Early Precambrian Research Articles

Prebiotic chiral transfer from self-aminoacylating ribozymes may favor either handedness

Modern life is essentially homochiral, containing D-sugars in nucleic acid backbones and L-amino acids in proteins. Since coded proteins are theorized to have developed from a prebiotic RNA World, the homochirality of L-amino acids observed in all known life presumably resulted from chiral transfer from a homochiral D-RNA World. This transfer would have been mediated by aminoacyl-RNAs defining the genetic code. Previous work on aminoacyl transfer using tRNA mimics has suggested that aminoacylation using D-RNA may be inherently biased toward reactivity with L-amino acids, implying a deterministic path from a D-RNA World to L-proteins. Using a model system of self-aminoacylating D-ribozymes and epimerizable activated amino acid analogs, we test the chiral selectivity of 15 ribozymes derived from an exhaustive search of sequence space. All of the ribozymes exhibit detectable selectivity, and a substantial fraction react preferentially to produce the D-enantiomer of the product. Furthermore, chiral preference is conserved within sequence families. These results are consistent with the transfer of chiral information from RNA to proteins but do not support an intrinsic bias of D-RNA for L-amino acids. Different aminoacylation structures result in different directions of chiral selectivity, such that L-proteins need not emerge from a D-RNA World.

Covalent Linkages Used in Prebiotic Chemistry for RNA‐Templated Amino Acid Transfer and Peptide Synthesis

AbstractBiological translation is a universal process taking place in the ribosome. It involves the synthesis of a protein with a particular sequence from the information encoded in a messenger RNA and the amino acids carried by transfer RNAs with the assistance of specific enzymes. However, the origin of translation in the prebiotic world and, thus, in the absence of enzymes is difficult to envisage. Past and recent studies proposed different prebiotic models, following top‐down and bottom‐up approaches, for the origin and evolution of a primitive ribosome. The bottom‐up models made use of distinct covalent linkages to connect RNA strands with amino acids and peptides. In this review, I focus on the covalent linkages used in these prebiotic models: acyl phosphate mixed anhydrides, phosphoramidates and ureas. I describe their syntheses under prebiotically plausible reaction conditions, as well as include their main conventional preparation methods. I also comment on their properties and chemical stabilities in aqueous solution. Finally, I examine the functions of the described covalent linkages in prebiotic processes involving RNA‐templated amino acid transfer and peptide synthesis.

Earth's core-mantle boundary shaped by the crystallization of a hydrous terrestrial magma ocean.

Enigmatic anomalous structures of Earth's lowermost mantle may have been incubated at the hydrous magma ocean of the Hadean eon.

On the Re-Creation of Protoribosome Analogues in the Lab.

The evolution of the translation system is a fundamental issue in the quest for the origin of life. A feasible evolutionary scenario necessitates the autonomous emergence of a protoribosome capable of catalyzing the synthesis of the initial peptides. The peptidyl transferase center (PTC) region in the modern ribosomal large subunit is believed to retain a vestige of such a prebiotic non-coded protoribosome, which would have self-assembled from random RNA chains, catalyzed peptide bond formation between arbitrary amino acids, and produced short peptides. Recently, three research groups experimentally demonstrated that several distinct dimeric constructs of protoribosome analogues, derived predicated on the approximate 2-fold rotational symmetry inherent in the PTC region, possess the ability to spontaneously fold, dimerize, and catalyze the formation of peptide bonds and of short peptides. These dimers are examined, aiming at retrieving information concerned with the characteristics of a prebiotic protoribosome. The analysis suggests preconditions for the laboratory re-creation of credible protoribosome analogues, including the preference of a heterodimer protoribosome, contradicting the common belief in the precedence of homodimers. Additionally, it derives a dynamic process which possibly played a role in the spontaneous production of the first bio-catalyzed peptides in the prebiotic world.

Adenosine Triphosphate: The Primordial Molecule That Controls Protein Homeostasis and Shapes the Genome-Proteome Interface.

