Emergent ribozyme behaviors in oxychlorine brines indicate a unique niche for molecular evolution on Mars.

Mars is a particularly attractive candidate among known astronomical objects to potentially host life. Results from space exploration missions have provided insights into Martian geochemistry that indicate oxychlorine species, particularly perchlorate, are ubiquitous features of the Martian geochemical landscape. Perchlorate presents potential obstacles for known forms of life due to its toxicity. However, it can also provide potential benefits, such as producing brines by deliquescence, like those thought to exist on present-day Mars. Here we show perchlorate brines support folding and catalysis of functional RNAs, while inactivating representative protein enzymes. Additionally, we show perchlorate and other oxychlorine species enable ribozyme functions, including homeostasis-like regulatory behavior and ribozyme-catalyzed chlorination of organic molecules. We suggest nucleic acids are uniquely well-suited to hypersaline Martian environments. Furthermore, Martian near- or subsurface oxychlorine brines, and brines found in potential lifeforms, could provide a unique niche for biomolecular evolution.

Sequential gentle hydration increases encapsulation in model protocells.

Small, spherical vesicles are a widely used chassis for the formation of model protocells and investigating the beginning of compartmentalized evolution. Various methods exist for their preparation, with one of the most common approaches being gentle hydration, where thin layers of lipids are hydrated with aqueous solutions and gently agitated to form vesicles. An important benefit to gentle hydration is that the method produces vesicles without introducing any organic contaminants, such as mineral oil, into the lipid bilayer. However, compared to other methods of liposome formation, gentle hydration is much less efficient at encapsulating aqueous cargo. Improving the encapsulation efficiency of gentle hydration would be of broad use for medicine, biotechnology, and protocell research. Here, we describe a method of sequentially hydrating lipid thin films to increase encapsulation efficiency. We demonstrate that sequential gentle hydration significantly improves encapsulation of water-soluble cargo compared to the traditional method, and that this improved efficiency is dependent on buffer composition. Similarly, we also demonstrate how this method can be used to increase concentrations of oleic acid, a fatty acid commonly used in origins of life research, to improve the formation of vesicles in aqueous buffer.

PACRAT: pathogen detection with aptamer-observed cascaded recombinase polymerase amplification-in vitro transcription.

The SARS-CoV-2 pandemic underscored the need for early, rapid, and widespread pathogen detection tests that are readily accessible. Many existing rapid isothermal detection methods use the recombinase polymerase amplification (RPA), which exhibits polymerase chain reaction (PCR)-like sensitivity, specificity, and even higher speed. However, coupling RPA to other enzymatic reactions has proven difficult. For the first time, we demonstrate that with tuning of buffer conditions and optimization of reagent concentrations, RPA can be cascaded into an in vitro transcription reaction, enabling detection using fluorescent aptamers in a one-pot reaction. We show that this reaction, which we term PACRAT (pathogen detection with aptamer-observed cascaded recombinase polymerase amplification-in vitro transcription) can be used to detect SARS-CoV-2 RNA with single-copy detection limits, Escherichia coli with single-cell detection limits, and 10-min detection times. Further demonstrating the utility of our one-pot, cascaded amplification system, we show PACRAT can be used for multiplexed detection of the pathogens SARS-CoV-2 and E. coli, along with multiplexed detection of two variants of SARS-CoV-2.

Controlled exchange of protein and nucleic acid signals from and between synthetic minimal cells.

Synthetic minimal cells are a class of bioreactors that have some, but not all, functions of live cells. Here, we report a critical step toward the development of a bottom-up minimal cell: cellular export of functional protein and RNA products. We used cell-penetrating peptide tags to translocate payloads across a synthetic cell vesicle membrane. We demonstrated efficient transport of active enzymes and transport of nucleic acid payloads by RNA-binding proteins. We investigated influence of a concentration gradient alongside other factors on the efficiency of the translocation, and we show a method to increase product accumulation in one location. We demonstrate the use of this technology to engineer molecular communication between different populations of synthetic cells, to exchange protein and nucleic acid signals. The synthetic minimal cell production and export of proteins or nucleic acids allows experimental designs that approach the complexity and relevancy of natural biological systems. A record of this paper's transparent peer review process is included in the supplemental information.

Sequential gentle hydration increases encapsulation in model protocells.

