Chapter 4: A Geological and Chemical Context for the Origins of Life on Early Earth.

Within the first billion years of Earth's history, the planet transformed from a hot, barren, and inhospitable landscape to an environment conducive to the emergence and persistence of life. This chapter will review the state of knowledge concerning early Earth's (Hadean/Eoarchean) geochemical environment, including the origin and composition of the planet's moon, crust, oceans, atmosphere, and organic content. It will also discuss abiotic geochemical cycling of the CHONPS elements and how these species could have been converted to biologically relevant building blocks, polymers, and chemical networks. Proposed environments for abiogenesis events are also described and evaluated. An understanding of the geochemical processes under which life may have emerged can better inform our assessment of the habitability of other worlds, the potential complexity that abiotic chemistry can achieve (which has implications for putative biosignatures), and the possibility for biochemistries that are vastly different from those on Earth.

Chapter 1: The Astrobiology Primer 3.0.

The Astrobiology Primer 3.0 (ABP3.0) is a concise introduction to the field of astrobiology for students and others who are new to the field of astrobiology. It provides an entry into the broader materials in this supplementary issue of Astrobiology and an overview of the investigations and driving hypotheses that make up this interdisciplinary field. The content of this chapter was adapted from the other 10 articles in this supplementary issue and thus represents the contribution of all the authors who worked on these introductory articles. The content of this chapter is not exhaustive and represents the topics that the authors found to be the most important and compelling in a dynamic and changing field.

Alternating co-synthesis of glycol nucleic acid (GNA) monomers with dicarboxylic acids via drying.

We report the co-polymerization of glycol nucleic acid (GNA) monomers with unsubstituted and substituted dicarboxylic acid linkers under plausible early Earth aqueous dry-down conditions. Both linear and branched co-polymers are produced. Mechanistic aspects of the reaction and potential roles of these polymers in prebiotic chemistry are discussed.

Spectroscopic and Biophysical Methods to Determine Differential Salt-Uptake by Primitive Membraneless Polyester Microdroplets.

α-Hydroxy acids are prebiotic monomers that undergo dehydration synthesis to form polyester gels, which assemble into membraneless microdroplets upon aqueous rehydration. These microdroplets are proposed as protocells that can segregate and compartmentalize primitive molecules/reactions. Different primitive aqueous environments with a variety of salts could have hosted chemistries that formed polyester microdroplets. These salts could be essential cofactors of compartmentalized prebiotic reactions or even directly affect protocell structure. However, fully understanding polyester-salt interactions remains elusive, partially due to technical challenges of quantitative measurements in condensed phases. Here, spectroscopic and biophysical methods are applied to analyze salt uptake by polyester microdroplets. Inductively coupled plasma mass spectrometry is applied to measure the cation concentration within polyester microdroplets after addition of chloride salts. Combined with methods to determine the effects of salt uptake on droplet turbidity, size, surface potential and internal water distribution, it was observed that polyester microdroplets can selectively partition salt cations, leading to differential microdroplet coalescence due to ionic screening effects reducing electrostatic repulsion forces between microdroplets. Through applying existing techniques to novel analyses related to primitive compartment chemistry and biophysics, this study suggests that even minor differences in analyte uptake can lead to significant protocellular structural change.

Primitive membraneless compartments as a window into the earliest cells.

What did the first cells on Earth look like? This is an unanswered mystery investigated by researchers in the origins of life field. While at some point cells must have developed membranes, genetic components, and catalytic cycles and catalysts, when the earliest cells developed these is not clear. One system which could shed light into the structure and function of the first cells on Earth is membraneless compartments generated from phase separation, perhaps before or as a precursor to the advent of membrane-bound compartmentalization. Here, we briefly comment on two prebiotically relevant membraneless compartment systems: coacervates and polyester microdroplets. This discussion seeks to highlight the current understanding of these systems and to pose unanswered questions as a challenge to the field at large.

A liquid crystal world for the origins of life.

Nucleic acids (NAs) in modern biology accomplish a variety of tasks, and the emergence of primitive nucleic acids is broadly recognized as a crucial step for the emergence of life. While modern NAs have been optimized by evolution to accomplish various biological functions, such as catalysis or transmission of genetic information, primitive NAs could have emerged and been selected based on more rudimental chemical-physical properties, such as their propensity to self-assemble into supramolecular structures. One such supramolecular structure available to primitive NAs are liquid crystal (LC) phases, which are the outcome of the collective behavior of short DNA or RNA oligomers or monomers that self-assemble into linear aggregates by combinations of pairing and stacking. Formation of NA LCs could have provided many essential advantages for a primitive evolving system, including the selection of potential genetic polymers based on structure, protection by compartmentalization, elongation, and recombination by enhanced abiotic ligation. Here, we review recent studies on NA LC assembly, structure, and functions with potential prebiotic relevance. Finally, we discuss environmental or geological conditions on early Earth that could have promoted (or inhibited) primitive NA LC formation and highlight future investigation axes essential to further understanding of how LCs could have contributed to the emergence of life.