Adenosine triphosphate (ATP) acts as the universal energy currency that drives various biological processes, while nucleic acids function to store and transmit genetic information for all living organisms. Liquid-liquid phase separation (LLPS) represents the common principle for the formation of membrane-less organelles (MLOs) composed of proteins rich in intrinsically disordered regions (IDRs) and nucleic acids. Currently, while IDRs are well recognized to facilitate LLPS through dynamic and multivalent interactions, the precise mechanisms by which ATP and nucleic acids affect LLPS still remain elusive. This review summarizes recent NMR results on the LLPS of human FUS, TDP-43, and the viral nucleocapsid (N) protein of SARS-CoV-2, as modulated by ATP and nucleic acids, revealing the following: (1) ATP binds to folded domains overlapping with nucleic-acid-binding interfaces; (2) ATP and nucleic acids interplay to biphasically modulate LLPS by competitively binding to overlapping pockets of folded domains and Arg/Lys within IDRs; (3) ATP energy-independently induces protein folding with the highest efficiency known so far. As ATP likely emerged in the prebiotic monomeric world, while LLPS represents a pivotal mechanism to concentrate and compartmentalize rare molecules for forming primordial cells, ATP appears to control protein homeostasis and shape genome-proteome interfaces throughout the evolutionary trajectory, from prebiotic origins to modern cells.

Neoarchean clockwise P-T trajectories with evolution sequence from HP to UHT metamorphism during decompression: Evidence from petrology, mineralogy, and phase equilibria modeling of mafic granulites in the Sittampundi Complex, southern India

The dispute of tectonic environments such as subduction-collision setting versus extension tectonic setting, is a common phenomenon in the Precambrian. Despite the ongoing controversy, the pressure–temperature (P–T) paths recorded by metamorphic rocks are often used as an indicator for distinguishing between subduction-collision and extension tectonic settings in the early Precambrian. It is widely acknowledged that high-pressure (HP) granulites may have formed in crustal-thickening-dominated collision/subduction environment. Conversely, ultra-high temperature (UHT) granulites may have formed in the elevated mantle heat flux environment. The Sittampundi Complex (SC), where HP granulites and UHT granulites coexist, plays a crucial role in unraveling the late-Neoarchean to early-Paleoproterozoic tectonic evolution of northern South Granulite-facies Terrane (NSGT) of India. In this study, we present new P–T paths from three HP mafic granulites overprinted by UHT granulite facies metamorphism in the SC and provide more data to unravel metamorphic evolution of NSGT. Phase equilibria modeling and traditional thermobarometry calculations illustrated that clockwise metamorphic pressure–temperature (P–T) paths were yielded, passing from 3.3 to 9.3 kbar/631–669 °C (prograde stage) through 11.9–18.5 kbar/828–986 °C (pressure-peak stage), and through 6.6–11.1 kbar/1019–1085 °C (temperature-peak stage) to 4.4–8.1 kbar/845–930 °C (retrograde stage). The HP mafic granulites exhibit clockwise P–T paths with a considerable heating process (ca. 150–180 °C) from the pressure-peak stage (HP-granulite sub-facies) to the temperature-peak stage (UHT-granulite sub-facies). Zircon U-Pb dating yields an age of 2513 ± 29 indicates that the metamorphism took place in the Neoarchean. The uncommon P–T paths from Neoarchean HP granulite in the NSGT suggest that during late Neoarchean, the NSGT had experienced the crustal-thickening dominated collision/subduction (assembly of micro-blocks of Dharwar Craton) and subsequently mantle diapirism process.

Continental subduction in Paleoproterozoic: Insights from ∼ 1.9 Ga nepheline syenite in the North China craton

Subduction of continent is known as a feature of modern plate tectonics, and it is still unclear whether such a process also occurred in the early Precambrian. Despite of ultra-high pressure metamorphism of terrestrial rocks as a proxy of continental subduction, magmatism produced during continental subduction is also a potential alternative. The North China craton is well-known for the occurrence of 1.9 Ga retrograde eclogite, which was likely originated from the subducting oceanic crust along the Trans-North China Orogen (TNCO). Here a nepheline syenite (the Aohu pluton), the first of the kind from the TNCO, is discovered in Yunzhong Mts. It comprises nepheline (∼20 vol%), albite (50–55 vol%), K-feldspar (5–10 vol%), biotite (∼10 vol%), and calcite (∼5 vol%), with minor accessory minerals. No associated mafic or silicic rocks are outcropped in the pluton. SHRIMP zircon U-Pb analyses yield a crystallization age of 1893 ± 13 Ma and a metamorphic overprinting of 1871 ± 17 Ma. The rocks have medium SiO2 contents (51.9–58.9 wt%), low MgO (0.6–1.6 wt%) and Mg# (24–34), but high Al2O3 (20.0–22.9 wt%) and extremely high K2O + Na2O contents (10.0–13.6 wt%; Na2O/K2O = 1.3–2.6). They are enriched in light rare earth elements (REEs) and large ion lithophile elements, but relatively depleted in heavy REEs and high field strengthen elements. They have extremely low compatible elements (e.g., Ni and Cr). They show high (87Sr/86Sr)i ratios (0.706–0.707) but low εNd(t) values (−5.3 to −6.0; Nd T2DM = ca. 2.7 Ga). Their zircon in-situ εHf(t) values have a wide range (0 to −5.7; Hf T2DM = 2.5–2.85 Ga). The absence of mafic component and the extremely low-Mg feature, as well as their trace element patterns and initial Sr-Nd isotopes being compatible with melts derived from the Archean mafic crustal enclaves in the region, support a continental crust origin for the Aohu nepheline syenite. Based on comparison and modeling, we suggest that its generation was most likely under ultra-high pressure condition. It is thus proposed that continental subduction (into the mantle) might have occurred in the TNCO during the Paleoproterozoic.

