The beginnings of man-made life.
Synthesis of constituents of nucleic acid and protein molecule for artificial production of life
NUCLEIC ACID↗
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Some recent work on prebiological synthesis of organic compounds.
Origin of constituents of nucleic acid and protein molecules, noting biological molecules synthesis under conditions similar to those prevailing in prebiotic Earth, following Oparin-Haldane hypothesis
ABIOGENESIS↗
Energetics of the lattice: packing elements in crystals of four-stranded intercalated cytosine-rich DNA molecules
Condensation of single molecules from solution into crystals represents a transition between distinct energetic states. In solution, the atomic interactions within the molecule dominate. In the crystalline state, however, a set of additional interactions are formed between molecules in close contact in the lattice--these are the packing interactions. The crystal structures of d(CCCT), d(TAACCC), d(CCCAAT), and d(AACCCC) have in common a four-stranded intercalated cytosine segment, built by stacked layers of cytosine.cytosine+ (C.C+) base pairs coming from two parallel duplexes that intercalate into each other with opposite polarity. The intercalated cytosine segments in these structures are similar in their geometry, even though the sequences crystallized in different space groups. In the crystals, adenine and thymine residues of the sequences are used to build the three-dimensional crystal lattice by elaborately interacting with symmetry-related molecules. The packing elements observed provide novel insight about the copious ways in which nucleic acid molecules can interact with each other--for example, when folded in more complicated higher order structures, such as mRNA and chromatin.
Non-NASA Center↗
Process to Selectively Distinguish Viable from Non-Viable Bacterial Cells
The combination of ethidium monoazide (EMA) and post-fragmentation, randomly primed DNA amplification technologies will enhance the analytical capability to discern viable from non-viable bacterial cells in spacecraft-related samples. Intercalating agents have been widely used since the inception of molecular biology to stain and visualize nucleic acids. Only recently, intercalating agents such as EMA have been exploited to selectively distinguish viable from dead bacterial cells. Intercalating dyes can only penetrate the membranes of dead cells. Once through the membrane and actually inside the cell, they intercalate DNA and, upon photolysis with visible light, produce stable DNA monoadducts. Once the DNA is crosslinked, it becomes insoluble and unable to be fragmented for post-fragmentation, randomly primed DNA library formation. Viable organisms DNA remains unaffected by the intercalating agents, allowing for amplification via post-fragmentation, randomly primed technologies. This results in the ability to carry out downstream nucleic acid-based analyses on viable microbes to the exclusion of all non-viable cells.
LaDuc, Myron T.↗
Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology
A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.
Non-NASA Center↗
Template switching between PNA and RNA oligonucleotides
The origin of the RNA world is not easily understood, as effective prebiotic syntheses of the components of RNA, the beta-ribofuranoside-5'-phosphates, are hard to envisage. Recognition of this difficulty has led to the proposal that other genetic systems, the components of which are more easily formed, may have preceded RNA. This raises the question of how transitions between one genetic system and another could occur. Peptide nucleic acid (PNA) resembles RNA in its ability to form double-helical complexes stabilized by Watson-Crick hydrogen bonding between adenine and thymine and between cytosine and guanine, but has a backbone that is held together by amide rather than by phosphodiester bonds. Oligonucleotides bases on RNA are known to act as templates that catalyse the non-enzymatic synthesis of their complements from activated mononucleotides, we now show that RNA oligonucleotides facilitate the synthesis of complementary PNA strands and vice versa. This suggests that a transition between different genetic systems can occur without loss of information.
NASA Discipline Exobiology↗
Asymmetric structure of five and six membered DNA hairpin loops
The tertiary structure of nucleic acid hairpins was elucidated by means of the accessibility of the single-strand-specific nuclease from mung bean. This molecular probe has proven especially useful in determining details of the structural arrangement of the nucleotides within a loop. In this study 3'-labeling is introduced to complement previously used 5'-labeling in order to assess and to exclude possible artifacts of the method. Both labeling procedures result in mutually consistent cleavage patterns. Therefore, methodological artifacts can be excluded and the potential of the nuclease as structural probe is increased. DNA hairpins with five and six membered loops reveal an asymmetric loop structure with a sharp bend of the phosphate-ribose backbone between the second and third nucleotide on the 3'-side of a loop. These hairpin structures differ from smaller loops with 3 or 4 members, which reveal this type of bend between the first and second 3' nucleotide, and resemble with respect to the asymmetry anticodon loops of tRNA.
NASA Discipline Exobiology↗
Microbial detection with low molecular weight RNA
The need to monitor microorganisms in the environment has increased interest in assays based on hybridization probes that target nucleic acids (e.g., rRNA). We report the development of liquid-phase assays for specific bacterial 5S rRNA sequences or similarly sized artificial RNAs (aRNAs) using molecular beacon technology. These beacons fluoresce only in the presence of specific target sequences, rendering as much as a 27-fold fluorescence enhancement. The assays can be used with both crude cell lysates and purified total RNA preparations. Minimal sample preparation (e.g., heating to promote leakage from cells) is sufficient to detect many Gram-negative bacteria. Using this approach it was possible to detect an aRNA-labeled Escherichia coli strain in the presence of a large background of an otherwise identical E. coli strain. Finally, by using a longer wavelength carboxytetramethylrhodamine beacon it was possible to reduce the fraction of the signal due to cellular autofluorescence to below 0.5%.
