RNA World Research Articles

Infrared Spectroscopy of RNA Nucleosides in a Wide Range of Temperatures

The RNA world hypothesis suggests that early cellular ancestors relied solely on RNA molecules for both genetic information storage and cellular functions. RNA, composed of four nucleosides—adenosine, guanosine, cytidine, and uridine—forms the basis of this theory. These nucleosides consist of purine nucleobases, adenine and guanine, and pyrimidine nucleobases, cytosine and uracil, bonded to ribose sugar. Notably, carbonaceous chondrite meteorites have revealed the presence of these bases and sugar, hinting at the potential existence of nucleosides in space. This study aims to present the infrared spectra of four RNA nucleosides commonly found in terrestrial biochemistry, facilitating their detection in space, especially in astrobiological and astrochemical contexts. Laboratory measurements involved obtaining mid- and far-IR spectra at three temperatures (−180 °C, room temperature, and +180 °C), followed by calculating molar extinction coefficients (ε) and integrated molar absorptivities (ψ) for corresponding bands. These spectral data, along with ε and ψ values, serve to provide quantitative insights into the presence and relative abundance of nucleosides in space and aid in their detection.

Membrane Catalyzed Formation of Nucleotide Clusters and Their Role in the Origins of Life: Insights from Molecular Simulations and Lattice Modeling.

One of the mysteries in studying the molecular "Origin of Life" is the emergence of RNA and RNA-based life forms, where nonenzymatic polymerization of nucleotides is a crucial hypothesis in formation of large RNA chains. The nonenzymatic polymerization can be mediated by various environmental settings, such as cycles of hydration and dehydration, temperature variations, and proximity to a variety of organizing matrices, such as clay, salt, fatty acids, lipid membrane, and mineral surface. In this work, we explore the influence of different phases of the lipid membrane toward nucleotide organization and polymerization in a simulated prebiotic setting. Our molecular simulations quantify the localization propensity of a mononucleotide, uridine monophosphate (UMP), in distinct membrane settings. We perform all-atom molecular dynamics (MD) simulations to estimate the role of the monophasic and biphasic membranes in modifying the behavior of UMPs localization and their clustering mechanism. Based on the interaction energy of mononucleotides with the membrane and their diffusion profile from our MD calculations, we developed a lattice-based model to explore the thermodynamic limits of the observations made from the MD simulations. The mathematical model substantiates our hypothesis that the lipid layers can act as unique substrates for "catalyzing" polymerization of mononucleotides due to the inherent spatiotemporal heterogeneity and phase change behavior.

High-Fidelity RNA Copying via 2',3'-Cyclic Phosphate Ligation.

Templated ligation offers an efficient approach to replicate long strands in an RNA world. The 2',3'-cyclic phosphate (>P) is a prebiotically available activation that also forms during RNA hydrolysis. Using gel electrophoresis and high-performance liquid chromatography, we found that the templated ligation of RNA with >P proceeds in simple low-salt aqueous solutions with 1 mM MgCl2 under alkaline pH ranging from 9 to 11 and temperatures from -20 to 25 °C. No additional catalysts were required. In contrast to previous reports, we found an increase in the number of canonical linkages to 50%. The reaction proceeds in a sequence-specific manner, with an experimentally determined ligation fidelity of 82% at the 3' end and 91% at the 5' end of the ligation site. With splinted oligomers, five ligations created a 96-mer strand, demonstrating a pathway for the ribozyme assembly. Due to the low salt requirements, the ligation conditions will be compatible with strand separation. Templated ligation mediated by 2',3'-cyclic phosphate in alkaline conditions therefore offers a performant replication and elongation reaction for RNA on early Earth.

An anticodon-sensing T-boxzyme generates the elongator nonproteinogenic aminoacyl-tRNA in situ of a custom-made translation system for incorporation.

In the hypothetical RNA world, ribozymes could have acted as modern aminoacyl-tRNA synthetases (ARSs) to charge tRNAs, thus giving rise to the peptide synthesis along with the evolution of a primitive translation apparatus. We previously reported a T-boxzyme, Tx2.1, which selectively charges initiator tRNA with N-biotinyl-phenylalanine (BioPhe) in situ in a Flexible In-vitro Translation (FIT) system to produce BioPhe-initiating peptides. Here, we performed in vitro selection of elongation-capable T-boxzymes (elT-boxzymes), using para-azido-l-phenylalanine (PheAZ) as an acyl-donor. We implemented a new strategy to enrich elT-boxzyme-tRNA conjugates that self-aminoacylated on the 3'-terminus selectively. One of them, elT32, can charge PheAZ onto tRNA in trans in response to its cognate anticodon. Further evolution of elT32 resulted in elT49, with enhanced aminoacylation activity. We have demonstrated the translation of a PheAZ-containing peptide in an elT-boxzyme-integrated FIT system, revealing that elT-boxzymes are able to generate the PheAZ-tRNA in response to the cognate anticodon in situ of a custom-made translation system. This study, together with Tx2.1, illustrates a scenario where a series of ribozymes could have overseen aminoacylation and co-evolved with a primitive RNA-based translation system.

The dynamic world of RNA: beyond translation to subcellular localization and function.

The dynamic world of RNA: beyond translation to subcellular localization and function.