An Aminoisoxazole‐Based Proto‐RNA

AbstractThe RNA world hypothesis predicts that life started with the development of replicating and catalytically active RNA, which evolved in a process of molecular evolution to increasingly complex chemical structures. RNA is, however, so complex that it has most likely formed from a precursor (proto‐RNA) that was more easily accessible in a prebiotic world. Recently, 3‐aminoisoxazoles (IO3) were identified as building blocks that can form under prebiotic conditions and can rearrange to give the nucleoside cytidine (C). The present study shows that the constitutional isomer 5‐aminoisoxazole (IO5) can undergo the same reaction to give uridine (U). Both compounds (IO3 and IO5), if embedded in RNA, react selectively to C and U, which are the main pyrimidine nucleosides of the genetic system. Importantly, the stereochemical outcome of the IO5 reaction in RNA depends on the neighboring bases. If they are β‐configured RNA nucleosides, the reaction proceeds with high selectivity to give exclusively the β‐configured U RNA base (anomeric control).

Origins of Genetic Coding: Self-Guided Molecular Self-Organisation.

The origin of genetic coding is characterised as an event of cosmic significance in which quantum mechanical causation was transcended by constructive computation. Computational causation entered the physico-chemical processes of the pre-biotic world by the incidental satisfaction of a condition of reflexivity between polymer sequence information and system elements able to facilitate their own production through translation of that information. This event, which has previously been modelled in the dynamics of Gene-Replication-Translation systems, is properly described as a process of self-guided self-organisation. The spontaneous emergence of a primordial genetic code between two-letter alphabets of nucleotide triplets and amino acids is easily possible, starting with random peptide synthesis that is RNA-sequence-dependent. The evident self-organising mechanism is the simultaneous quasi-species bifurcation of the populations of information-carrying genes and enzymes with aminoacyl-tRNA synthetase-like activities. This mechanism allowed the code to evolve very rapidly to the ~20 amino acid limit apparent for the reflexive differentiation of amino acid properties using protein catalysts. The self-organisation of semantics in this domain of physical chemistry conferred on emergent molecular biology exquisite computational control over the nanoscopic events needed for its self-construction.

On Earth’s habitability over the Sun’s main-sequence history: joint influence of space weather and Earth’s magnetic field evolution

ABSTRACT The aim of this study is to analyse the Earth habitability with respect to the direct exposition of the Earth atmosphere to the solar wind (SW) along the Sun’s evolution on the main sequence including the realistic evolution of the space weather conditions and the Earth magnetic field. The MHD code PLUTO in spherical coordinates is applied to perform parametric studies with respect to the SW dynamic pressure and the interplanetary magnetic field intensity for different Earth magnetic field configurations. Quiet space weather conditions may not impact the Earth habitability. On the other hand, the impact of interplanetary coronal mass ejections (ICME) could lead to the erosion of the primary Earth atmosphere during the Hadean eon. A dipolar field of 30 μT is strong enough to shield the Earth from the Eo-Archean age as well as 15 and 5 μT dipolar fields from the Meso-Archean and Meso-Proterozoic, respectively. Multipolar weak field period during the Meso-Proterozoic age may not be a threat for ICME-like space weather conditions if the field intensity is at least 15 μT and the ratio between the quadrupolar (Q) and dipolar (D) coefficients is $\frac{Q}{D} \le 0.5$. By contrast, the Earth habitability in the Phanerozoic eon (including the present time) can be hampered during multipolar low field periods with a strength of 5 μT and $\frac{Q}{D} \ge 0.5$ associated with geomagnetic reversals. Consequently, the effect of the SW should be considered as a possible driver of Earth’s habitability.