A Physicochemical Consideration of Prebiotic Microenvironments for Self-Assembly and Prebiotic Chemistry.

The origin of life on Earth required myriads of chemical and physical processes. These include the formation of the planet and its geological structures, the formation of the first primitive chemicals, reaction, and assembly of these primitive chemicals to form more complex or functional products and assemblies, and finally the formation of the first cells (or protocells) on early Earth, which eventually evolved into modern cells. Each of these processes presumably occurred within specific prebiotic reaction environments, which could have been diverse in physical and chemical properties. While there are resources that describe prebiotically plausible environments or nutrient availability, here, we attempt to aggregate the literature for the various physicochemical properties of different prebiotic reaction microenvironments on early Earth. We introduce a handful of properties that can be quantified through physical or chemical techniques. The values for these physicochemical properties, if they are known, are then presented for each reaction environment, giving the reader a sense of the environmental variability of such properties. Such a resource may be useful for prebiotic chemists to understand the range of conditions in each reaction environment, or to select the medium most applicable for their targeted reaction of interest for exploratory studies.

A material-based panspermia hypothesis: The potential of polymer gels and membraneless droplets.

The Panspermia hypothesis posits that either life's building blocks (molecular Panspermia) or life itself (organism-based Panspermia) may have been interplanetarily transferred to facilitate the origins of life (OoL) on a given planet, complementing several current OoL frameworks. Although many spaceflight experiments were performed in the past to test for potential terrestrial organisms as Panspermia seeds, it is uncertain whether such organisms will likely "seed" a new planet even if they are able to survive spaceflight. Therefore, rather than using organisms, using abiotic chemicals as seeds has been proposed as part of the molecular Panspermia hypothesis. Here, as an extension of this hypothesis, we introduce and review the plausibility of a polymeric material-based Panspermia seed (M-BPS) as a theoretical concept, where the type of polymeric material that can function as a M-BPS must be able to: (1) survive spaceflight and (2) "function", i.e., contingently drive chemical evolution toward some form of abiogenesis once arriving on a foreign planet. We use polymeric gels as a model example of a potential M-BPS. Polymeric gels that can be prebiotically synthesized on one planet (such as polyester gels) could be transferred to another planet via meteoritic transfer, where upon landing on a liquid bearing planet, can assemble into structures containing cellular-like characteristics and functionalities. Such features presupposed that these gels can assemble into compartments through phase separation to accomplish relevant functions such as encapsulation of primitive metabolic, genetic and catalytic materials, exchange of these materials, motion, coalescence, and evolution. All of these functions can result in the gels' capability to alter local geochemical niches on other planets, thereby allowing chemical evolution to lead to OoL events.

Sequencing the origins of life.

One goal of origins of life research is to understand how primitive informational and catalytic biopolymers emerged and evolved. Recently, a number of sequencing techniques have been applied to analysis of replicating and evolving primitive biopolymer systems, providing a sequence-specific and high-resolution view of primitive chemical processes. Here, we review application of sequencing techniques to analysis of synthetic and primitive nucleic acids and polypeptides. This includes next-generation sequencing of primitive polymerization and evolution processes, followed by discussion of other novel biochemical techniques that could contribute to sequence analysis of primitive biopolymer driven chemical systems. Further application of sequencing to origins of life research, perhaps as a life detection technology, could provide insight into the origin and evolution of informational and catalytic biopolymers on early Earth or elsewhere.

Connecting primitive phase separation to biotechnology, synthetic biology, and engineering.

One aspect of the study of the origins of life focuses on how primitive chemistries assembled into the first cells on Earth and how these primitive cells evolved into modern cells. Membraneless droplets generated from liquid-liquid phase separation (LLPS) are one potential primitive cell-like compartment; current research in origins of life includes study of the structure, function, and evolution of such systems. However, the goal of primitive LLPS research is not simply curiosity or striving to understand one of life's biggest unanswered questions, but also the possibility to discover functions or structures useful for application in the modern day. Many applicational fields, including biotechnology, synthetic biology, and engineering, utilize similar phaseseparated structures to accomplish specific functions afforded by LLPS. Here, we briefly review LLPS applied to primitive compartment research and then present some examples of LLPS applied to biomolecule purification, drug delivery, artificial cell construction, waste and pollution management, and flavor encapsulation. Due to a significant focus on similar functions and structures, there appears to be much for origins of life researchers to learn from those working on LLPS in applicational fields, and vice versa, and we hope that such researchers can start meaningful cross-disciplinary collaborations in the future.

A General LC-MS-Based Method for Direct and De Novo Sequencing of RNA Mixtures Containing both Canonical and Modified Nucleotides.