Small, spherical vesicles are a widely used chassis for the formation of model protocells and investigating the beginning of compartmentalized evolution. Various methods exist for their preparation, with one of the most common approaches being gentle hydration, where thin layers of lipids are hydrated with aqueous solutions and gently agitated to form vesicles. An important benefit to gentle hydration is that the method produces vesicles without introducing any organic contaminants, such as mineral oil, into the lipid bilayer. However, compared to other methods of liposome formation, gentle hydration is much less efficient at encapsulating aqueous cargo. Improving the encapsulation efficiency of gentle hydration would be of broad use for medicine, biotechnology, and protocell research. Here, we describe a method of sequentially hydrating lipid thin films to increase encapsulation efficiency. We demonstrate that sequential gentle hydration significantly improves encapsulation of water-soluble cargo compared to the traditional method, and that this improved efficiency is dependent on buffer composition. Similarly, we also demonstrate how this method can be used to increase concentrations of oleic acid, a fatty acid commonly used in origins of life research, to improve the formation of vesicles in aqueous buffer.

A gene expression control technology for cell-free systems and synthetic cells via targeted gene silencing and transfection.

Synthetic cells, expressing proteins using cell-free transcription-translation (TXTL), is a technology utilized for a variety of applications, such as investigating natural gene pathways, metabolic engineering, drug development or bioinformatics. For all these purposes, the ability to precisely control gene expression is essential. Various strategies to control gene expression in TXTL have been developed; however, further advancements on gene-specific and straightforward regulation methods are still needed. Here, we present a method of control of gene expression in TXTL using a "silencing oligo": a short oligonucleotide, designed with a particular secondary structure, that binds to the target messenger RNA. We demonstrated that silencing oligo inhibits protein expression in TXTL in a sequence-dependent manner. We showed that silencing oligo activity is associated with RNase H activity in bacterial TXTL. To complete the gene expression control toolbox for synthetic cells, we also engineered a first transfection system. We demonstrated the transfection of various payloads, enabling the introduction of RNA and DNA of different lengths to synthetic cell liposomes. Finally, we combined the silencing oligo and the transfection technologies, demonstrating control of gene expression by transfecting silencing oligo into synthetic minimal cells.

Trumpet is an operating system for simple and robust cell-free biocomputing.

Biological computation is becoming a viable and fast-growing alternative to traditional electronic computing. Here we present a biocomputing technology called Trumpet: Transcriptional RNA Universal Multi-Purpose GatE PlaTform. Trumpet combines the simplicity and robustness of the simplest in vitro biocomputing methods, adding signal amplification and programmability, while avoiding common shortcomings of live cell-based biocomputing solutions. We have demonstrated the use of Trumpet to build all universal Boolean logic gates. We have also built a web-based platform for designing Trumpet gates and created a primitive processor by networking several gates as a proof-of-principle for future development. The Trumpet offers a change of paradigm in biocomputing, providing an efficient and easily programmable biological logic gate operating system.

New Aequorea Fluorescent Proteins for Cell-Free Bioengineering.

Recently, a new subset of fluorescent proteins has been identified from the Aequorea species of jellyfish. These fluorescent proteins were characterized in vivo; however, there has not been validation of these proteins within cell-free systems. Cell-free systems and technology development is a rapidly expanding field, encompassing foundational research, synthetic cells, bioengineering, biomanufacturing, and drug development. Cell-free systems rely heavily on fluorescent proteins as reporters. Here we characterize and validate this new set of Aequorea proteins for use in a variety of cell-free and synthetic cell expression platforms.

Parasites, Infections, and Inoculation in Synthetic Minimal Cells.

Synthetic minimal cells provide a controllable and engineerable model for biological processes. While much simpler than any live natural cell, synthetic cells offer a chassis for investigating the chemical foundations of key biological processes. Herein, we show a synthetic cell system with host cells, interacting with parasites and undergoing infections of varying severity. We demonstrate how the host can be engineered to resist infection, we investigate the metabolic cost of carrying resistance, and we show an inoculation that immunizes the host against pathogens. Our work expands the synthetic cell engineering toolbox by demonstrating host-pathogen interactions and mechanisms for acquiring immunity. This brings synthetic cell systems one step closer to providing a comprehensive model of complex, natural life.

T7Max transcription system.

BACKGROUND: Efficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. In addition, the yields of transcription in cell-free systems lag behind transcriptional efficiency of live cells. Most commonly used in vitro translation systems utilize T7 RNA polymerase, which is also the enzyme included in many commercial kits. RESULTS: Here we present characterization of a variant of T7 RNA polymerase promoter that acts to significantly increase the yields of gene expression within in vitro systems. We have demonstrated that T7Max increases the yield of translation in many types of commonly used in vitro protein expression systems. We also demonstrated increased protein expression yields from linear templates, allowing the use of T7Max driven expression from linear templates. CONCLUSIONS: The modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase- based in vitro protein expression system.

Expanding luciferase reporter systems for cell-free protein expression.

