The ability of single-chain bolalipids (or bolaamphiphiles) to self-assemble into vesicular structures remains poorly characterized. Here, we report the synthesis and self-assembly behavior of a new class of proline-based single-chain lipids, a symmetric bipolar molecule (2Pro-C18:1) and, for comparison, a unipolar analog (1Pro-C18:1), both bearing an unsaturated C18 hydrocarbon chain. Confocal fluorescence microscopy, encapsulation assays, and fluorescence spectroscopy demonstrate that both amphiphiles form stable vesicles capable of entrapping small molecules. Vesicle formation occurs over a broad range of pH values, with the bolalipid favoring higher pH, consistent with differences in apparent pKa determined from zeta potential measurements. At the air-water interface, 2Pro-C18:1 exhibits lower surface activity than 1Pro-C18:1, and vibrational sum frequency generation spectroscopy is consistent with a bent or U-shaped interfacial conformation for the bolalipid, in contrast to a more extended geometry for the unipolar analog. Both lipids display dynamic exchange between interfacial and bulk-associated aggregates at higher concentrations, with 2Pro-C18:1 exhibiting more complex concentration-dependent behavior. Taken together, these results demonstrate that single-chain bolalipids can form stable, dynamic aggregates with bulk and interfacial properties that are governed primarily by hydrophobic chain effects modulated by headgroup architecture. These findings provide insight into molecular design principles for functional amphiphilic systems.

Macromolecular assembly between biomolecules dictates the material state of chemically complex aqueous dispersions such as the cytoplasm. The formation of protein precipitates, fibers, or liquid droplets have been associated with metabolic regulation and disease. However, the effect of metabolic flux on the material properties of aqueous dispersions remains underexplored. Here, we use the protometabolic reduction of NAD+ to NADH by pyruvate to study the effect of NADH production on the phase separation properties of polyarginine. We show that reduction of NAD+ in the presence of polyarginine can tune the material properties of the dispersion between precipitates, homogeneous solution, and liquid droplets depending on the buffer concentration. In situ droplet formation results in 2-3 times higher reaction rate and NADH yield, compared to homogeneous solution. Our study provides a setting for coupling protometabolism to active protocell environments in the absence of enzymes and sheds light on the self-regulation of metabolic flux on mediating biomolecular phase separation.

The transition from unregulated, prebiotic chemistry to metabolic-like systems capable of supporting an evolving protocell has remained difficult to explain. One hypothesis is that early catalysts began to prune the chemical landscape in a manner that facilitated the emergence of modern-day enzymes. As enzymes frequently rely on the intrinsic reactivity of metal ions, it follows that these early catalysts may have been metal ions coordinated to prebiotic peptides that have remained as core structures within extant proteins. Here, we demonstrate that UV light directly selects for the types of metal-binding peptide motifs found in biology. This is because bare cysteine is much more susceptible to photolysis than cysteine bound by a metal ion. Therefore, peptides with greater affinity for environmentally available metal ions, such as Fe2+ or Zn2+, are more stable. Our results are supported by mass spectrometry, calorimetry, X-ray absorption, NMR spectroscopy, transient absorption pump probe spectroscopy, and excited-state quantum-chemical calculations. Photostability arises from the ability of the metal ion to engage transiently generated reactive radical centers in a manner that prevents subsequent degradative processes. The data are consistent with the enrichment of a restricted set of high affinity, extant-like metallopeptides in surficial environments on the early Earth.

Greater concentrations of hydrosulfide lead to the prebiotic formation of higher nuclearity Fe–S peptides, culminating in a putative nitrogenase-like [6Fe–9S] cluster. Higher nuclearity clusters are more stable with lower reduction potential.

For life to emerge on Earth, peptides must first have formed without the aid of enzymes — but how? Reactions of sulfur-containing molecules might have been key. For life to emerge on Earth, peptides must first have formed without the aid of enzymes — but how? Reactions of sulfur-containing molecules might have been key.