Mass spectrometry (MS)-based sequencing has advantages in direct sequencing of RNA, compared to cDNA-based RNA sequencing methods, as it is completely independent of enzymes and base complementarity errors in sample preparation. In addition, it allows for sequencing of different RNA modifications in a single study, rather than just one specific modification type per study. However, many technical challenges remain in de novo MS sequencing of RNA, making it difficult to MS sequence mixed RNAs or to differentiate isomeric modifications such as pseudouridine (Ψ) from uridine (U). Our recent study incorporates a two-dimensional hydrophobic end labeling strategy into MS-based sequencing (2D-HELS MS Seq) to systematically address the current challenges in MS sequencing of RNA, making it possible to directly and de novo sequence purified single RNA and mixed RNA containing both canonical and modified nucleotides. Here, we describe the method to sequence representative single-RNA and mixed-RNA oligonucleotides, each with a different sequence and/or containing modified nucleotides such as Ψ and 5-methylcytosine (m5C), using 2D-HELS MS Seq.

Origin of Species before Origin of Life: The Role of Speciation in Chemical Evolution.

Speciation, an evolutionary process by which new species form, is ultimately responsible for the incredible biodiversity that we observe on Earth every day. Such biodiversity is one of the critical features which contributes to the survivability of biospheres and modern life. While speciation and biodiversity have been amply studied in organismic evolution and modern life, it has not yet been applied to a great extent to understanding the evolutionary dynamics of primitive life. In particular, one unanswered question is at what point in the history of life did speciation as a phenomenon emerge in the first place. Here, we discuss the mechanisms by which speciation could have occurred before the origins of life in the context of chemical evolution. Specifically, we discuss that primitive compartments formed before the emergence of the last universal common ancestor (LUCA) could have provided a mechanism by which primitive chemical systems underwent speciation. In particular, we introduce a variety of primitive compartment structures, and associated functions, that may have plausibly been present on early Earth, followed by examples of both discriminate and indiscriminate speciation affected by primitive modes of compartmentalization. Finally, we discuss modern technologies, in particular, droplet microfluidics, that can be applied to studying speciation phenomena in the laboratory over short timescales. We hope that this discussion highlights the current areas of need in further studies on primitive speciation phenomena while simultaneously proposing directions as important areas of study to the origins of life.

Incorporation of Basic α-Hydroxy Acid Residues into Primitive Polyester Microdroplets for RNA Segregation.

Nucleic acid segregation and compartmentalization were likely essential functions that primitive compartment systems resolved during evolution. Recently, polyester microdroplets generated from dehydration synthesis of various α-hydroxy acids (αHA) were suggested as potential primitive compartments. Some of these droplets can differentially segregate and compartmentalize organic dyes, proteins, and nucleic acids. However, the previously studied polyester microdroplets included limited αHA chemical diversity, which may not reflect the chemical diversity available in the primitive Earth environment. Here, we increased the chemical diversity of polyester microdroplet systems by combinatorially adding an αHA monomer with a basic side chain, 4-amino-2-hydroxybutyric acid (4a2h), which was incorporated with different ratios of other αHAs containing uncharged side chains to form combinatorial heteropolyesters via dehydration synthesis. Incorporation of 4a2h in the polymers resulted in the assembly of some polyester microdroplets able to segregate fluorescent RNA or potentially acquire intrinsic fluorescent character, suggesting that minor modifications of polyester composition can significantly impact the functional properties of primitive compartments. This study suggests one process by which primitive chemical systems can increase diversity of compartment "phenotype" through simple modifications in their chemical composition.

Prebiotic oligomerization and self-assembly of structurally diverse xenobiological monomers.

Prebiotic chemists often study how modern biopolymers, e.g., peptides and nucleic acids, could have originated in the primitive environment, though most contemporary biomonomers don't spontaneously oligomerize under mild conditions without activation or catalysis. However, life may not have originated using the same monomeric components that it does presently. There may be numerous non-biological (or "xenobiological") monomer types that were prebiotically abundant and capable of facile oligomerization and self-assembly. Many modern biopolymers degrade abiotically preferentially via processes which produce thermodynamically stable ring structures, e.g. diketopiperazines in the case of proteins and 2', 3'-cyclic nucleotide monophosphates in the case of RNA. This weakness is overcome in modern biological systems by kinetic control, but this need not have been the case for primitive systems. We explored here the oligomerization of a structurally diverse set of prebiotically plausible xenobiological monomers, which can hydrolytically interconvert between cyclic and acyclic forms, alone or in the presence of glycine under moderate temperature drying conditions. These monomers included various lactones, lactams and a thiolactone, which varied markedly in their stability, propensity to oligomerize and apparent modes of initiation, and the oligomeric products of some of these formed self-organized microscopic structures which may be relevant to protocell formation.

