Future of the Search for Life: Workshop Report.

The 2-week, virtual Future of the Search for Life science and engineering workshop brought together more than 100 scientists, engineers, and technologists in March and April 2022 to provide their expert opinion on the interconnections between life-detection science and technology. Participants identified the advances in measurement and sampling technologies they believed to be necessary to perform in situ searches for life elsewhere in our Solar System, 20 years or more in the future. Among suggested measurements for these searches, those pertaining to three potential indicators of life termed "dynamic disequilibrium," "catalysis," and "informational polymers" were identified as particularly promising avenues for further exploration. For these three indicators, small breakout groups of participants identified measurement needs and knowledge gaps, along with corresponding constraints on sample handling (acquisition and processing) approaches for a variety of environments on Enceladus, Europa, Mars, and Titan. Despite the diversity of these environments, sample processing approaches all tend to be more complex than those that have been implemented on missions or envisioned for mission concepts to date. The approaches considered by workshop breakout groups progress from nondestructive to destructive measurement techniques, and most involve the need for fluid (especially liquid) sample processing. Sample processing needs were identified as technology gaps. These gaps include technology and associated sampling strategies that allow the preservation of the thermal, mechanical, and chemical integrity of the samples upon acquisition; and to optimize the sample information obtained by operating suites of instruments on common samples. Crucially, the interplay between science-driven life-detection strategies and their technological implementation highlights the need for an unprecedented level of payload integration and extensive collaboration between scientists and engineers, starting from concept formulation through mission deployment of life-detection instruments and sample processing systems.

The Role of the CuCl Active Complex in the Stereoselectivity of the Salt-Induced Peptide Formation Reaction: Insights from Density Functional Theory Calculations.

The salt-induced peptide formation (SIPF) reaction is a prebiotically plausible mechanism for the spontaneous polymerization of amino acids into peptides on early Earth. Experimental investigations of the SIPF reaction have found that in certain conditions, the l enantiomer is more reactive than the d enantiomer, indicating its potential role in the rise of biohomochirality. Previous work hypothesized that the distortion of the CuCl active complex toward a tetrahedral-like structure increases the central chirality on the Cu ion, which amplifies the inherent parity-violating energy differences between l- and d-amino acid enantiomers, leading to stereoselectivity. Computational evaluations of this theory have been limited to the protonated-neutral l + l forms of the CuCl active complex. Here, density functional theory methods were used to compare the energies and geometries of the homochiral (l + l and d + d) and heterochiral (l + d) CuCl-amino acid complexes for both the positive-neutral and neutral-neutral forms for alanine, valine, and proline. Significant energy differences were not observed between different chiral active complexes (i.e., d + d, l + l vs. l + d), and the distortions of active complexes between stereoselective systems and non-selective systems were not consistent, indicating that the geometry of the active complex is not the primary driver of the observed stereoselectivity of the SIPF reaction.

Diverse organic-mineral associations in Jezero crater, Mars.

The presence and distribution of preserved organic matter on the surface of Mars can provide key information about the Martian carbon cycle and the potential of the planet to host life throughout its history. Several types of organic molecules have been previously detected in Martian meteorites1 and at Gale crater, Mars2-4. Evaluating the diversity and detectability of organic matter elsewhere on Mars is important for understanding the extent and diversity of Martian surface processes and the potential availability of carbon sources1,5,6. Here we report the detection of Raman and fluorescence spectra consistent with several species of aromatic organic molecules in the Máaz and Séítah formations within the Crater Floor sequences of Jezero crater, Mars. We report specific fluorescence-mineral associations consistent with many classes of organic molecules occurring in different spatial patterns within these compositionally distinct formations, potentially indicating different fates of carbon across environments. Our findings suggest there may be a diversity of aromatic molecules prevalent on the Martian surface, and these materials persist despite exposure to surface conditions. These potential organic molecules are largely found within minerals linked to aqueous processes, indicating that these processes may have had a key role in organic synthesis, transport or preservation.