As minimal cells or protocells are dramatically simpler than modern unicells it is possible to quantitatively estimate free energy changes for every process in the lifecycle of a protocell and compare these with estimates of the free energy changes for lifecycles in modern unicells. We present quantitative estimates of all metabolic changes in part by new density function theory (DFT) estimations, in part by compiling previously measured or estimated free energy changes, and in part by new thermodynamic calculations for all self-assembly, vesicle bending, and fission energies.

In living cells, a complex mixture of biomolecules is assembled within and across membranes. This non-equilibrium state is maintained by sophisticated protein machinery, which imports food molecules, removes waste products and orchestrates cell division. However, it remains unclear how this complex cellular machinery emerged and evolved. Here we show how the molecular contents of a cell can be coupled in a coordinated way to non-equilibrium heat flow. A temperature difference across a water-filled pore assembled the core components of a modern cell, which could then activate the gene expression. The mechanism arose from the interplay of convection and thermophoresis, both driven by the same heat source. The cellular machinery of protein synthesis from DNA via RNA was triggered as a direct result of the concentration of cell components. The same non-equilibrium setting continued to attract food molecules from an adjacent fluid stream, keeping the cellular molecules in a confined pocket protected against diffusion. Our results show how a simple non-equilibrium physical process can assemble the many different molecules of a cell and trigger its basic functions. The framework provides a membrane-free environment to bridge the long evolutionary times from an RNA world to a protein-based cell-like proto-metabolism.

Nicotinamide adenine dinucleotide (NAD+) is a redox active molecule that is universally found in biology. Despite the importance and simplicity of this molecule, few reports exist that investigate which molecular features are important for the activity of this ribodinucleotide. By exploiting the nonenzymatic reduction and oxidation of NAD+ by pyruvate and methylene blue, respectively, we were able to identify key molecular features necessary for the intrinsic activity of NAD+ through kinetic analysis. Such features may explain how NAD+ could have been selected early during the emergence of life. Simpler molecules, such as nicotinamide, that lack an anomeric carbon are incapable of accepting electrons from pyruvate. The phosphate moiety inhibits activity in the absence of metal ions but facilitates activity at physiological pH and model prebiotic conditions by recruiting catalytic Mg2+. Reduction proceeds through consecutive single electron transfer events. Of the derivatives tested, including nicotinamide mononucleotide, nicotinamide riboside, 3-(aminocarbonyl)-1-(2,3-dihydroxypropyl)pyridinium, 1-methylnicotinamide, and nicotinamide, only NAD+ and nicotinamide mononucleotide would be capable of efficiently accepting and donating electrons within a nonenzymatic electron transport chain. The data are consistent with early metabolic chemistry exploiting NAD+ or nicotinamide mononucleotide and not simpler molecules.