NASA Discipline Life Sciences Technologies↗
Emergence of a replicating species from an in vitro RNA evolution reaction
The technique of self-sustained sequence replication allows isothermal amplification of DNA and RNA molecules in vitro. This method relies on the activities of a reverse transcriptase and a DNA-dependent RNA polymerase to amplify specific nucleic acid sequences. We have modified this protocol to allow selective amplification of RNAs that catalyze a particular chemical reaction. During an in vitro RNA evolution experiment employing this modified system, a unique class of "selfish" RNAs emerged and replicated to the exclusion of the intended RNAs. Members of this class of selfish molecules, termed RNA Z, amplify efficiently despite their inability to catalyze the target chemical reaction. Their amplification requires the action of both reverse transcriptase and RNA polymerase and involves the synthesis of both DNA and RNA replication intermediates. The proposed amplification mechanism for RNA Z involves the formation of a DNA hairpin that functions as a template for transcription by RNA polymerase. This arrangement links the two strands of the DNA, resulting in the production of RNA transcripts that contain an embedded RNA polymerase promoter sequence.
Non-NASA Center↗
Optimization of single-base-pair mismatch discrimination in oligonucleotide microarrays
The discrimination between perfect-match and single-base-pair-mismatched nucleic acid duplexes was investigated by using oligonucleotide DNA microarrays and nonequilibrium dissociation rates (melting profiles). DNA and RNA versions of two synthetic targets corresponding to the 16S rRNA sequences of Staphylococcus epidermidis (38 nucleotides) and Nitrosomonas eutropha (39 nucleotides) were hybridized to perfect-match probes (18-mer and 19-mer) and to a set of probes having all possible single-base-pair mismatches. The melting profiles of all probe-target duplexes were determined in parallel by using an imposed temperature step gradient. We derived an optimum wash temperature for each probe and target by using a simple formula to calculate a discrimination index for each temperature of the step gradient. This optimum corresponded to the output of an independent analysis using a customized neural network program. These results together provide an experimental and analytical framework for optimizing mismatch discrimination among all probes on a DNA microarray.
NASA Discipline Life Sciences Technologies↗
A molecular description of the evolution of resistance
BACKGROUND: In vitro evolution has been used to obtain nucleic acid molecules with interesting functional properties. The evolution process usually is carried out in a stepwise manner, involving successive rounds of selection, amplification and mutation. Recently, a continuous in vitro evolution system was devised for RNAs that catalyze the ligation of oligonucleotide substrates, allowing the evolution of catalytic function to be studied in real time. RESULTS: Continuous in vitro evolution of an RNA ligase ribozyme was carried out in the presence of a DNA enzyme that was capable of cleaving, and thereby inactivating, the ribozyme. The DNA concentration was increased steadily over 33.5 hours of evolution, reaching a final concentration that would have been sufficient to inactivate the starting population in one second. The evolved population of ribozymes developed resistance to the DNA enzyme, reducing their vulnerability to cleavage by 2000-fold but retaining their own catalytic function. Based on sequencing and kinetic analysis of the ribozymes, two mechanisms are proposed for this resistance. One involves three nucleotide substitutions, together with two compensatory mutations, that alter the site at which the DNA enzyme binds the ribozyme. The other involves enhancement of the ribozyme's ability to bind its own substrate in a way that protects it from cleavage by the DNA enzyme. CONCLUSIONS: The ability to direct the evolution of an enzyme's biochemical properties in response to the behavior of another macromolecule provides insight into the evolution of resistance and may be useful in developing enzymes with novel or enhanced function.
Non-NASA Center↗
DNA-directed mutations. Leading and lagging strand specificity
The fidelity of replication has evolved to reproduce B-form DNA accurately, while allowing a low frequency of mutation. The fidelity of replication can be compromised, however, by defined order sequence DNA (dosDNA) that can adopt unusual or non B-DNA conformations. These alternative DNA conformations, including hairpins, cruciforms, triplex DNAs, and slipped-strand structures, may affect enzyme-template interactions that potentially lead to mutations. To analyze the effect of dosDNA elements on spontaneous mutagenesis, various mutational inserts containing inverted repeats or direct repeats were cloned in a plasmid containing a unidirectional origin of replication and a selectable marker for the mutation. This system allows for analysis of mutational events that are specific for the leading or lagging strands during DNA replication in Escherichia coli. Deletions between direct repeats, involving misalignment stabilized by DNA secondary structure, occurred preferentially on the lagging strand. Intermolecular strand switch events, correcting quasipalindromes to perfect inverted repeats, occurred preferentially during replication of the leading strand.