Aqueous alteration processes in Jezero crater, Mars-implications for organic geochemistry.

The Perseverance rover landed in Jezero crater, Mars, in February 2021. We used the Scanning Habitable Environments with Raman and Luminescence for Organics and Chemicals (SHERLOC) instrument to perform deep-ultraviolet Raman and fluorescence spectroscopy of three rocks within the crater. We identify evidence for two distinct ancient aqueous environments at different times. Reactions with liquid water formed carbonates in an olivine-rich igneous rock. A sulfate-perchlorate mixture is present in the rocks, which probably formed by later modifications of the rocks by brine. Fluorescence signatures consistent with aromatic organic compounds occur throughout these rocks and are preserved in minerals related to both aqueous environments.

Visualization and identification of single meteoritic organic molecules by atomic force microscopy.

Using high-resolution atomic force microscopy (AFM) with CO-functionalized tips, we atomically resolved individual molecules from Murchison meteorite samples. We analyzed powdered Murchison meteorite material directly, as well as processed extracts that we prepared to facilitate characterization by AFM. From the untreated Murchison sample, we resolved very few molecules, as the sample contained mostly small molecules that could not be identified by AFM. By contrast, using a procedure based on several trituration and extraction steps with organic solvents, we isolated a fraction enriched in larger organic compounds. The treatment increased the fraction of molecules that could be resolved by AFM, allowing us to identify organic constituents and molecular moieties, such as polycyclic aromatic hydrocarbons and aliphatic chains. The AFM measurements are complemented by high-resolution mass spectrometry analysis of Murchison fractions. We provide a proof of principle that AFM can be used to image and identify individual organic molecules from meteorites and propose a method for extracting and preparing meteorite samples for their investigation by AFM. We discuss the challenges and prospects of this approach to study extraterrestrial samples based on single-molecule identification.

COSPAR Sample Safety Assessment Framework (SSAF).

The Committee on Space Research (COSPAR) Sample Safety Assessment Framework (SSAF) has been developed by a COSPAR appointed Working Group. The objective of the sample safety assessment would be to evaluate whether samples returned from Mars could be harmful for Earth's systems (e.g., environment, biosphere, geochemical cycles). During the Working Group's deliberations, it became clear that a comprehensive assessment to predict the effects of introducing life in new environments or ecologies is difficult and practically impossible, even for terrestrial life and certainly more so for unknown extraterrestrial life. To manage expectations, the scope of the SSAF was adjusted to evaluate only whether the presence of martian life can be excluded in samples returned from Mars. If the presence of martian life cannot be excluded, a Hold & Critical Review must be established to evaluate the risk management measures and decide on the next steps. The SSAF starts from a positive hypothesis (there is martian life in the samples), which is complementary to the null-hypothesis (there is no martian life in the samples) typically used for science. Testing the positive hypothesis includes four elements: (1) Bayesian statistics, (2) subsampling strategy, (3) test sequence, and (4) decision criteria. The test sequence capability covers self-replicating and non-self-replicating biology and biologically active molecules. Most of the investigations associated with the SSAF would need to be carried out within biological containment. The SSAF is described in sufficient detail to support planning activities for a Sample Receiving Facility (SRF) and for preparing science announcements, while at the same time acknowledging that further work is required before a detailed Sample Safety Assessment Protocol (SSAP) can be developed. The three major open issues to be addressed to optimize and implement the SSAF are (1) setting a value for the level of assurance to effectively exclude the presence of martian life in the samples, (2) carrying out an analogue test program, and (3) acquiring relevant contamination knowledge from all Mars Sample Return (MSR) flight and ground elements. Although the SSAF was developed specifically for assessing samples from Mars in the context of the currently planned NASA-ESA MSR Campaign, this framework and the basic safety approach are applicable to any other Mars sample return mission concept, with minor adjustments in the execution part related to the specific nature of the samples to be returned. The SSAF is also considered a sound basis for other COSPAR Planetary Protection Category V, restricted Earth return missions beyond Mars. It is anticipated that the SSAF will be subject to future review by the various MSR stakeholders.