Central to the quest of understanding the emergence of life is to uncover the role of metals, particularly iron, in shaping prebiotic chemistry. Iron, as the most abundant of the accessible transition metals on the prebiotic Earth, played a pivotal role in early biochemical processes and continues to be indispensable to modern biology. Here, we discuss our recent contributions to probing the plausibility of prebiotic complexes with iron, including heme and iron-sulfur clusters, in mediating chemistry beneficial to a protocell. Laboratory experiments and spectroscopic findings suggest plausible pathways, often facilitated by UV light, for the synthesis of heme and iron-sulfur clusters. Once formed, heme displays catalytic, peroxidase-like activity when complexed with amphiphiles. This activity could have been beneficial in two ways. First, heme could have catalytically removed a molecule (H2O2) that could have had degradative effects on a protocell. Second, heme could have helped in the synthesis of the building blocks of life by coupling the reduction of H2O2 with the oxidation of organic substrates. The necessity of amphiphiles to avoid the formation of inactive complexes of heme is telling, as the modern-day electron transport chain possesses heme embedded within a lipid membrane. Conversely, prebiotic iron-sulfur peptides have yet to be reported to partition into lipid membranes, nor have simple iron-sulfur peptides been found to be capable of participating in the synthesis of organic molecules. Instead, iron-sulfur peptides span a wide range of reduction potentials complementary to the reduction potentials of hemes. The reduction potential of iron-sulfur peptides can be tuned by the type of iron-sulfur cluster formed, e.g., [2Fe-2S] versus [4Fe-4S], or by the substitution of ligands to the metal center. Since iron-sulfur clusters easily form upon stochastic encounters between iron ions, hydrosulfide, and small organic molecules possessing a thiolate, including peptides, the likelihood of soluble iron-sulfur clusters seems to be high. What remains challenging to determine is if iron-sulfur peptides participated in early prebiotic chemistry or were recruited later when protocellular membranes evolved that were compatible with the exploitation of electron transfer for the storage of energy as a proton gradient. This problem mirrors in some ways the difficulty in deciphering the origins of metabolism as a whole. Chemistry that resembles some facets of extant metabolism must have transpired on the prebiotic Earth, but there are few clues as to how and when such chemistry was harnessed to support a (proto)cell. Ultimately, unraveling the roles of hemes and iron-sulfur clusters in prebiotic chemistry promises to deepen our understanding of the origins of life on Earth and aids the search for life elsewhere in the universe.

HapX and SreA are transcription factors that regulate the response of the fungus Aspergillus fumigatus to the availability of iron. During iron starvation, HapX represses genes involved in iron consuming pathways and upon a shift to iron excess, HapX activates these same genes. SreA blocks the expression of genes needed for iron uptake during periods of iron availability. Both proteins possess cysteine-rich regions (CRR) that are hypothesized to be necessary for the sensing of iron levels. However, the contribution of each of these domains to the function of the protein has remained unclear. Here, the ability of peptide analogs of each CRR is determined to bind an iron-sulfur cluster in vitro. UV-vis and resonance Raman (RR) spectroscopies reveal that each CRR is capable of coordinating a [2Fe-2S] cluster with comparable affinities. The iron-sulfur cluster coordinated to the CRR-B domain of HapX displays particularly high stability. The data are consistent with HapX and SreA mediating responses to cellular iron levels through the direct coordination of [2Fe-2S] clusters. The high stability of the CRR-B peptide may also find use as a starting point for the development of new green catalysts.

Synthetic biology is a broad field with a unifying theme of learning through building. At IUPAB2024 in Kyoto, Japan, five researchers gave lectures focusing on in vitro synthetic biology, where the built objects were not living cells but in vitro systems composed of biomolecules. The talks sparked lively discussions on the knowledge gained about living cells from in vitro reconstructions.

While many proposals of paper-based diagnostics utilize cell-free gene expression systems, these assays oftentimes suffer from the need for temperature cycling and high operational costs, particularly for developing countries. Here, we explore and report the experimental conditions for the colorimetric detection of viral RNA with an in vitro transcription/translation assay that uses crude E. coli extracts at room temperature where the signal amplification is aided by body heat. Clinically-relevant concentrations of RNA (ca. 600 copies/test) were detected from synthetic RNA samples. The activation of cell-free gene expression was achieved using toehold-switch-mediated riboregulatory elements that are specific to RNA sequences. The colorimetric output was generated by the α-complementation of β-galactosidase ω-fragment (LacZω) with cell-free expressed LacZα, using an X-gal analogue as a substrate. The estimated cost of a single reaction is as low as 0.26 euro/test, which may help to facilitate the accessibility of the diagnostic kit in developing countries. With future optimizations and bacterial strain engineering, production costs can be even further brought down, and the test times can be shortened.

