthermophilic Archaea to cope with extreme temperatures and sudden temperature changes. Among others, we discovered that hyperthermophiles dynamically adjust the extent and chemical identity of posttranscriptionally installed rRNA modifications in adaptation to changes in growth temperature.
In the RNA world, improvements to RNA replication would permit more complex functional RNA molecules to evolve, which would in turn select for further improvements to replication in a bootstrapping process that presaged the emergence of complex cellular life and genetically encoded proteins. Only vestigial traces of the RNA world are left in modern life, but polymerase ribozymes have been evolved de novo in the laboratory. To improve these ribozymes’ activity and study RNA-based life in the laboratory, polymerases have been evolved in vitro to synthesize functional RNA molecules from RNA templates. As polymerase activity improves, the complexity of the functional RNA target can be increased, driving further improvements in polymerase activity in a bootstrapping process analogous to the evolution of genome complexity in the RNA world. Over more than sixty generations of in vitro evolution, polymerases have been selected to synthesize first simple ligand-binding RNA aptamers, then simple self-cleaving ribozymes, and most recently a 97-nucleotide ligase ribozyme that is related to the catalytic core of the polymerase itself and more than half its length. Over these generations, polymerases have accumulated more than 30 mutations and have undergone a major structural rearrangement of the catalytic core. Polymerases first improved efficiency by increasing rates of nucleotide incorporation by more than 300 fold and lowering the binding constant for primer-template duplexes from more than 3 mM to less than 1 µM. In the most recent generations, under selection to synthesize complex ribozymes that are more easily inactivated by mutation, polymerase accuracy has improved, enabling synthesis of 50-fold more unmutated RNA products from the most complex RNA templates. As polymerase activity improves further, it may soon be possible to extend selection toward polymerase synthesis of component pieces of itself. Once a polymerase achieves self-replication, long-term propagation of the polymerase and its autonomous Darwinian evolution would represent a reconstruction of RNA life, and enable the direct study of the RNA world in the laboratory.
On the path to modern protein-dominated biology, life on earth has gone through multiple stages. Yet, even before life appeared, numerous amino acids have been accessible through prebiotic chemistry. It is therefore conceivable that the chemical functionality of amino acids or even short peptides has been utilized since the earliest stages of life. Later, the emergence of DNA-encoded protein synthesis greatly facilitated the use of peptides. Evolution eventually led to the modern universal genetic code, likely starting initially with a smaller set of early amino acids. Numerous studies have speculated on the genesis of amino acids and the history of the genetic code. Nevertheless, the evolutionary connection between primordial amino acids generated by prebiotic chemistry and today’s highly functional proteins composed of the standard amino acids is still largely lacking experimental support.
We are investigating the functional capabilities of polypeptides with limited amino acid compositions. Our experiments will examine the ability of primitive alphabets, composed of substantially fewer than twenty different amino acids, to support structured proteins and enable simple functions such as cofactor binding. We will address how the chemical diversity of random polypeptides influences structure and function, and if a minimum alphabet is necessary to confer a biological function. By generating tangible empirical data in a field of research that has been largely dominated by theoretical approaches, this project has the potential to provide critical insights into the history of the standard amino acid alphabet.
Newton, Morrone, Lee, Seelig, B. (2019) Genetic code evolution investigated through synthesis and characterisation of proteins from reduced alphabet libraries. ChemBioChem. (20) 846-856.
A major challenge in biocatalysis is the development of novel enzymes for chemical reactions beyond nature’s synthetic repertoire. A successful strategy employs artificial metalloenzymes, which are designed rationally to combine the catalytic properties of a metal cofactor with the chiral environment of a protein scaffold that provides stereoselectivity (1). These systems are genetically encodable and therefore amenable to optimization by directed evolution. This technique mimics natural selection in the laboratory through iterative cycles of mutagenesis and screening (2).
We recently developed a de novo protein scaffold with femtomolar affinity for lanthanides, where metal binding can be observed by sensitizing the element-specific luminescence (3). Lanthanide ions are not only potent Lewis acid catalysts; one of them, cerium(III/IV), also promotes photoredox chemistry. The metal ions are incorporated by dative anchoring, which exploits direct coordination by natural amino acids of the protein. We now work on turning these de novo metalloproteins into a biocatalytic platform for synthetically valuable reactions. Furthermore, we aim to study smaller de novo metallopeptides in the context of prebiotic catalysis by evolving them to drive challenging transformations in metabolic reaction cycles.
(1) Schwizer, F.; Okamoto, Y.; Heinisch, T.; Gu, Y.; Pellizzoni, M. M.; Lebrun, V.; Reuter, R.; Kohler, V.; Lewis, J. C.; Ward, T. R. Artificial Metalloenzymes: Reaction Scope and Optimization Strategies. Chem. Rev. 2018, 118, 142-231.
(2) Zeymer, C.; Hilvert, D. Directed Evolution of Protein Catalysts. Annu. Rev. Biochem. 2018, 87, 131-157.
(3) Caldwell, S. J.; Haydon, I. C.; Piperidou, N.; Huang, P. S.; Bick, M. J.; Sjöström, H. S.; Hilvert, D.; Baker, D.; Zeymer, C. Tight and specific lanthanide binding in a de novo TIM barrel with a large internal cavity designed by symmetric domain fusion. Proc. Natl. Acad. Sci. U.S.A. 2020, 117, 30362-30369.