Calibration of the SHERLOC Deep Ultraviolet Fluorescence-Raman Spectrometer on the Perseverance Rover.

We describe the wavelength calibration of the spectrometer for the scanning of habitable environments with Raman and luminescence for organics and chemicals (SHERLOC) instrument onboard NASA's Perseverance Rover. SHERLOC utilizes deep ultraviolet Raman and fluorescence (DUV R/F) spectroscopy to enable analysis of samples from the Martian surface. SHERLOC employs a 248.6 nm deep ultraviolet laser to generate Raman-scattered photons and native fluorescence emission photons from near-surface material to detect and classify chemical and mineralogical compositions. The collected photons are focused on a charge-coupled device and the data are returned to Earth for analysis. The compact DUV R/F spectrometer has a spectral range from 249.9 nm to 353.6 nm (∼200 cm-1 to 12 000 cm-1) (with a spectral resolution of 0.296 nm (∼40 cm-1)). The compact spectrometer uses a custom design to project a high-resolution Raman spectrum and a low-resolution fluorescence spectrum on a single charge-coupled device. The natural spectral separation enabled by deep ultraviolet excitation enables wavelength separation of the Raman/fluorescence spectra. The SHERLOC spectrometer was designed to optimize the resolution of the Raman spectral region and the wavelength range of the fluorescence region. The resulting illumination on the charge-coupled device is curved, requiring a segmented, nonlinear wavelength calibration in order to understand the mineralogy and chemistry of Martian materials.

Real-Time Culture-Independent Microbial Profiling Onboard the International Space Station Using Nanopore Sequencing.

For the past two decades, microbial monitoring of the International Space Station (ISS) has relied on culture-dependent methods that require return to Earth for analysis. This has a number of limitations, with the most significant being bias towards the detection of culturable organisms and the inherent delay between sample collection and ground-based analysis. In recent years, portable and easy-to-use molecular-based tools, such as Oxford Nanopore Technologies' MinION™ sequencer and miniPCR bio's miniPCR™ thermal cycler, have been validated onboard the ISS. Here, we report on the development, validation, and implementation of a swab-to-sequencer method that provides a culture-independent solution to real-time microbial profiling onboard the ISS. Method development focused on analysis of swabs collected in a low-biomass environment with limited facility resources and stringent controls on allowed processes and reagents. ISS-optimized procedures included enzymatic DNA extraction from a swab tip, bead-based purifications, altered buffers, and the use of miniPCR and the MinION. Validation was conducted through extensive ground-based assessments comparing current standard culture-dependent and newly developed culture-independent methods. Similar microbial distributions were observed between the two methods; however, as expected, the culture-independent data revealed microbial profiles with greater diversity. Protocol optimization and verification was established during NASA Extreme Environment Mission Operations (NEEMO) analog missions 21 and 22, respectively. Unique microbial profiles obtained from analog testing validated the swab-to-sequencer method in an extreme environment. Finally, four independent swab-to-sequencer experiments were conducted onboard the ISS by two crewmembers. Microorganisms identified from ISS swabs were consistent with historical culture-based data, and primarily consisted of commonly observed human-associated microbes. This simplified method has been streamlined for high ease-of-use for a non-trained crew to complete in an extreme environment, thereby enabling environmental and human health diagnostics in real-time as future missions take us beyond low-Earth orbit.

Off Earth Identification of Bacterial Populations Using 16S rDNA Nanopore Sequencing.