Iron-sulfur clusters are ancient cofactors that could have played a role in the prebiotic chemistry leading to the emergence of protometabolism. Previous research has shown that certain iron-sulfur clusters can form from prebiotically plausible components, such as cysteine-containing oligopeptides. However, it is unclear if these iron-sulfur clusters could have survived in prebiotically plausible environments. To begin exploring this possibility, we tested the stability of iron-sulfur clusters coordinated to a tripeptide and to N-acetyl-L-cysteine methyl ester in a variety of solutions meant to mimic prebiotically plausible environments. We also assessed the impact of individual chemical components on stability. We find that iron-sulfur clusters form over a wide variety of conditions but that the type of iron-sulfur cluster formed is strongly impacted by the chemical environment and the coordinating scaffold. These findings support the general hypothesis that iron-sulfur clusters were present on the prebiotic Earth and that different types of iron-sulfur cluster predominated in different environments.

Abstract Multi‐analyte liquid biopsies represent an emerging opportunity for non‐invasive cancer assessment. We developed ONCE (One Aliquot for Circulating Elements), an approach for the isolation of extracellular vesicles (EV) and cell‐free DNA (cfDNA) from a single aliquot of blood. We assessed ONCE performance to classify HER2‐positive early‐stage breast cancer (BrCa) patients by combining EV‐associated RNA (EV‐RNA) and cfDNA signals on n = 64 healthy donors (HD) and non–metastatic BrCa patients. Specifically, we isolated EV‐enriched samples by a charge‐based (CB) method and investigated EV‐RNA and cfDNA by next‐generation sequencing (NGS) and by digital droplet PCR (ddPCR). Sequencing of cfDNA and EV‐RNA from HER2‐ and HER2+ patients demonstrated concordance with in situ molecular analyses of matched tissues. Combined analysis of the two circulating analytes by ddPCR showed increased sensitivity in ERBB2/HER2 detection compared to single nucleic acid components. Multi‐analyte liquid biopsy prediction performance was comparable to tissue‐based sequencing results from TCGA. Also, imaging flow cytometry analysis revealed HER2 protein on the surface of EV isolated from the HER2+ BrCa plasma, thus corroborating the potential relevance of studying EV as companion analyte to cfDNA. This data confirms the relevance of combining cfDNA and EV‐RNA for HER2 cancer assessment and supports ONCE as a valuable tool for multi‐analytes liquid biopsies’ clinical implementation.

Mitochondria must maintain adequate amounts of metabolites for protective and biosynthetic functions. However, how mitochondria sense the abundance of metabolites and regulate metabolic homeostasis is not well understood. In this work, we focused on glutathione (GSH), a critical redox metabolite in mitochondria, and identified a feedback mechanism that controls its abundance through the mitochondrial GSH transporter, SLC25A39. Under physiological conditions, SLC25A39 is rapidly degraded by mitochondrial protease AFG3L2. Depletion of GSH dissociates AFG3L2 from SLC25A39, causing a compensatory increase in mitochondrial GSH uptake. Genetic and proteomic analyses identified a putative iron-sulfur cluster in the matrix-facing loop of SLC25A39 as essential for this regulation, coupling mitochondrial iron homeostasis to GSH import. Altogether, our work revealed a paradigm for the autoregulatory control of metabolic homeostasis in organelles.

There is currently no plausible path for the emergence of a self-replicating protocell, because prevalent formulations of model protocells are built with fatty acid vesicles that cannot withstand the concentrations of Mg2+ needed for the function and replication of nucleic acids. Although prebiotic chelates increase the survivability of fatty acid vesicles, the resulting model protocells are incapable of growth and division. Here, we show that protocells made of mixtures of cyclophospholipids and fatty acids can grow and divide in the presence of Mg2+-citrate. Importantly, these protocells retain encapsulated nucleic acids during growth and division, can acquire nucleotides from their surroundings, and are compatible with the nonenzymatic extension of an RNA oligonucleotide, chemistry needed for the replication of a primitive genome. Our work shows that prebiotically plausible mixtures of lipids form protocells that are active under the conditions necessary for the emergence of Darwinian evolution.