NASA Discipline Radiation Health↗
From inanimate matter to living systems
Current understandings of the origin and evolution of life on earth from inaminate matter are reviewed. Approaches to the study of early life and its origin are considered, and it is noted that whereas the inference of origins from knowledge at hand is the approach favored by most theoreticians, only the laboratory simulation of the assembly of precellular polymers to protocells under geologically relevant conditions is capable of indicating the way in which life began. Progress in simulating the step-by-step emergence of a protocell composed of ordered macromolecules and having numerous protobiological activities through stages characterized by primordial matter, amino acids, proteinoids, protocells, light-active protocells and nucleic-acid instructed cells is discussed, with particular emphasis on experiments with proteinoid microspheres formed from self-ordered copolyamino acids. The subsequent development of ordered, protometabolic, infrastructured protoreproductive protocells is examined noting the importance of the formation of lysine-rich proteinoids with catalytic activities. Attention is also given to questions of the point of the actual emergence of life and scientific and creationist objections to the theory developed.
Fox, S. W.↗
Enzymatic DNA molecules
The present invention discloses deoxyribonucleic acid enzymes--catalytic or enzymatic DNA molecules--capable of cleaving nucleic acid sequences or molecules, particularly RNA, in a site-specific manner, as well as compositions including same. Methods of making and using the disclosed enzymes and compositions are also disclosed.
Joyce, Gerald F.↗
Carbonaceous meteorites as a source of sugar-related organic compounds for the early Earth
The much-studied Murchison meteorite is generally used as the standard reference for organic compounds in extraterrestrial material. Amino acids and other organic compounds important in contemporary biochemistry are thought to have been delivered to the early Earth by asteroids and comets, where they may have played a role in the origin of life. Polyhydroxylated compounds (polyols) such as sugars, sugar alcohols and sugar acids are vital to all known lifeforms-they are components of nucleic acids (RNA, DNA), cell membranes and also act as energy sources. But there has hitherto been no conclusive evidence for the existence of polyols in meteorites, leaving a gap in our understanding of the origins of biologically important organic compounds on Earth. Here we report that a variety of polyols are present in, and indigenous to, the Murchison and Murray meteorites in amounts comparable to amino acids. Analyses of water extracts indicate that extraterrestrial processes including photolysis and formaldehyde chemistry could account for the observed compounds. We conclude from this that polyols were present on the early Earth and therefore at least available for incorporation into the first forms of life.
Biogenesis↗
Development of Solid-State Nanopore Technology for Life Detection
Biomarkers for life on Earth are an important starting point to guide the search for life elsewhere. However, the search for life beyond Earth should incorporate technologies capable of recognizing an array of potential biomarkers beyond what we see on Earth, in order to minimize the risk of false negatives from life detection missions. With this in mind, charged linear polymers may be a universal signature for life, due to their ability to store information while also inherently reducing the tendency of complex tertiary structure formation that significantly inhibit replication. Thus, these molecules are attractive targets for biosignature detection as potential "self-sustaining chemical signatures." Examples of charged linear polymers, or polyelectrolytes, include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) as well as synthetic polyelectrolytes that could potentially support life, including threose nucleic acid (TNA) and other xenonucleic acids (XNAs). Nanopore analysis is a novel technology that has been developed for singlemolecule sequencing with exquisite single nucleotide resolution which is also well-suited for analysis of polyelectrolyte molecules. Nanopore analysis has the ability to detect repeating sequences of electrical charges in organic linear polymers, and it is not molecule- specific (i.e. it is not restricted to only DNA or RNA). In this sense, it is a better life detection technique than approaches that are based on specific molecules, such as the polymerase chain reaction (PCR), which requires that the molecule being detected be composed of DNA.
Bywaters, K. B.↗
Search for the Universal Ancestors
By its nature, the study of the origins of life is multidisciplinary, requiring contributions from astronomers, biologists, chemists, geologists, physicists, and many others. Partial answers are provided to many questions about organic chemical evolution and the origin of life. It is observed that the gaps in our knowledge concerning the steps from the nonliving to the living are numerous. Among these gaps are: (1) a solar system formation with its accumulation of raw materials; (2) the synthesis of the life forming monomers, such as the amino acids, nucleotides, and lipids; (3) the condensation of these monomers into useful polymers, such as proteins and nucleic acids; (4) the sequestering of these materials into droplets of proteinoid or membrane-like structures; and (5) the development of a chemical memory (the genetic code) to pass on to the progeny the information acquired.
Hartman, H.↗
The Enantiomeric Ratios of Meteoritic Organic Compounds: Their Possible Roles in the Origin of Life
This talk will give an overview of the enantiomer (mirror-image) ratios of organic compounds in meteorites and also describe the results of the present work in my lab. The primary focus will be on sugar derivatives (sugar acids) of carbonaceous meteorites. Our work begins to address questions associated with chirality, i.e., the origins of homochirality. On Earth, biological monomers (amino acids, sugars, etc.) are usually found with one of the enantiomers more abundant than the other. However, biological polymers (proteins, nucleic acids, etc.) are only composed of one enantiomer i.e., they are homochiral. There are hints in meteorites that some organic molecules may also exist in homochiral forms. The talk will address questions such as: did extraterrestrial sources aid in the beginning of this homochirality? Do the increasing size and apparent enantiomer excesses of some meteoritic compounds also extend to larger meteoritic compounds and polymers?
Cooper, George↗