Reviewed are three decades of synthetic biology research in our laboratory that has generated alternatives to standard DNA and RNA as possible informational systems to support Darwinian evolution, ...and therefore life, and to understand their natural history, on Earth and throughout the cosmos. From this, we have learned that: • the core structure of nucleic acids appears to be a natural outcome of non-biological chemical processes probably in constrained, intermittently irrigated, sub-aerial aquifers on the surfaces of rocky planets like Earth and/or Mars approximately 4.36 ± 0.05 billion years ago; • however, this core is not unique. Synthetic biology has generated many different molecular systems able to support the evolution of molecular information; • these alternatives to standard DNA and RNA support biotechnology, including DNA synthesis, human diagnostics, biomedical research and medicine; • in particular, they support laboratory
evolution (LIVE) with performance to generate catalysts at least 10
-10
fold better than standard DNA libraries, enhancing access to receptors and catalysts on demand. Coupling nanostructures to the products of LIVE with expanded DNA offers new approaches for disease therapy; and • nevertheless, a polyelectrolyte structure and size regular building blocks are required for
informational polymer to support Darwinian evolution. These features serve as universal and agnostic biosignatures, useful for seeking life throughout the Solar System. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.
According to a current “RNA first” model for the origin of life, RNA emerged in some form on early Earth to become the first biopolymer to support Darwinism here. Threose nucleic acid (TNA) and other ...polyelectrolytes are also considered as the possible first Darwinian biopolymer(s). This model is being developed by research pursuing a “Discontinuous Synthesis Model” (DSM) for the formation of RNA and/or TNA from precursor molecules that might have been available on early Earth from prebiotic reactions, with the goal of making the model less discontinuous. In general, this is done by examining the reactivity of isolated products from proposed steps that generate those products, with increasing complexity of the reaction mixtures in the proposed mineralogical environments. Here, we report that adenine, diaminopurine, and hypoxanthine nucleoside phosphates and a noncanonical pyrimidine nucleoside (zebularine) phosphate can be formed from the direct coupling reaction of cyclic carbohydrate phosphates with the free nucleobases. The reaction is stereoselective, giving only the β-anomer of the nucleotides within detectable limits. For purines, the coupling is also regioselective, giving the N-9 nucleotide for adenine as a major product. In the DSM, phosphorylated carbohydrates are presumed to have been available via reactions explored previously Krishnamurthy R, Guntha S, Eschenmoser A (2000) Angew Chem Int Ed 39:2281–2285, while nucleobases are presumed to have been available from hydrogen cyanide and other nitrogenous species formed in Earth’s primitive atmosphere.
RNA has been called a "prebiotic chemist's nightmare" because of its combination of large size, carbohydrate building blocks, bonds that are thermodynamically unstable in water, and overall intrinsic ...instability. However, a discontinuous synthesis model is well-supported by experimental work that might produce RNA from atmospheric CO(2), H(2)O, and N(2). For example, electrical discharge in such atmospheres gives formaldehyde (HCHO) in large amounts and glycolaldehyde (HOCH(2)CHO) in small amounts. When rained into alkaline aquifers generated by serpentinizing rocks, these substances were undoubtedly converted to carbohydrates including ribose. Likewise, atmospherically generated HCN was undoubtedly converted in these aquifers to formamide and ammonium formate, precursors for RNA nucleobases. Finally, high reduction potentials maintained by mantle-derived rocks and minerals would allow phosphite to be present in equilibrium with phosphate, mobilizing otherwise insoluble phosphorus for the prebiotic synthesis of phosphite and phosphate esters after oxidation. So why does the community not view this discontinuous synthesis model as compelling evidence for the RNA-first hypothesis for the origin of life? In part, the model is deficient because no experiments have joined together those steps without human intervention. Further, many steps in the model have problems. Some are successful only if reactive compounds are presented in a specific order in large amounts. Failing controlled addition, the result produces complex mixtures that are inauspicious precursors for biology, a situation described as the "asphalt problem". Many bonds in RNA are thermodynamically unstable with respect to hydrolysis in water, creating a "water problem". Finally, some bonds in RNA appear to be "impossible" to form under any conditions considered plausible for early Earth. To get a community-acceptable "RNA first" model for the origin of life, the discontinuous synthesis model must be developed. In particular, the model must be refined so that it yields oligomeric RNA from CO(2), H(2)O, and N(2) without human intervention. This Account describes our efforts in this direction. Our hypothesis centers on a geological model that synthesizes RNA in a prebiotic intermountain dry valley (not in a marine environment). This valley receives high pH run-off from a watershed rich in serpentinizing olivines and eroding borate minerals. The runoff contains borate-stabilized carbohydrates, formamide, and ammonium formate. As atmospheric CO(2) dissolves in the subaerial aquifer, the pH of the aquifer is lowered. In the desert valley, evaporation of water, a solvent with a nucleophilic "background reactivity", leaves behind formamide, a solvent with an electrophilic "background reactivity". As a result, nucleobases, formylated nucleobases, and formylated carbohydrates, including formylated ribose, can form. Well-known chemistry transforms these structures into nucleosides, nucleotides, and partially formylated oligomeric RNA.