Luciferases are often used as a sensitive, versatile reporter in cell-free transcription-translation (TXTL) systems, for research and practical applications such as engineering genetic parts, validating genetic circuits, and biosensor outputs. Currently, only two luciferases (Firefly and Renilla) are commonly used without substrate cross-talk. Here we demonstrate the expansion of the cell-free luciferase reporter system, with two orthogonal luciferase reporters: N. nambi luciferase (Luz) and LuxAB. These luciferases do not have cross-reactivity with the Firefly and Renilla substrates. We also demonstrate a substrate regeneration pathway for one of the new luciferases, enabling long-term time courses of protein expression monitoring in the cell-free system. Furthermore, we reduced the number of genes required in TXTL expression, by engineering a cell extract containing part of the luciferase enzymes. Our findings lead to an expanded platform with multiple orthogonal luminescence translation readouts for in vitro protein expression.

Akaby-Cell-free protein expression system for linear templates.

Cell-free protein expression is increasingly becoming popular for biotechnology, biomedical and research applications. Among cell-free systems, the most popular one is based on Escherichia coli (E. coli). Endogenous nucleases in E. coli cell-free transcription-translation (TXTL) degrade the free ends of DNA, resulting in inefficient protein expression from linear DNA templates. RecBCD is a nuclease complex that plays a major role in nuclease activity in E. coli, with the RecB subunit possessing the actual nuclease activity. We created a RecB knockout of an E. coli strain optimized for cell-free expression. We named this new strain Akaby. We demonstrated that Akaby TXTL successfully reduced linear DNA degradations, rescuing the protein expression efficiency from the linear DNA templates. The practicality of Akaby for TXTL is an efficient, simple alternative for linear template expression in cell-free reactions. We also use this work as a model protocol for modifying the TXTL source E. coli strain, enabling the creation of TXTL systems with other custom modifications.

DNA G-quadruplexes are uniquely stable in the presence of denaturants and monovalent cations.

Ions in the Hofmeister series exhibit varied effects on biopolymers. Those classed as kosmotropes generally stabilize secondary structure, and those classed as chaotropes generally destabilize secondary structure. Here, we report that several anionic chaotropes exhibit unique effects on one DNA secondary structure - a G quadruplex. These chaotropes exhibit the expected behaviour (destabilization of secondary structure) in two other structural contexts: a DNA duplex and i-Motifs. Uniquely among secondary structures, we observe that G quadruplexes are comparatively insensitive to the presence of anionic chaotropes, but not other denaturants. Further, the presence of equimolar NaCl provided greater mitigation of the destabilization caused by other non-anionic denaturants. These results are consistent with the presence of monovalent cations providing an especially pronounced stabilizing effect to G quadruplexes when studied in denaturing solution conditions.

Switchable DNA-Based Peroxidases Controlled by a Chaotropic Ion.

Here we demonstrate a switchable DNA electron-transfer catalyst, enabled by selective destabilization of secondary structure by the denaturant, perchlorate. The system is comprised of two strands, one of which can be selectively switched between a G-quadruplex and duplex or single-stranded conformations. In the G-quadruplex state, it binds hemin, enabling peroxidase activity. This switching ability arises from our finding that perchlorate, a chaotropic Hofmeister ion, selectively destabilizes duplex over G-quadruplex DNA. By varying perchlorate concentration, we show that the DNA structure can be switched between states that do and do not catalyze electron-transfer catalysis. State switching can be achieved in three ways: thermally, by dilution, or by concentration.

Programmable Fusion and Differentiation of Synthetic Minimal Cells.

Synthetic cells can mimic the intricate complexities of live cells, while mitigating the level of noise that is present natural systems; however, many crucial processes still need to be demonstrated in synthetic cells to use them to comprehensively study and engineer biology. Here we demonstrate key functionalities of synthetic cells previously available only to natural life: differentiation and mating. This work presents a toolset for engineering combinatorial genetic circuits in synthetic cells. We demonstrate how progenitor populations can differentiate into new lineages in response to small molecule stimuli or as a result of fusion, and we provide practical demonstration of utility for metabolic engineering. This work provides a tool for bioengineering and for natural pathway studies, as well as paving the way toward the construction of live artificial cells.

Isothermal SARS-CoV-2 Diagnostics: Tools for Enabling Distributed Pandemic Testing as a Means of Supporting Safe Reopenings.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, poses grave threats to both the global economy and health. The predominant diagnostic screens in use for SARS-CoV-2 detection are molecular techniques such as nucleic acid amplification tests. In this Review, we compare current and emerging isothermal diagnostic methods for COVID-19. We outline the molecular and serological techniques currently being used to detect SARS-CoV-2 infection, past or present, in patients. We also discuss ongoing research on isothermal techniques, CRISPR-mediated detection assays, and point-of-care diagnostics that have potential for use in SARS-CoV-2 detection. Large-scale viral testing during a global pandemic presents unique challenges, chief among them the simultaneous need for testing supplies, durable equipment, and personnel in many regions worldwide, with each of these regions possessing testing needs that vary as the pandemic progresses. The low-cost isothermal technologies described in this Review provide a promising means by which to address these needs and meet the global need for testing of symptomatic individuals as well as provide a possible means for routine testing of asymptomatic individuals, providing a potential means of safely enabling reopenings and early monitoring of outbreaks.

