Hannes Mutschler

All living organisms must maintain a balanced pool of DNA building blocks to replicate and propagate their genetic information. In living cells, ribonucleotide reductase (RNR) is the central enzyme responsible for dNTP synthesis. In contrast, minimal protein-based cell-free systems, are powerful platforms for reconstituting biological processes in vitro, but lack the endogenous metabolic pathways required to autonomously replicate their DNA and rely entirely on externally supplied dNTPs. This dependency prevents cell-free systems from achieving the metabolic autonomy required for self-sufficient genetic replication. In this work, we successfully integrated RNR’s redox activity and complex allosteric regulation for in situ synthesis of all dNTPs from endogenous NTP pools. We combined RNR’s activity to DNA synthesis and propagation of the genetic information encoding a self-contained minimal DNA replication machinery. The replication products retain the genetic information and enable re-booting of self-encoded RNR-dependent DNA synthesis. Using this strategy, cell-free systems with self-sufficient dNTP metabolism may open new avenues toward completely autonomous synthetic cells.

Synthetic cells emulate fundamental biological behaviors, like growth, metabolism, and mobility, but have lacked genotype-driven selection, which is essential for Darwinian evolution. Here, we introduce libraries of short DNA sequences as genotypes into fuel-dependent peptide-RNA-based coacervate droplets, serving as synthetic cells. By sequencing, we identify sequences that partition in the droplets, revealing strong preferences for guanine-rich and adenine-rich motifs. These sequences affect the synthetic cell phenotype—adenine-rich sequences shorten droplet lifetimes through hybridization. In contrast, guanine-rich sequences kinetically trap droplets via peptide interactions, altering dissolution rates and morphology. This study demonstrates how genotype affects phenotype in synthetic cells, establishing essential design principles for achieving Darwinian evolution in minimal protocellular systems.

DNA aptamers are short, single-stranded DNA molecules that bind specifically to a range of targets such as proteins, cells, and small molecules. Typically, they are utilized in the development of therapeutic agents, diagnostics, drug delivery systems, and biosensors. Although aptamers perform well in controlled extracellular environments, their intracellular use has been less explored due to challenges of expressing them in vivo. In this study, we employed the bacterial retron system Eco2 to express a DNA light-up aptamer in Escherichia coli . Our data confirms that structure-guided insertion of the aptamer domain into the non-coding region of the retron enables reverse transcription and biosynthesis of functional aptamer constructs in bacteria. The purified DNA aptamer synthesized under intracellular conditions shows comparable activity to a chemically synthesized control. Our findings demonstrate that retrons can be used to express short DNA aptamers within living cells, potentially broadening and optimizing their application in intracellular settings.

DNA aptamers are short, single-stranded DNA molecules that bind specifically to a range of targets such as proteins, cells, and small molecules. Typically, they are utilized in the development of therapeutic agents, diagnostics, drug delivery systems, and biosensors. Although aptamers perform well in controlled extracellular environments, their intracellular use has been less explored due to challenges of expressing them in vivo. In this study, we employed the bacterial retron system Eco2, to express a DNA light-up aptamer in Escherichia coli. Our data confirms that structure-guided insertion of the aptamer domain into the non-coding region of the retron enables reverse transcription and biosynthesis of functional aptamer constructs in bacteria. The purified DNA aptamer synthesized under intracellular conditions shows comparable activity to a chemically synthesized control. Our findings demonstrate that retrons can be used to express short DNA aptamers within living cells, potentially broadening and optimizing their application in intracellular settings.

Ribonucleotide reductases (RNRs) convert all four ribonucleotides to deoxyribonucleotides, providing essential building blocks for DNA biosynthesis and repair through radical‐based catalysis. These functions are key to cellular proliferation and have made RNRs well established targets for antimicrobial and antiviral drugs and combination chemotherapies. Here, we describe a novel highly sensitive one‐pot enzymatic assay, which amplifies RNR activity by coupling it to the synthesis of a fluorogenic RNA aptamer. We validated this approach by testing RNR activity under dNTP‐limiting conditions to emulate RNR's complex allosteric regulatory patterns and by detecting the dose‐ and time‐dependent inhibition of RNR by hydroxyurea. This unique assay builds on previous high‐throughput screening assays for investigation of RNR's catalytic mechanisms by improving sensitivity and reducing readout timeframes. Impact statement: Ribonucleotide reductases (RNRs) are essential for controlling cellular dNTP supply and are major targets in cancer, antiviral, and antimicrobial therapy. FLARE is a novel single‐tube, real‐time RNR assay, coupling dNTP synthesis to the transcription of a fluorogenic aptamer for continuous monitoring of activity, regulation, and inhibition using standard microplate readers.