The plausibility of any model in science comes from the extent of its interconnections to other models that are grounded in different premises and reasoning. Focusing research on paradoxes in those ...models, logic whereby they appear to generate unacceptable conclusions from seemingly indisputable premises, helps find those interconnections.
The next goals in the development of a synthetic biology that uses artificial genetic systems will require chemistry-biology combinations that allow the amplification of DNA containing any number of ...sequential and nonsequential nonstandard nucleotides. This amplification must ensure that the nonstandard nucleotides are not unidirectionally lost during PCR amplification (unidirectional loss would cause the artificial system to revert to an all-natural genetic system). Further, technology is needed to sequence artificial genetic DNA molecules. The work reported here meets all three of these goals for a six-letter artificially expanded genetic information system (AEGIS) that comprises four standard nucleotides (G, A, C, and T) and two additional nonstandard nucleotides (Z and P). We report polymerases and PCR conditions that amplify a wide range of GACTZP DNA sequences having multiple consecutive unnatural synthetic genetic components with low (0.2% per theoretical cycle) levels of mutation. We demonstrate that residual mutation processes both introduce and remove unnatural nucleotides, allowing the artificial genetic system to evolve as such, rather than revert to a wholly natural system. We then show that mechanisms for these residual mutation processes can be exploited in a strategy to sequence "six-letter" GACTZP DNA. These are all not yet reported for any other synthetic genetic system.
This Account describes work done in these laboratories that has used synthetic, physical organic, and biological chemistry to understand the roles played by the nucleobases, sugars, and phosphates of ...DNA in the molecular recognition processes central to genetics. The number of nucleobases has been increased from 4 to 12, generating an artificially expanded genetic information system. This system is used today in the clinic to monitor the levels of HIV and hepatitis C viruses in patients, helping to manage patient care. Work with uncharged phosphate replacements suggests that a repeating charge is a universal feature of genetic molecules operating in water and will be found in extraterrestrial life (if it is ever encountered). The use of ribose may reflect prebiotic processes in the presence of borate-containing minerals, which stabilize ribose formed from simple organic precursors. A new field, synthetic biology, is emerging on the basis of these experiments, where chemistry mimics biological processes as complicated as Darwinian evolution.
To the astrobiologist, Enceladus offers easy access to a potential subsurface biosphere via the intermediacy of a plume of water emerging directly into space. A direct question follows: If we were to ...collect a sample of this plume, what in that sample, through its presence or its absence, would suggest the presence and/or absence of life in this exotic locale? This question is, of course, relevant for life detection in any aqueous lagoon that we might be able to sample. This manuscript reviews physical chemical constraints that must be met by a genetic polymer for it to support Darwinism, a process believed to be required for a chemical system to generate properties that we value in biology. We propose that the most important of these is a repeating backbone charge; a Darwinian genetic biopolymer must be a "polyelectrolyte." Relevant to mission design, such biopolymers are especially easy to recover and concentrate from aqueous mixtures for detection, simply by washing the aqueous mixtures across a polycharged support. Several device architectures are described to ensure that, once captured, the biopolymer meets two other requirements for Darwinism, homochirality and a small building block "alphabet." This approach is compared and contrasted with alternative biomolecule detection approaches that seek homochirality and constrained alphabets in non-encoded biopolymers. This discussion is set within a model for the history of the terran biosphere, identifying points in that natural history where these alternative approaches would have failed to detect terran life. Key Words: Enceladus-Life detection-Europa-Icy moon-Biosignatures-Polyelectrolyte theory of the gene. Astrobiology 17, 840-851.