The MinION sequencer has made in situ sequencing feasible in remote locations. Following our initial demonstration of its high performance off planet with Earth-prepared samples, we developed and tested an end-to-end, sample-to-sequencer process that could be conducted entirely aboard the International Space Station (ISS). Initial experiments demonstrated the process with a microbial mock community standard. The DNA was successfully amplified, primers were degraded, and libraries prepared and sequenced. The median percent identities for both datasets were 84%, as assessed from alignment of the mock community. The ability to correctly identify the organisms in the mock community standard was comparable for the sequencing data obtained in flight and on the ground. To validate the process on microbes collected from and cultured aboard the ISS, bacterial cells were selected from a NASA Environmental Health Systems Surface Sample Kit contact slide. The locations of bacterial colonies chosen for identification were labeled, and a small number of cells were directly added as input into the sequencing workflow. Prepared DNA was sequenced, and the data were downlinked to Earth. Return of the contact slide to the ground allowed for standard laboratory processing for bacterial identification. The identifications obtained aboard the ISS, Staphylococcus hominis and Staphylococcus capitis, matched those determined on the ground down to the species level. This marks the first ever identification of microbes entirely off Earth, and this validated process could be used for in-flight microbial identification, diagnosis of infectious disease in a crewmember, and as a research platform for investigators around the world.

Analysis of amino acids, hydroxy acids, and amines in CR chondrites.

The abundances, relative distributions, and enantiomeric and isotopic compositions of amines, amino acids, and hydroxy acids in Miller Range (MIL) 090001 and MIL 090657 meteorites were determined. Chiral distributions and isotopic compositions confirmed that most of the compounds detected were indigenous to the meteorites and not the result of terrestrial contamination. Combined with data in the literature, suites of these compounds have now been analyzed in a set of six CR chondrites, spanning aqueous alteration types 2.0-2.8. Amino acid abundances ranged from 17 to 3300 nmol g-1 across the six CRs; hydroxy acid abundances ranged from 180 to 1800 nmol g-1; and amine abundances ranged from 40 to 2100 nmol g-1. For amino acids and amines, the weakly altered chondrites contained the highest abundances, whereas hydroxy acids were most abundant in the more altered CR2.0 chondrite. Because water contents in the meteorites are orders of magnitude greater than soluble organics, synthesis of hydroxy acids, which requires water, may be less affected by aqueous alteration than amines and amino acids that require nitrogen-bearing precursors. Two chiral amino acids that were plausibly extraterrestrial in origin were present with slight enantiomeric excesses: L-isovaline (~10% excess) and D-ß-amino-n-butyric acid (~9% excess); further studies are needed to verify that the chiral excess in the latter compound is truly extraterrestrial in origin. The isotopic compositions of compounds reported here did not reveal definitive links between the different compound classes such as common synthetic precursors, but will provide a framework for further future in-depth analyses.

The Search for Chiral Asymmetry as a Potential Biosignature in our Solar System.

The search for evidence of extraterrestrial life in our Solar System is currently guided by our understanding of terrestrial biology and its associated biosignatures. The observed homochirality in all life on Earth, that is, the predominance of "left-handed" or l-amino acids and "right-handed" or d-sugars, is a unique property of life that is crucial for molecular recognition, enzymatic function, information storage and structure and is thought to be a prerequisite for the origin or early evolution of life. Therefore, the detection of l- or d-enantiomeric excesses (ee) of chiral amino acids and sugars could be a powerful indicator for extant or extinct life on another world. However, studies of primitive meteorites have revealed they contain extraterrestrial amino acids and sugar acids (aldonic acids) with large enantiomeric excesses of the same chirality as terrestrial biology resulting from nonbiological processes, complicating the use of chiral asymmetry by itself as a definitive biosignature. Here we review our current knowledge of the distributions and enantiomeric and isotopic compositions of amino acids and polyols found in meteorites compared to terrestrial biology and propose a set of criteria for future life detection missions that can be used to help establish the origin of chiral asymmetry.

Radiation Tolerance of Nanopore Sequencing Technology for Life Detection on Mars and Europa.

The search for life beyond Earth is a key motivator in space exploration. Informational polymers, like DNA and RNA, are key biosignatures for life as we know it. The MinION is a miniature DNA sequencer based on versatile nanopore technology that could be implemented on future planetary missions. A critical unanswered question is whether the MinION and its protein-based nanopores can withstand increased radiation exposure outside Earth's shielding magnetic field. We evaluated the effects of ionizing radiation on the MinION platform - including flow cells, reagents, and hardware - and discovered limited performance loss when exposed to ionizing doses comparable to a mission to Mars. Targets with harsher radiation environments, like Europa, would require improved radiation resistance via additional shielding or design refinements.