Feldspar Megacrysts as a Source of Information on Crustal Contamination of Basaltic Melt

The paper discusses data on the elemental composition and 87Sr/86Sr, and δ18O isotopic ratios of feldspar megacrysts collected from lava flows, tuffs, and cinders of three volcanic fields in the Baikal rift system: Iya–Uda, Vitim, and Khamar-Daban, which are located within the early Precambrian, Riphean, and Paleozoic crustal blocks, respectively. Megacrysts are hosted in trachybasalts in the Iya–Uda and Khamar-Daban fields and in basanites in the Vitim field. Megacrysts belong to the following three compositional groups of minerals: (i) plagioclase in lavas of the Iya–Uda field, (ii) anorthoclase in lava flows, tuffs, and cinders of the Khamar-Daban and Vitim fields, and (iii) sanidine in the Vitim field. Elemental and isotope data suggest that megacrysts crystallized in volcanic chambers at different depth levels: anorthoclase crystallized from the most primitive magma with mantle-derived isotopic signatures at subcrustal depth levels, plagioclases were produced in deep crustal chambers during the interaction between mantle-derived magma and crustal rocks, and sanidine was captured from the upper crustal rocks.

Concerning the discussion about the Bug series of the Middle Bug (Ukrainian shield)

The materials of several articles and reports published in 2022 by the authors of this work and V.P. Kirilyuk (Kirilyuk, 2022 a,b), who discussed the structure and stratigraphy of the Вug area granulite complex of the Ukrainian Shield. The authors, relying on their geological, geophysical and geochemical studies, isotopic geochronology data, as well as on the world experience in studying the Early Precambrian complexes of the Canadian and Australian shields, argue that during 3.8—1.7 Ga, the Bug area granulite complex underwent at least a threefold structural metamorphic processing. Its modern structural plan, as well as, to a large extent, the composition and structural and textural features of metamorphic rocks, are of Early Proterozoic age. Therefore, its division into suites of primary sedimentary-volcanogenic rocks and their vertical distribution by age does not make sense. Only a division into two series is possible — the Dniester-Bug and the Bug, between which there is a tectonic contact. Contrary to this, V.P. Kirilyuk, relying on the experience of his stratigenic-metamorphogenic studies and the authority of the well-known school of L.I. Salopa, E.M. Lazko et al., defends the point of view on the stratigenic nature of the Bug area granulite complex, in which the primacy and stability of the position of strata-formations relative to each other were preserved throughout the entire time. According to V.P. Kirilyuk, there are no breaks and disagreements between the suites, so he proposes to abandon the identification of the early Precambrian USh Bug Group. And the seven suites identified by him, the youngest of which is 3.8 billion years old, are proposed to be attributed to a single Dniester-Bug series. The authors of both alternative points of view apply to the National Stratigraphic Committee of Ukraine with a proposal to revise the stratigraphic scheme of the Early Precambrian of the USh.

Formation of ammonia through serpentinization in the Hadean Eon

Formation of ammonia through serpentinization in the Hadean Eon

Formation of Amino Acids and Carboxylic Acids in Weakly Reducing Planetary Atmospheres by Solar Energetic Particles from the Young Sun.

Life most likely started during the Hadean Eon; however, the environmental conditions which contributed to the complexity of its chemistry are poorly known. A better understanding of various environmental conditions, including global (heliospheric) and local (atmospheric, surface, and oceanic), along with the internal dynamic conditions of the early Earth, are required to understand the onset of abiogenesis. Herein, we examine the contributions of galactic cosmic rays (GCRs) and solar energetic particles (SEPs) associated with superflares from the young Sun to the formation of amino acids and carboxylic acids in weakly reduced gas mixtures representing the early Earth's atmosphere. We also compare the products with those introduced by lightning events and solar ultraviolet light (UV). In a series of laboratory experiments, we detected and characterized the formation of amino acids and carboxylic acids via proton irradiation of a mixture of carbon dioxide, methane, nitrogen, and water in various mixing ratios. These experiments show the detection of amino acids after acid hydrolysis when 0.5% (v/v) of initial methane was introduced to the gas mixture. In the set of experiments with spark discharges (simulation of lightning flashes) performed for the same gas mixture, we found that at least 15% methane was required to detect the formation of amino acids, and no amino acids were detected in experiments via UV irradiation, even when 50% methane was used. Carboxylic acids were formed in non-reducing gas mixtures (0% methane) by proton irradiation and spark discharges. Hence, we suggest that GCRs and SEP events from the young Sun represent the most effective energy sources for the prebiotic formation of biologically important organic compounds from weakly reducing atmospheres. Since the energy flux of space weather, which generated frequent SEPs from the young Sun in the first 600 million years after the birth of the solar system, was expected to be much greater than that of GCRs, we conclude that SEP-driven energetic protons are the most promising energy sources for the prebiotic production of bioorganic compounds in the atmosphere of the Hadean Earth.