Rapid deployment of smartphone-based augmented reality tools for field and online education in structural biology.

Structural biology education commonly employs molecular visualization software, such as PyMol, RasMol, and VMD, to allow students to appreciate structure-function relationships in biomolecules. In on-ground, classroom-based education, these programs are commonly used on University-owned devices with software preinstalled. Remote education typically involves the use of student-owned devices, which complicates the use of such software, owing to the fact that (a) student devices have differing configurations (e.g., Windows vs MacOS) and processing power, and (b) not all student devices are suitable for use with such software. Smartphones are near-ubiquitous devices, with smartphone ownership exceeding personal computer ownership, according to a recent survey. Here, we show the use of a smartphone-based augmented reality app, Augment, in a structural biology classroom exercise, which students installed independently without IT support. Post-lab attitudinal survey results indicate positive student experiences with this app. Based on our experiences, we suggest that smartphone-based molecular visualization software, such as that used in this exercise, is a powerful educational tool that is particularly well-suited for use in remote education.

Highly specific, multiplexed isothermal pathogen detection with fluorescent aptamer readout.

Isothermal, cell-free, synthetic biology-based approaches to pathogen detection leverage the power of tools available in biological systems, such as highly active polymerases compatible with lyophilization, without the complexity inherent to live-cell systems, of which nucleic acid sequence based amplification (NASBA) is well known. Despite the reduced complexity associated with cell-free systems, side reactions are a common characteristic of these systems. As a result, these systems often exhibit false positives from reactions lacking an amplicon. Here we show that the inclusion of a DNA duplex lacking a promoter and unassociated with the amplicon fully suppresses false positives, enabling a suite of fluorescent aptamers to be used as NASBA tags (Apta-NASBA). Apta-NASBA has a 1 pM detection limit and can provide multiplexed, multicolor fluorescent readout. Furthermore, Apta-NASBA can be performed using a variety of equipment, for example, a fluorescence microplate reader, a qPCR instrument, or an ultra-low-cost Raspberry Pi-based 3D-printed detection platform using a cell phone camera module, compatible with field detection.

Methods for thermal denaturation studies of nucleic acids in complex with fluorogenic dyes.

Thermal denaturation is a common technique in the biophysical study of nucleic acids. These experiments are typically performed by monitoring the increase in absorbance (hyperchromism) of a sample at 260nm with temperature (Mergny & Lacroix, 2003; Puglisi & Tinoco, 1989). This wavelength is chosen as nucleic acids of mixed sequence typically exhibit their maximum absorbance here. Exceptions exist, however, some noncanonical nucleic acid structures exhibit differing spectral changes with temperature, resulting in other wavelengths being convenient reporters of secondary structure. In the case of nucleic acids that bind visible light-absorbing ligands, such as fluorogenic aptamers, another wavelength can be a convenient reporter of secondary structure stability and RNA-ligand recognition. As it can be difficult, if not impossible, to know which wavelength to employ a priori, we have developed a system for obtaining the full UV-visible spectrum of a sample at each wavelength, allowing for the subsequent extraction of the absorbance-temperature profile at the desired wavelength. Here, we describe the apparatus and software used to do so. We also describe another technique for the use of a qPCR instrument for measuring secondary structure stability of fluorescent nucleic acid-ligand complexes.

Catalysis of Template-Directed Nonenzymatic RNA Copying by Iron(II).

The study of nonenzymatic template-directed RNA copying is the experimental basis for the search for chemistry and reaction conditions consistent with prebiotic RNA replication. The most effective model systems for RNA copying have to date required a high concentration of Mg2+. Recently, Fe2+, which was abundant on the prebiotic anoxic Earth, was shown to promote the folding of RNA in a manner similar to the case of Mg2+, as a result of the two cations having similar interactions with phosphate groups. These observations raise the question of whether Fe2+ could have promoted RNA copying on the prebiotic Earth. Here, we demonstrate that Fe2+ is a better catalyst and promotes faster nonenzymatic RNA primer extension and ligation than Mg2+ when using 2-methylimidazole activated nucleotides in slightly acidic to neutral pH solutions. Thus, it appears likely that Fe2+ could have facilitated RNA replication and evolution in concert with other metal cations on the prebiotic Earth.