Insights into Abiotically-Generated Amino Acid Enantiomeric Excesses Found in Meteorites.

Biology exhibits homochirality, in that only one of two possible molecular configurations (called enantiomers) is used in both proteins and nucleic acids. The origin of this phenomenon is currently unknown, as nearly all known abiotic mechanisms for generating these compounds result in equal (racemic) mixtures of both enantiomers. However, analyses of primitive meteorites have revealed that a number of amino acids of extraterrestrial origin are present in enantiomeric excess, suggesting that there was an abiotic route to synthesize amino acids in a non-racemic manner. Here we review the amino acid contents of a range of meteorites, describe mechanisms for amino acid formation and their potential to produce amino acid enantiomeric excesses, and identify processes that could have amplified enantiomeric excesses.

Nanopore DNA Sequencing and Genome Assembly on the International Space Station.

We evaluated the performance of the MinION DNA sequencer in-flight on the International Space Station (ISS), and benchmarked its performance off-Earth against the MinION, Illumina MiSeq, and PacBio RS II sequencing platforms in terrestrial laboratories. Samples contained equimolar mixtures of genomic DNA from lambda bacteriophage, Escherichia coli (strain K12, MG1655) and Mus musculus (female BALB/c mouse). Nine sequencing runs were performed aboard the ISS over a 6-month period, yielding a total of 276,882 reads with no apparent decrease in performance over time. From sequence data collected aboard the ISS, we constructed directed assemblies of the ~4.6 Mb E. coli genome, ~48.5 kb lambda genome, and a representative M. musculus sequence (the ~16.3 kb mitochondrial genome), at 100%, 100%, and 96.7% consensus pairwise identity, respectively; de novo assembly of the E. coli genome from raw reads yielded a single contig comprising 99.9% of the genome at 98.6% consensus pairwise identity. Simulated real-time analyses of in-flight sequence data using an automated bioinformatic pipeline and laptop-based genomic assembly demonstrated the feasibility of sequencing analysis and microbial identification aboard the ISS. These findings illustrate the potential for sequencing applications including disease diagnosis, environmental monitoring, and elucidating the molecular basis for how organisms respond to spaceflight.

Meteoritic Amino Acids: Diversity in Compositions Reflects Parent Body Histories.

The analysis of amino acids in meteorites dates back over 50 years; however, it is only in recent years that research has expanded beyond investigations of a narrow set of meteorite groups (exemplified by the Murchison meteorite) into meteorites of other types and classes. These new studies have shown a wide diversity in the abundance and distribution of amino acids across carbonaceous chondrite groups, highlighting the role of parent body processes and composition in the creation, preservation, or alteration of amino acids. Although most chiral amino acids are racemic in meteorites, the enantiomeric distribution of some amino acids, particularly of the nonprotein amino acid isovaline, has also been shown to vary both within certain meteorites and across carbonaceous meteorite groups. Large l-enantiomeric excesses of some extraterrestrial protein amino acids (up to ∼60%) have also been observed in rare cases and point to nonbiological enantiomeric enrichment processes prior to the emergence of life. In this Outlook, we review these recent meteoritic analyses, focusing on variations in abundance, structural distributions, and enantiomeric distributions of amino acids and discussing possible explanations for these observations and the potential for future work.

The elusive quest for RNA knots.

Physical entanglement, and particularly knots arise spontaneously in equilibrated polymers that are sufficiently long and densely packed. Biopolymers are no exceptions: knots have long been known to occur in proteins as well as in encapsidated viral DNA. The rapidly growing number of RNA structures has recently made it possible to investigate the incidence of physical knots in this type of biomolecule, too. Strikingly, no knots have been found to date in the known RNA structures. In this Point of View Article we discuss the absence of knots in currently available RNAs and consider the reasons why knots in RNA have not yet been found, despite the expectation that they should exist in Nature. We conclude by singling out a number of RNA sequences that, based on the properties of their predicted secondary structures, are good candidates for knotted RNAs.

