Validation of Cell-Free Protein Synthesis Aboard the International Space Station.

Cell-free protein synthesis (CFPS) is a rapidly maturing in vitro gene expression platform that can be used to transcribe and translate nucleic acids at the point of need, enabling on-demand synthesis of peptide-based vaccines and biotherapeutics as well as the development of diagnostic tests for environmental contaminants and infectious agents. Unlike traditional cell-based systems, CFPS platforms do not require the maintenance of living cells and can be deployed with minimal equipment; therefore, they hold promise for applications in low-resource contexts, including spaceflight. Here, we evaluate the performance of the cell-free platform BioBits aboard the International Space Station by expressing RNA-based aptamers and fluorescent proteins that can serve as biological indicators. We validate two classes of biological sensors that detect either the small-molecule DFHBI or a specific RNA sequence. Upon detection of their respective analytes, both biological sensors produce fluorescent readouts that are visually confirmed using a hand-held fluorescence viewer and imaged for quantitative analysis. Our findings provide insights into the kinetics of cell-free transcription and translation in a microgravity environment and reveal that both biosensors perform robustly in space. Our findings lay the groundwork for portable, low-cost applications ranging from point-of-care health monitoring to on-demand detection of environmental hazards in low-resource communities both on Earth and beyond.

Benchtop 19F Nuclear Magnetic Resonance (NMR) Spectroscopy Provides Mechanistic Insight into the Biginelli Condensation toward the Chemical Synthesis of Novel Trifluorinated Dihydro- and Tetrahydropyrimidinones as Antiproliferative Agents.

Benchtop nuclear magnetic resonance (NMR) spectroscopy has enabled the monitoring and optimization of chemical transformations while simultaneously providing kinetic, mechanistic, and structural insight into reaction pathways with quantitative precision. Moreover, benchtop NMR proton lock capabilities further allow for rapid and convenient monitoring of various organic reactions in real time, as the use of deuterated solvents is not required. The complementary role of 19F NMR-based kinetic monitoring in the fluorination of bioactive compounds has many benefits in the drug discovery process since fluorinated motifs additionally improve drug pharmacology. In this study, 19F NMR spectroscopy was utilized to monitor the synthesis of novel trifluorinated analogs of monastrol, a small molecule dihydropyrimidinone kinesin-Eg5 inhibitor, and to probe the mechanism of the Biginelli cyclocondensation, a multicomponent reaction used to synthesize dihydropyrimidinone and tetrahydropyrimidinones through a Bronsted- or Lewis-acid catalyzed cyclocondensation between ethyl acetoacetate, thiourea, and an aryl aldehyde. In the present study, a trifluorinated ketoester serves a dual purpose as being the source of the trifluoromethyl group in our fluorinated dihydropyrimidinones and as a spectroscopic handle for real-time reaction monitoring and tracking of reactive intermediates by 19F NMR. Further, upon extending this workflow to a diverse array of 3- and 4-substituted aryl aldehydes, we were able to derive Hammett linear free energy relationships (LFER) to determine stereoelectronic effects of para- and meta-substituted aryl aldehydes to corresponding reaction rates and mechanistic routes. In addition, we used density functional theory (DFT) calculations to corroborate our experimental results through the thermodynamic values of key intermediates in each mechanism. Finally, these studies culminate in the synthesis of a novel trifluorinated analog of monastrol and its subsequent biological evaluation in vitro. More broadly, we show an application of benchtop 19F NMR spectroscopy as an analytical tool in the real-time investigation of a mechanistically and chemically complex multicomponent reaction mixture.

Uncovering the functional diversity of rare CRISPR-Cas systems with deep terascale clustering.

Microbial systems underpin many biotechnologies, including CRISPR, but the exponential growth of sequence databases makes it difficult to find previously unidentified systems. In this work, we develop the fast locality-sensitive hashing-based clustering (FLSHclust) algorithm, which performs deep clustering on massive datasets in linearithmic time. We incorporated FLSHclust into a CRISPR discovery pipeline and identified 188 previously unreported CRISPR-linked gene modules, revealing many additional biochemical functions coupled to adaptive immunity. We experimentally characterized three HNH nuclease-containing CRISPR systems, including the first type IV system with a specified interference mechanism, and engineered them for genome editing. We also identified and characterized a candidate type VII system, which we show acts on RNA. This work opens new avenues for harnessing CRISPR and for the broader exploration of the vast functional diversity of microbial proteins.