Iron–sulfur clusters are ubiquitous cofactors that mediate central biological processes. However, despite their long history, these metallocofactors remain challenging to investigate when coordinated to small (≤ six amino acids) oligopeptides in aqueous solution. In addition to being often unstable in vitro, iron–sulfur clusters can be found in a wide variety of forms with varied characteristics, which makes it difficult to easily discern what is in solution. This difficulty is compounded by the dynamics of iron–sulfur peptides, which frequently coordinate multiple types of clusters simultaneously. To aid investigations of such complex samples, a summary of data from multiple techniques used to characterize both iron–sulfur proteins and peptides is provided. Although not all spectroscopic techniques are equally insightful, it is possible to use several, readily available methods to gain insight into the complex composition of aqueous solutions of iron–sulfur peptides.

Infections caused by drug-resistant bacteria, particularly Gram-negative organisms, are increasingly difficult to treat using antibiotics. A potential alternative is "phage therapy", in which phages infect and lyse the bacterial host. However, phage therapy poses serious drawbacks and safety concerns, such as the risk of genetic transduction of antibiotic resistance genes, inconsistent pharmacokinetics, and unknown evolutionary potential. In contrast, metallic nanoparticles possess precise, tunable properties, including efficient conversion of electronic excitation into heat. In this work, we demonstrate that engineered phage-nanomaterial conjugates that target the Gram-negative pathogen Pseudomonas aeruginosa are highly effective as a treatment of infected wounds in mice. Photothermal heating, performed as a single treatment (15 min) or as two treatments on consecutive days, rapidly reduced the bacterial load and released Zn2+ to promote wound healing. The phage-nanomaterial treatment was significantly more effective than systemic standard-of-care antibiotics, with a >10× greater reduction in bacterial load and ∼3× faster healing as measured by wound size reduction when compared to fluoroquinolone treatment. Notably, the phage-nanomaterial was also effective against a P. aeruginosa strain resistant to polymyxins, a last-line antibiotic therapy. Unlike these antibiotics, the phage-nanomaterial showed no detectable toxicity or systemic effects in mice, consistent with the short duration and localized nature of phage-nanomaterial treatment. Our results demonstrate that phage therapy controlled by inorganic nanomaterials can be a safe and effective antimicrobial strategy in vivo.

NMR data of the paper "Protometabolic Reduction of NAD+ with α-Keto Acids" from Basak et al.

Wet-dry cycles driven by heating to high temperatures are frequently invoked for the prebiotic synthesis of peptides. Similarly, iron-sulfur clusters are often cited as an example of an ancient catalyst that helped prune early chemical systems into metabolic-like pathways. Because extant iron-sulfur clusters are metallocofactors of protein enzymes and nearly ubiquitous across biology, a reasonable hypothesis is that prebiotic iron-sulfur peptides formed on the early Earth. However, iron-sulfur clusters are coordinated by multiple cysteine residues, and the stability of cysteines to the heat steps of wet-dry cycles has not been determined. It, therefore, has remained unclear if the peptides needed to stabilize the formation of iron-sulfur clusters could have formed. If not, then iron-sulfur-dependent activity may have emerged later, when milder, more biological-like peptide synthesis machinery took hold. Here, we report the thermal stability of cysteine-containing peptides. We show that temperatures of 150 °C lead to the rapid degradation of cysteinyl peptides. However, the presence of Mg2+ at environmentally reasonable concentrations leads to significant protection. Thiophilic metal ions also protect against degradation at 150 °C but require concentrations not frequently observed in the environment. Nevertheless, cysteine-containing peptides are stable at lower, prebiotically plausible temperatures in seawater, carbonate lake, and ferrous lake conditions. The data are consistent with the persistence of cysteine-containing peptides on the early Earth in environments rich in metal ions. High concentrations of Mg2+ are common intra- and extra-cellularly, suggesting that the protection afforded by Mg2+ may reflect conditions that were present on the prebiotic Earth.