Protoenzymes: The Case of Hyperbranched Polymer-Scaffolded ZnS Nanocrystals.

Enzymes are biological catalysts that are comprised of small-molecule, metal, or cluster catalysts augmented by biopolymeric scaffolds. It is conceivable that early in chemical evolution, ancestral enzymes opted for simpler, easier to assemble scaffolds. Herein, we describe such possible protoenzymes: hyperbranched polymer-scaffolded metal-sulfide nanocrystals. Hyperbranched polyethyleneimine (HyPEI) and glycerol citrate polymer-supported ZnS nanocrystals (NCs) are formed in a simple process. Transmission electron microscopy (TEM) analyses of HyPEI-supported NCs reveal spherical particles with an average size of 10 nm that undergo only a modest aggregation over a 14-day incubation. The polymer-supported ZnS NCs are shown to possess a high photocatalytic activity in an eosin B photodegradation assay, making them an attractive model for the study of the origin of life under the "Zn world" theory dominated by a photocatalytic proto-metabolic redox reaction network. The catalyst, however, could be easily adapted to apply broadly to different protoenzymatic systems.

Liquid Crystal Coacervates Composed of Short Double-Stranded DNA and Cationic Peptides.

Phase separation of nucleic acids and proteins is a ubiquitous phenomenon regulating subcellular compartment structure and function. While complex coacervation of flexible single-stranded nucleic acids is broadly investigated, coacervation of double-stranded DNA (dsDNA) is less studied because of its propensity to generate solid precipitates. Here, we reverse this perspective by showing that short dsDNA and poly-l-lysine coacervates can escape precipitation while displaying a surprisingly complex phase diagram, including the full set of liquid crystal (LC) mesophases observed to date in bulk dsDNA. Short dsDNA supramolecular aggregation and packing in the dense coacervate phase are the main parameters regulating the global LC-coacervate phase behavior. LC-coacervate structure was characterized upon variations in temperature and monovalent salt, DNA, and peptide concentrations, which allow continuous reversible transitions between all accessible phases. A deeper understanding of LC-coacervates can gain insights to decipher structures and phase transition mechanisms within biomolecular condensates, to design stimuli-responsive multiphase synthetic compartments with different degrees of order and to exploit self-assembly driven cooperative prebiotic evolution of nucleic acids and peptides.

Direct Sequencing of tRNA by 2D-HELS-AA MS Seq Reveals Its Different Isoforms and Dynamic Base Modifications.

Post-transcriptional modifications are intrinsic to RNA structure and function. However, methods to sequence RNA typically require a cDNA intermediate and are either not able to sequence these modifications or are tailored to sequence one specific nucleotide modification only. Interestingly, some of these modifications occur with <100% frequency at their particular sites, and site-specific quantification of their stoichiometries is another challenge. Here, we report a direct method for sequencing tRNAPhe without cDNA by integrating a two-dimensional hydrophobic RNA end-labeling strategy with an anchor-based algorithm in mass spectrometry-based sequencing (2D-HELS-AA MS Seq). The entire tRNAPhe was sequenced and the identity, location, and stoichiometry of all eleven different RNA modifications was determined, five of which were not 100% modified, including a 2'-O-methylated G (Gm) in the wobble anticodon position as well as an N2, N2-dimethylguanosine (m22G), a 7-methylguanosine (m7G), a 1-methyladenosine (m1A), and a wybutosine (Y), suggesting numerous post-transcriptional regulations in tRNA. Two truncated isoforms at the 3'-CCA tail of the tRNAPhe (75 nt with a 3'-CC tail (80% abundance) and 74 nt with a 3'-C tail (3% abundance)) were identified in addition to the full-length 3'-CCA-tailed tRNAPhe (76 nt, 17% abundance). We discovered a new isoform with A-G transitions/editing at the 44 and 45 positions in the tRNAPhe variable loop, and discuss possible mechanisms related to the emergence and functions of the isoforms with these base transitions or editing. Our method revealed new isoforms, base modifications, and RNA editing as well as their stoichiometries in the tRNA that cannot be determined by current cDNA-based methods, opening new opportunities in the field of epitranscriptomics.

Biological phase separation: cell biology meets biophysics.

Progress in development of biophysical analytic approaches has recently crossed paths with macromolecule condensates in cells. These cell condensates, typically termed liquid-like droplets, are formed by liquid-liquid phase separation (LLPS). More and more cell biologists now recognize that many of the membrane-less organelles observed in cells are formed by LLPS caused by interactions between proteins and nucleic acids. However, the detailed biophysical processes within the cell that lead to these assemblies remain largely unexplored. In this review, we evaluate recent discoveries related to biological phase separation including stress granule formation, chromatin regulation, and processes in the origin and evolution of life. We also discuss the potential issues and technical advancements required to properly study biological phase separation.