ARTICLE DOI: 10.1038/s41467-017-02639-1 OPEN Wet-dry cycles enable the parallel origin of canonical and non-canonical nucleosides by continuous synthesis Sidney Becker 1, Christina Schneider 1, Hidenori Okamura1, Antony Crisp 1, Tynchtyk Amatov 1, Milan Dejmek2 & Thomas Carell 1 1234567890():,; The molecules of life were created by a continuous physicochemical process on an early Earth. In this hadean environment, chemical transformations were driven by fluctuations of the naturally given physical parameters established for example by wet–dry cycles. These conditions might have allowed for the formation of (self)-replicating RNA as the fundamental biopolymer during chemical evolution. The question of how a complex multistep chemical synthesis of RNA building blocks was possible in such an environment remains unanswered. Here we report that geothermal fields could provide the right setup for establishing wet–dry cycles that allow for the synthesis of RNA nucleosides by continuous synthesis. Our model provides both the canonical and many ubiquitous non-canonical purine nucleosides in parallel by simple changes of physical parameters such as temperature, pH and concentration. The data show that modified nucleosides were potentially formed as competitor molecules. They could in this sense be considered as molecular fossils. 1 Center for Integrated Protein Science Munich CiPSM at the Department of Chemistry, Ludwig-Maximilians-Universität München, 81377 Munich, Germany. 2 Institute of Organic Chemistry and Biochemistry ASCR, 16610 Prague 6, Czech Republic. Correspondence and requests for materials should be addressedto T.C. (email: [email protected]) NATURE COMMUNICATIONS | (2018) 9:163 | DOI: 10.1038/s41467-017-02639-1 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02639-1 he molecules of life originated around 4 billion years ago Herein, we report a robust synthetic pathway, which is purely under conditions governed by the composition of the based on fluctuations of physicochemical parameters such as pH, T ’ 1, 2 – Earth s crust and atmosphere at that time . Molecules concentration, and temperature, driven by wet dry cycles. These such as nucleic acids and amino acids must have formed by a fluctuations enable the direct enrichment or purification of all continuous physicochemical process, in which greater structural reaction intermediates that are directly used for the next synthetic complexity was generated based on fluctuations of the naturally steps. As such, a continuous synthesis is established. Our results given physical parameters3. Geothermal fields, for example, could show that RNA building blocks can indeed be formed in a pre- have established such fluctuations by wet–dry cycles that may biotically plausible geochemical environment without sophisti- have driven chemical transformations, which ultimately allowed cated isolation and purification procedures. The chemical – the emergence of life4 8. The appearance of (self)-replicating scenario presented here supports the hypothesis that life may RNA was certainly of central importance for the transition from have originated in a hydrothermal milieu on land rather than in a – an abiotic world to biology9 11. We need to consider, however, deep sea environment. The key assembly step in our pathway is that an early genetic polymer might have been structurally dif- the formation of variously substituted 5-nitroso-pyrimidines ferent from contemporary RNA. This involves differences (nitrosoPys) that can be converted into formamidopyrimidines regarding the sugar configuration (e.g., pyranosyl RNA) or the (FaPys) in the presence of formic acid and elementary metals (Ni presence of other nucleobases12, 13. Selection pressure led in this or Fe). When combined with ribose, the FaPy compounds react to scenario to the chemical evolution of RNA. Contemporary RNA give a set of purine nucleosides. This chemical pathway delivers molecules contain four canonical nucleosides (A, G, C, U), which not only the canonical purine nucleosides but concomitantly establish the sequence information. In addition, >120 non- many of the ubiquitously present non-canonical relatives, sug- canonical nucleosides are present, which govern a diverse set of gesting their origin as prebiotic competitor nucleosides (A, ms2A, 14 2 2 2 1 properties such as correct folding, e.g., to enable catalysis .In m A, DA, G, m G, m 2G, m G). Since chemical evolution fact, the genetic system of all known life is dependent on modified depended on those molecules that were available on early Earth, nucleosides. Many of these non-canonical nucleosides are found these non-canonical nucleosides may