Membrane-free complex coacervate microdroplets are compelling models for primitive compartmentalisation with the ability to form from biological molecules. However, understanding how molecular interactions can influence physicochemical properties and catalytic activity of membrane-free compartments is still in its infancy. This is important for defining the function of membrane-free compartments during the origin of life as well as in modern biology. Here, we use RNA-peptide coacervate microdroplets prepared with prebiotically relevant amino acids and a minimal hammerhead ribozyme. This is a model system to probe the relationship between coacervate composition, its properties and ribozyme activity. We show that ribozyme catalytic activity is inhibited within the coacervate compared to buffer solution, whilst variations in peptide sequence can modulate rates and yield of the ribozyme within the coacervate droplet by up to 15-fold. The apparent ribozyme rate constant is anti-correlated with its concentration and correlated to its diffusion coefficient within the coacervates. Our results provide a relationship between the physicochemical properties of the coacervate microenvironment and the catalytic activity of the ribozyme where membrane-free compartments could provide a selection pressure to drive molecular evolution on prebiotic earth.

Synthetic cells emulate fundamental biological behaviors, such as growth, metabolism, and evolution, under non-equilibrium conditions, but have lacked genotype-driven selection, which is essential for Darwinian evolution. Here, we introduce short DNA sequences as genotypes into fuel-dependent, peptide-RNA-based coacervate droplets. By sequencing random DNA libraries, we identify sequence-dependent partitioning into droplets, revealing strong preferences for guanine-rich and adenine-rich motifs. Adenine-rich sequences shortened the droplet lifetimes through RNA hybridization, whereas guanine-rich sequences kinetically trapped droplets via peptide interactions, altering dissolution rates and morphology. This study demonstrates genotype-phenotype coupling in synthetic cells, establishing essential design principles for achieving Darwinian evolution in minimal protocellular systems.

Synthetic cells emulate fundamental biological behaviors, such as growth, metabolism, and evolution, under non-equilibrium conditions, but have lacked genotype-driven selection, which is essential for Darwinian evolution. Here, we introduce short DNA sequences as genotypes into fuel-dependent, peptide-RNA-based coacervate droplets. By sequencing random DNA libraries, we identify sequence-dependent partitioning into droplets, revealing strong preferences for guanine-rich and adenine-rich motifs. Adenine-rich sequences shortened the droplet lifetimes through RNA hybridization, whereas guanine-rich sequences kinetically trapped droplets via peptide interactions, altering dissolution rates and morphology. This study demonstrates genotype-phenotype coupling in synthetic cells, establishing essential design principles for achieving Darwinian evolution in minimal protocellular systems.

Group II introns (G2Is) are large self‐splicing ribozymes with emerging potential in biotechnological applications. Despite growing interest, their complexity has so far precluded efforts to design them from scratch. While computational approaches have enabled the design of small RNA catalysts, methods for engineering large ribozymes remain underdeveloped. Herein, the RNA inverse folding algorithm aRNAque is used to design G2Is from scratch, yielding three novel self‐splicing ribozymes with unusually stable structures. The designed intron Arq.I2 is revealed to be an unexpectedly proficient ribozyme in vitro, self‐splicing at a rate comparable to the fastest known G2Is. While most G2Is are believed to be inactive under intracellular conditions in the absence of maturase proteins, it is shown that Arq.I2 self‐splices in Escherichia coli cells. The results demonstrate that highly active variants of large and complex ribozymes can be designed de novo with relative ease using existing inverse folding algorithms, paving the way for the design of bespoke ribozymes derived from G2Is for the development of biotechnological tools.