Defining life Benner, Steven A
Astrobiology,
12/2010, Letnik:
10, Številka:
10
Journal Article
Recenzirano
Odprti dostop
Any definition is intricately connected to a theory that gives it meaning. Accordingly, this article discusses various definitions of life held in the astrobiology community by considering their ...connected "theories of life." These include certain "list" definitions and a popular definition that holds that life is a "self-sustaining chemical system capable of Darwinian evolution." We then act as "anthropologists," studying what scientists do to determine which definition-theories of life they constructively hold as they design missions to seek non-terran life. We also look at how constructive beliefs about biosignatures change as observational data accumulate. And we consider how a definition centered on Darwinian evolution might itself be forced to change as supra-Darwinian species emerge, including in our descendents, and consider the chances of our encountering supra-Darwinian species in our exploration of the Cosmos. Last, we ask what chemical structures might support Darwinian evolution universally; these structures might be universal biosignatures.
Artificially expanded genetic information systems (AEGISs) are unnatural forms of DNA that increase the number of independently replicating nucleotide building blocks. To do this, AEGIS pairs are ...joined by different arrangements of hydrogen bond donor and acceptor groups, all while retaining their Watson–Crick geometries. We report here a unique case where AEGIS DNA has been used to execute a systematic evolution of ligands by exponential enrichment (SELEX) experiment. This AEGIS–SELEX was designed to create AEGIS oligonucleotides that bind to a line of breast cancer cells. AEGIS–SELEX delivered an AEGIS aptamer (ZAP-2012) built from six different kinds of nucleotides (the standard G, A, C, and T, and the AEGIS nonstandard P and Z nucleotides, the last having a nitro functionality not found in standard DNA). ZAP-2012 has a dissociation constant of 30 nM against these cells. The affinity is diminished or lost when Z or P (or both) is replaced by standard nucleotides and compares well with affinities of standard GACT aptamers selected against cell lines using standard SELEX. The success of AEGIS–SELEX relies on various innovations, including (i) the ability to synthesize GACTZP libraries, (ii) polymerases that PCR amplify GACTZP DNA with little loss of the AEGIS nonstandard nucleotides, and (iii) technologies to deep sequence GACTZP DNA survivors. These results take the next step toward expanding the power and utility of SELEX and offer an AEGIS–SELEX that could possibly generate receptors, ligands, and catalysts having sequence diversities nearer to that displayed by proteins.
Expanding the number of nucleotides in DNA increases the information density of functional DNA molecules, creating nanoassemblies that cannot be invaded by natural DNA/RNA in complex biological ...systems. Here, we show how six‐letter GACTZP DNA contributes this property in two parts of a nanoassembly: 1) in an aptamer evolved from a six‐letter DNA library to selectively bind liver cancer cells; and 2) in a six‐letter self‐assembling GACTZP nanotrain that carries the drug doxorubicin. The aptamer‐nanotrain assembly, charged with doxorubicin, selectively kills liver cancer cells in culture, as the selectivity of the aptamer binding directs doxorubicin into the aptamer‐targeted cells. The assembly does not kill untransformed cells that the aptamer does not bind. This architecture, built with an expanded genetic alphabet, is reminiscent of antibodies conjugated to drugs, which presumably act by this mechanism as well, but with the antibody replaced by an aptamer.
Crazy train: Folded DNA molecules were evolved or designed from a six‐letter DNA library to perform two non‐genetic functions: binding selectively to liver cancer cells and binding the toxic drug doxorubicin. Combined into an aptamer‐nanotrain conjugate (6N‐Apt‐NTr‐Dox), these selectively kill liver cancer cells. This selective drug delivery is reminiscent of antibody–drug conjugates, but without proteins.
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