Nuclear pore complexes regulate the selective exchange of RNA and proteins across the nuclear envelope in eukaryotic cells. Biomimetic strategies offer new opportunities to investigate this remarkable transport phenomenon. Here, we show selective transport of proteins across individual biomimetic nuclear pore complexes at the single-molecule level. Each biomimetic complex is constructed by covalently tethering either Nup98 or Nup153 (phenylalanine-glycine (FG) nucleoporins) to a solid-state nanopore. Individual translocation events are monitored using ionic current measurements with sub-millisecond temporal resolution. Transport receptors (Impβ) proceed with a dwell time of ∼2.5 ms for both Nup98- and Nup153-coated pores, whereas the passage of non-specific proteins is strongly inhibited with different degrees of selectivity. For pores up to ∼25 nm in diameter, Nups form a dense and low-conducting barrier, whereas they adopt a more open structure in larger pores. Our biomimetic nuclear pore complex provides a quantitative platform for studying nucleocytoplasmic transport phenomena at the single-molecule level in vitro.

Replication of DNA-encoded information and its conversion into functional proteins are universal properties of life. In an effort toward the construction of a synthetic minimal cell, we implement here the DNA replication machinery of the Φ29 virus in a cell-free gene expression system. Amplification of a linear DNA template by self-encoded, de novo synthesized Φ29 proteins is demonstrated. Complete information transfer is confirmed as the copied DNA can serve as a functional template for gene expression, which can be seen as an autocatalytic DNA replication cycle. These results show how the central dogma of molecular biology can be reconstituted and form a cycle in vitro. Finally, coupled DNA replication and gene expression is compartmentalized inside phospholipid vesicles providing the chassis for evolving functions in a prospective synthetic cell relying on the extant biology.

The absence of ‘shovel-ready’ anti-coronavirus drugs during vaccine development has exceedingly worsened the SARS-CoV-2 pandemic. Furthermore, new vaccine-resistant variants and coronavirus outbreaks may occur in the near future, and we must be ready to face this possibility. However, efficient antiviral drugs are still lacking to this day, due to our poor understanding of the mode of incorporation and mechanism of action of nucleotides analogs that target the coronavirus polymerase to impair its essential activity. Here, we characterize the impact of remdesivir (RDV, the only FDA-approved anti-coronavirus drug) and other nucleotide analogs (NAs) on RNA synthesis by the coronavirus polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. We reveal that the location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We show that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into backtrack as far as 30 nt, which may appear as termination in traditional ensemble assays. SARS-CoV-2 is able to evade the endogenously synthesized product of the viperin antiviral protein, ddhCTP, though the polymerase incorporates this NA well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases.

DNA-guided cell-free protein synthesis using a minimal set of purified components has emerged as a versatile platform in constructive biology. The E. coli-based PURE (Protein synthesis Using Recombinant Elements) system offers the basic protein synthesis factory in a prospective minimal cell relying on extant molecules. However, it becomes urgent to improve the system's performance, and to build a mechanistic computational model that can help interpret and predict gene expression dynamics. Herein, we utilized all three commercially available PURE system variants: PURExpress, PUREfrex and PUREfrex2.0. We monitored apparent kinetics of mRNA and protein synthesis by fluorescence spectroscopy at different concentrations of DNA template. Analysis of polysome distributions by atomic force microscopy, combined with a stochastic model of translation, revealed inefficient usage of ribosomes, consistent with the idea that translation initiation is a limiting step. This preliminary dataset was used to formulate hypotheses regarding possible mechanisms impeding robust gene expression. Next, we challenged these hypotheses by devising targeted experiments aimed to alleviate the current limitations of PUREfrex. We identified depletion of key initiation factors by translationally inactive mRNA as a possible inhibitory mechanism. This adverse process could partly be remedied by targeted mRNA degradation, whereas addition of more IFs and of the hrpA RNA helicase had no substantial effects. Moreover, depletion of tRNAs as peptidyl-tRNAs can become limiting in PUREfrex (but not in PURExpress), which can be alleviated by addition of peptidyl-tRNAhydrolase (PTH). We attempted to build a new model for PURE system dynamics integrating all experimental observations. Although a satisfying global fit can be obtained in specific conditions (with PTH), a unifying system's level model is still missing.

The biosynthesis of proteins from genomic DNA is a universal process in every living organism. Building a synthetic cell using separate biological parts hence implies to reconstitute a minimal gene expression apparatus and to compartmentalize it in a cell-mimicking environment. Previous studies have demonstrated that the PURE (Protein synthesis Using Recombinant Elements) system could be functionally encapsulated inside lipid vesicles. However, quantitative insights on functional consequences of spatial confinement of PURE system reactions remain scarce, which has hampered the full exploitation of gene-expressing liposomes as the fundamental unit to build an artificial cell. We report on direct imaging of tens of thousands of gene-expressing liposomes per sample allowing us to assess sub-population features in a statistically relevant manner. Both the vesicle size (diameter <10 μm) and lipid composition (mixture of phospholipids with zwitterionic and negatively charged headgroups, including cardiolipin) are compatible with the properties of bacterial cells. Therefore, our liposomes provide a suitable chassis to host the Escherichia coli-derived PURE translation machinery and other bacterial processes in future developments. The potential of highcontent imaging to identify rare phenotypes is demonstrated by the fact that a subset of the liposome population exhibits a remarkably high yield of synthesized protein or a prolonged expression lifespan that surpasses the performance of ensemble liposome-averaged and bulk reactions. Among the three commercial PURE systems tested, PUREfrex2.0 offers the most favorable phenotypes displaying both high yield and long protein synthesis lifespan. Moreover, probing membrane permeability reveals a large heterogeneity amongst liposomes. In situ expression and membrane embedding of the poreforming connexin leads to a characteristic permeability time profile, while increasing the fraction of permeable liposomes in the population. We see diversity in gene expression dynamics and membrane permeability as an opportunity to complement a rational design approach aiming at further implementing biological functions in liposome-based synthetic cells.