Nanopore sequencing in microgravity.

Rapid DNA sequencing and analysis has been a long-sought goal in remote research and point-of-care medicine. In microgravity, DNA sequencing can facilitate novel astrobiological research and close monitoring of crew health, but spaceflight places stringent restrictions on the mass and volume of instruments, crew operation time, and instrument functionality. The recent emergence of portable, nanopore-based tools with streamlined sample preparation protocols finally enables DNA sequencing on missions in microgravity. As a first step toward sequencing in space and aboard the International Space Station (ISS), we tested the Oxford Nanopore Technologies MinION during a parabolic flight to understand the effects of variable gravity on the instrument and data. In a successful proof-of-principle experiment, we found that the instrument generated DNA reads over the course of the flight, including the first ever sequenced in microgravity, and additional reads measured after the flight concluded its parabolas. Here we detail modifications to the sample-loading procedures to facilitate nanopore sequencing aboard the ISS and in other microgravity environments. We also evaluate existing analysis methods and outline two new approaches, the first based on a wave-fingerprint method and the second on entropy signal mapping. Computationally light analysis methods offer the potential for in situ species identification, but are limited by the error profiles (stays, skips, and mismatches) of older nanopore data. Higher accuracies attainable with modified sample processing methods and the latest version of flow cells will further enable the use of nanopore sequencers for diagnostics and research in space.

A plausible simultaneous synthesis of amino acids and simple peptides on the primordial Earth.

Following his seminal work in 1953, Stanley Miller conducted an experiment in 1958 to study the polymerization of amino acids under simulated early Earth conditions. In the experiment, Miller sparked a gas mixture of CH4, NH3, and H2O, while intermittently adding the plausible prebiotic condensing reagent cyanamide. For unknown reasons, an analysis of the samples was not reported. We analyzed the archived samples for amino acids, dipeptides, and diketopiperazines by liquid chromatography, ion mobility spectrometry, and mass spectrometry. A dozen amino acids, 10 glycine-containing dipeptides, and 3 glycine-containing diketopiperazines were detected. Miller's experiment was repeated and similar polymerization products were observed. Aqueous heating experiments indicate that Strecker synthesis intermediates play a key role in facilitating polymerization. These results highlight the potential importance of condensing reagents in generating diversity within the prebiotic chemical inventory.

1-Aza-niumyl-cyclo-butane-1-carboxyl-ate monohydrate.

In the title compound, C5H9NO2·H2O, the amino acid is in the usual zwitterionic form involving the α-carboxyl-ate group. The cyclo-butane backbone of the amino acid is disordered over two conformations, with occupancies of 0.882 (7) and 0.118 (7). In the crystal, N-H⋯O and O-H⋯O hydrogen bonds link the zwitterions [with the water molecule involved as both acceptor (with the NH3 (+)) and donor (through a single carboxylate O from two different aminocyclobutane carb-oxylate moities)], resulting in a two-dimensional layered structure lying parallel to (100).

Amino acid analysis in micrograms of meteorite sample by nanoliquid chromatography-high-resolution mass spectrometry.

Amino acids and their enantiomers in a 360 microgram sample of Murchison meteorite were unambiguously identified and quantified using chemical derivatization and nanoliquid chromatography coupled to nanoelectrospray ionization high resolution orbitrap mass spectrometry techniques. The distribution and abundance of amino acids were similar to past studies of Murchison meteorite but the samples used here were three orders of magnitude lower. The analytical method was also highly sensitive, and some amino acid reference standards were successfully detected at a level of ∼200 attomoles (on column). These results may open up the possibility for investigating other less studied, sample-limited extraterrestrial samples (e.g., micrometeorites, interplanetary dust particles, and cometary particles) for biologically-relevant organic molecules.