be considered to be mole- today in all three domains of life, which indicates that they were cular fossils, which maintained their essential life-supporting present early on during the development of life. For the ubiqui- character until the present day. tous non-canonical nucleosides we may assume that they were already formed as competitors in parallel with the canonical ones on the early Earth15. So far, however, a geochemical scenario that Results would allow for the parallel formation of canonical and non- Selective crystallization of an organic salt. The chemical sce- canonical RNA building blocks by a continuous process is not nario that leads to a continuous synthesis of RNA building blocks known. All reported multistep chemical models so far rely on by just fluctuations of physical parameters is shown in Figs. 1 and tightly controlled laboratory conditions and the isolation and 2a. The scenario starts with an aqueous solution of malononitrile purification of central reaction intermediates by sophisticated – 1 and different amidinium salts 2a-d (HCl or H2SO4 salts, 400 methods1, 16 19. mM), both recognized prebiotic compounds18. In addition, River landscape in a geothermal environment OH O CO2 + H2O O O Sugar HO OH formation H H OH OH R1 Ribose H O R2 CO 2 N N 2 O Hydrolysis O CH4 N N R3 H2N H HO H N2 Ribose H2O 1 H R N N NH 2 6a-h Atmosphere CO O N R 2 N2 + CH4 HCN N 1 Pool of RNA H2S H2N R N2 3 building blocks NH H2N N R 3 H2N N 2a-d 5a-h Malononitrile and Formamido- Reduction amidine derivatives pyrimidine (FaPy) 0.5 N2 + CO2 1 O NH OR HNO 2 Day-night Wet 2 N N R2 in H2O NaNO2 or seasonal N Dry HNO / NaNO H NR CO NO 3 3 2 cycles N N 2a-d H N N R3 NaNO2 3 2 4a-i Organic salt Nitrosopyrimidine (nitrosoPy) Fig. 1 RNA nucleoside formation pathway. A geothermal environment provides the right set up for the depicted transformations by establishing wet–dry cycles. The prebiotic starting materials are produced from a prebiotic atmosphere and washed into an aqueous environment (e.g. by rain). Major atmospheric components are written in larger letters, whereas minor components are written in smaller letters. Transformations are taking place in different environments, illustrated by various rivers (in light blue). Each environment provides the right setup for different chemistries, leading to several different chemical transformations. This geochemical setup leads to a set of canonical and non-canonical RNA building blocks by continuous synthesis (6a, 1 1 2 3 2 1 2 3 1 2 3 2 1 2 3 2 1 m G: R = O, R = Me, R = NH2; 6b,ms A: R = NH, R = H, R = SMe; 6c,A:R = NH, R = H, R = H; 6d,m G: R = O, R = H, R = NHMe; 6e,m 2G: R = 2 3 1 2 3 1 2 3 2 1 2 3 O, R = H, R = N(Me)2; 6f,G:R = O, R = H, R = NH2; 6g, DA: R = NH, R = H, R = NH2; 6h,mA: R = NH, R = H, R = Me) 2 NATURE COMMUNICATIONS | (2018) 9:163 | DOI: 10.1038/s41467-017-02639-1 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02639-1 ARTICLE a Temperature pH Crystalized Crystalized 4g-i Mixture of Organic salt Selective One-pot prebiotic formation hydrolysis of R3 conversion molecules O NH Aminolysis to FaPys N R2 of R1 = SMe, N R3 3 2 Hot R = O R Basic 1 N H2N N R N R3 R1 4a-d O N N N R2 N O 70 °C O 3 pH 7 1 R OH 3 H2N N R O R 2 NH2 2 HN R OH OH O– N R N N N H2N R 1 1 H2N N R 2a-d H2N N R N N 5a-h O 4b-f Cold 3 N N – Acidic S O 4b-d, 4f Nucleoside Na+ NO – O 2 OH Solid state formation O Cl– NH nitroso-pyrimidine upon ribose OH formation condensation H2N R Complexity b Physical enrichment I pH ~ 4 T = 45 °C pH ~ 4 T = 8 °C – NH NO2 O 2b, R = SMe H2N R OH 2a-d 2a, R = NHMe Na+ N N 1 Dry O– N Cl– O 2c, R = NH O– 2 S N N NH2 O OH 3 H2N R 2a-d Dilute prebiotic mixture Crystal formation 2d, R = Me Fig. 2 Chemical complexity created by physical fluctuations. a Relative changes of temperature (in blue) and pH (in red) are shown for each synthetic step for the continuous synthesis of purine RNA building blocks from small organic and inorganic molecules. Several wet–dry cycles establish fluctuations of the depicted physical parameters that enable the physical enrichment of intermediates. Gray backgrounds denote compounds that are enriched by crystallization from an aqueous solution. b Formation of an organic salt consisting of amidine derivatives 2a-d and (hydroxyimino)malononitrile 3. The salt is selectively crystalized by concentrating a dilute mixture of organic and inorganic compounds by slow evaporation. The crystal structures of the four crystalized organic salts are depicted (Supplementary Tables 1–4) sodium nitrite and acetic acid are present to establish a slightly The robustness and ease of crystallization establishes a first acidic pH of around 4.