REGIONAL METAMORPHISM AND STRATIGRAPHY OF THE BASEMENT OF THE UKRAINIAN SHIELD

Stratigraphic complexes of the Lower Precambrian are everywhere metamorphosed. Therefore, the stratigraphic subdivision of the Lower Precambrian has always been inextricably linked with the study of metamorphism. For some time, the degree of metamorphism of the complexes was even used as an indication of their relative age. With the beginning of the use of isotopic dating, this sign was not confirmed, after which the degree of metamorphism was no longer taken into account in the stratigraphic dismemberment of the shields basement. The degree of metamorphism of the complexes was no longer taken into account for a long time when compiling official stratigraphic schemes of the Precambrian of the Ukrainian Shield, which, in the opinion of many geologists, led to distortions of the real stratigraphy of the basement of this region. The authors of the article believe that the degree of metamorphism can still be used in the development of the stratigraphy of the Ukrainian Shield and, above all, in the stratigraphic study of individual megablocks. The possibility of such use of metamorphism is considered in the cycle of publications. This is the second article in the cycle. The first article describes the stratigraphy and metamorphism of all megablocks of the Ukrainian Shield. A number of conclusions have been made about the regularities of the manifestation of metamorphism. It is shown that older stratigenic complexes in each of the megablocks are characterized by higher temperature metamorphism. This pattern provides a basis for establishing the relative stratigraphic sequence of complexes within individual megablocks based on the degree of their metamorphism. At the same time, the distinctive features of the composition and metamorphism of the stratigenic complexes, according to the authors, are a reflection of the successive stages of the geological development of the Ukrainian Shield in the Early Precambrian and can serve as the basis for compiling of the regional stratigraphic scheme on a historical and geological basis. In this second article of the cycle, modern approaches to the geochronological periodization of the Precambrian are considered: a) geochronometric, adopted for the International “The Geological Time Scale” and b) historical-geological, which is the basis of the “General Stratigraphic Scale of the Lower Precambrian of Russia”. The current “Correlation Chronostratigraphic Scheme of the Early Precambrian of the Ukrainian Shield” is based on the geochronometric approach, which the authors, like many other researchers, consider unacceptable for practical use. The article proposes a variant of the regional stratigraphic scheme of the Ukrainian Shield on a historicalgeological basis, compiled at the level of complexes, which in the final version of the scheme can be divided into series and suites.

Evolution of the composition of hydrothermal mineral-forming fluid of ore deposits in early Precambrian of the Ukrainian Shield

The aim of the study, the results of which are presented in this article, is to study changes in the composition of hydrothermal fluid ore systems of the early Precambrian on the example of gold and uranium ore deposits of the Ukrainian shield in connection with the evolution of exo- and endogenous processes in the outer shells of the Earth. The method used is a complex isotope-geochemical study of the composition of gas-liquid inclusions of hydrothermal fluid of ore systems. Based on experimental isotope-geochemical studies of the fluid of gas-liquid inclusions in the minerals of Precambrian deposits of uranium and gold of the Serednoprydniprovsky and Ingul megablocks of the Ukrainian Shield, a consistent change in the quantitative and isotopic composition of hydrothermal fluids of mineral formation from Mesoarchean to Paleoproterozoic was established. The content of H2O and CO2 was investigated and the isotopic composition of carbon CO2 fluids in quartz, pyrite, and feldspar were studied. The age range of the studied deposits is 3200—1750 million years. A decrease in the carbon dioxide content in the hydrothermal mineral-forming fluid of the Precambrian occurred in the Neoarchean, that is, before the Great Oxygen Event (Great Oxidation Event) — an increase in the oxygen content in the atmosphere in the Paleoproterozoic and is probably associated with the processes of exogenous and endogenous hydration of ultramafic rocks during the formation of the protocontinental crust. Molar fraction of CO2 in the mineral-forming fluid, in the Precambrian correlates to some extent with atmospheric pressure. The increase in the content of the carbon-12 isotope in the Paleoproterozoic in the mineral-forming fluid occurred due to the oxidation of organic matter with an increase in the oxygen content in the atmosphere.

Manifestation of Supramolecular Chirality during Adsorption on CsCuCl3 and γ-Glycine Crystals

The chirality of biopolymers and its emergence from the racemic prebiotic world is one of the key mysteries of science. There are many versions on how the total chiral balance breaking occurred, but they all face an insoluble challenge—the impossibility of a total shift of the chiral balance towards the formation of biopolymers based only on D-sugars and L-amino acids. A possible solution to this problem lies in the asymmetric autocatalysis on chiral crystals. Since the reaction is heterogeneous, it is important to study the features of adsorption on the surface of crystals. In this paper, the adsorption of limonene, α-pinene, and menthol enantiomers on γ-glycine and CsCuCl3 crystals was studied. Single-crystal X-ray crystallography, SEM, and porosimetry were used as auxiliary methods. The t-test was used to determine the reliability of chiral recognition. It was shown that both crystals were capable of chiral recognition at high coverages. The mechanism of supramolecular chiral recognition was identical to that of the chiral crystals studied previously. However, neither γ-glycine nor CsCuCl3 showed chiral recognition with respect to all enantiomers. In fact, γ-glycine crystals showed recognition for limonene enantiomers, and very high recognition in the case of menthol enantiomers. CsCuCl3 crystals showed the capability to recognize enantiomers of α-pinenes only. This led to the conclusion that the recognition of enantiomers by a supramolecular chiral surface is not universal.

A window into prebiotic worlds?

Zircon s reveal the geochemistry of Earth's earliest hydrothermal systems.

Organic carbon generation in 3.5-billion-year-old basalt-hosted seafloor hydrothermal vent systems.

Carbon is the key element of life, and its origin in ancient sedimentary rocks is central to questions about the emergence and early evolution of life. The oldest well-preserved carbon occurs with fossil-like structures in 3.5-billion-year-old black chert. The carbonaceous matter, which is associated with hydrothermal chert-barite vent systems originating in underlying basaltic-komatiitic lavas, is thought to be derived from microbial life. Here, we show that 3.5-billion-year-old black chert vein systems from the Pilbara Craton, Australia contain abundant residues of migrated organic carbon. Using younger analogs, we argue that the black cherts formed during precipitation from silica-rich, carbon-bearing hydrothermal fluids in vein systems and vent-proximal seafloor sediments. Given the volcanic setting and lack of organic-rich sediments, we speculate that the vent-mound systems contain carbon derived from rock-powered organic synthesis in the underlying mafic-ultramafic lavas, providing a glimpse of a prebiotic world awash in terrestrial organic compounds.

Insight into the structures of unusual base pairs in RNA complexes containing a primer/template/adenosine ligand.

In the prebiotic RNA world, the self-replication of RNA without enzymes can be achieved through the utilization of 2-aminoimidazole activated nucleotides as efficient substrates. The mechanism of RNA nonenzymatic polymerization has been extensively investigated biophysically and structurally by using the model of an RNA primer/template complex which is bound by the imidazolium-bridged or triphosphate-bridged diguanosine intermediate. However, beyond the realm of the guanosine substrate, the structural insight into how alternative activated nucleotides bind and interact with the RNA primer/template complex remains unexplored, which is important for understanding the low reactivity of adenosine and uridine substrates in RNA primer extension, as well as its relationship with the structures. Here we use crystallography as a method and determine a series of high-resolution structures of RNA primer/template complexes bound by ApppG, a close analog of the dinucleotide intermediate containing adenosine and guanosine. The structures show that ApppG ligands bind to the RNA template through both Watson-Crick and noncanonical base pairs, with the primer 3'-OH group far from the adjacent phosphorus atom of the incoming substrate. The structures indicate that when adenosine is included in the imidazolium-bridged intermediate, the complexes are likely preorganized in a suboptimal conformation, making it difficult for the primer to in-line attack the substrate. Moreover, by co-crystallizing the RNA primer/template with chemically activated adenosine and guanosine monomers, we successfully observe the slow formation of the imidazolium-bridged intermediate (Ap-AI-pG) and the preorganized structure for RNA primer extension. Overall, our studies offer a structural explanation for the slow rate of RNA primer extension when using adenosine-5'-phosphoro-2-aminoimidazolide as a substrate during nonenzymatic polymerization.