The RNA world: from experimental laboratory to "in silico" approach. Part 1: User friendly RNA expression databases portals

The RNA world: from experimental laboratory to "in silico" approach. Part 1: User friendly RNA expression databases portals

RNA-catalyzed evolution of catalytic RNA

An RNA polymerase ribozyme that was obtained by directed evolution can propagate a functional RNA through repeated rounds of replication and selection, thereby enabling Darwinian evolution. Earlier versions of the polymerase did not have sufficient copying fidelity to propagate functional information, but a new variant with improved fidelity can replicate the hammerhead ribozyme through reciprocal synthesis of both the hammerhead and its complement, with the products then being selected for RNA-cleavage activity. Two evolutionary lineages were carried out in parallel, using either the prior low-fidelity or the newer high-fidelity polymerase. The former lineage quickly lost hammerhead functionality as the population diverged toward random sequences, whereas the latter evolved new hammerhead variants with improved fitness compared to the starting RNA. The increase in fitness was attributable to specific mutations that improved the replicability of the hammerhead, counterbalanced by a small decrease in hammerhead activity. Deep sequencing analysis was used to follow the course of evolution, revealing the emergence of a succession of variants that progressively diverged from the starting hammerhead as fitness increased. This study demonstrates the critical importance of replication fidelity for maintaining heritable information in an RNA-based evolving system, such as is thought to have existed during the early history of life on Earth. Attempts to recreate RNA-based life in the laboratory must achieve further improvements in replication fidelity to enable the fully autonomous Darwinian evolution of RNA enzymes as complex as the polymerase itself.

Architecture of an RNA polymerase ribozyme illuminates the RNA World

'Ribo-organisms' of the primordial RNA World would have needed ribozymes that catalyze RNA replication. McRae, Wan, Kristoffersen et al. recently revealed how these RNA replicases might have functioned by solving the structure of an artificial polymerase ribozyme. This work illustrates how complex RNA structures evolve, with implications for the origins of life.

Three Biopolymers and Origin of Life Scenarios.

To track down the possible roots of life, various models for the initial living system composed of different combinations of the three extant biopolymers, RNA, DNA, and proteins, are presented. The suitability of each molecular set is assessed according to its ability to emerge autonomously, sustain, and evolve continuously towards life as we know it. The analysis incorporates current biological knowledge gained from high-resolution structural data and large sequence datasets, together with experimental results concerned with RNA replication and with the activity demonstrated by standalone constructs of the ribosomal Peptidyl Transferase Center region. The scrutiny excludes the DNA-protein combination and assigns negligible likelihood to the existence of an RNA-DNA world, as well as to an RNA world that contained a replicase made of RNA. It points to the precedence of an RNA-protein system, whose model of emergence suggests specific processes whereby a coded proto-ribosome ribozyme, specifically aminoacylated proto-tRNAs and a proto-polymerase enzyme, could have autonomously emerged, cross-catalyzing the formation of each other. This molecular set constitutes a feasible starting point for a continuous evolutionary path, proceeding via natural processes from the inanimate matter towards life as we know it.

Constraint on an RNA world on montmorillonite clay

Constraint on an RNA world on montmorillonite clay

Influence of the Weak Nuclear Force on Metal-Promoted Autocatalytic Strecker Synthesis of Amino Acids: Formation of a Chiral Pool of Precursors for Prebiotic Peptide and Protein Synthesis.

Natural chiral amino acids typically adopt an L structural configuration. While a preference for specific molecular chiralities is observed throughout biology and cellular chemistry, the origins of this preference are unclear. In a previous report the origin of enantiomeric selectivity was analyzed in terms of an "RNA World" model, and a pathway to a chiral preference for d-ribose was proposed based on the autocatalytic transformation of glyceraldehyde as a precursor to the formation of sugars. Metal-ion-promoted catalysis allows the parity non-conserving (PNC) weak nuclear interaction to influence the chirality of a nascent chiral carbon center. Since the PNC effect is the only natural property with an inherent handedness, it is an obvious candidate to influence enantiomeric preference from a catalytic reaction performed over geologically relevant time scales. The PNC influence requires and emphasizes the important role of catalytic metal ions in primordial chemistry. In this study, the impact of geologically available divalent calcium and higher Z alkaline earth elements are examined as mediators of chiral preference. Detailed calculations of the magnitude of the effect are presented, including the influence of time, temperature, pH, and metal ion identity. It is concluded that metal ions can direct chiral preference for amino acid synthesis via a metal-promoted autocatalytic Strecker reaction within a relatively short geological timeframe, thereby providing a pool of l-amino acids for catalytic chemistry evolving either from an RNA-world model of molecular evolution or alternative pathways to protein synthesis.

From RNA world to RNA-peptide world: A review

How life starts from small molecules to RNAs and further to modern life is an unanswered question. Cyanosulfitic chemistry established the synthesis of building blocks, including 12 proteinogenic amino acids, 4 ribo- and deoxyribo-nucleosides, and phospholipids, from hydrogen cyanide and hydrogen sulfite under prebiotically plausible conditions. Later on, the non-enzymatic monomer extension of nucleotides provided a plausible pathway from mononucleotides to RNAs giving rise to the RNA world. RNA is one of the key components for the origin of life, firstly, the sequence information can be heritage by template copying reaction. Secondly, RNA is able to fold into a secondary structure which has the capability to catalyze chemical reactions. The RNA world scenario has perfectly overcome the chicken-egg problem, but it still cannot explain why peptides are involved in modern life. Most recently, with the establishment of the reaction between RNA and peptides, the trajectory to the RNA-peptide world theory has opened up a new era of the origin of life research. Here I will discuss the current results relevant to the RNA world to RNA-peptide world theory.

The Evolutionary Theory along the Phylogenetic Tree of Unicellular Organisms

In parallel to the advancement of molecular biology suggesting the evolutionary route from the RNA world to the DNA-RNA-protein world through the RNA-protein world, the analysis on the nucleotide base changes in ribosomal RNA genes revels that the divergence of prokaryote and eukaryote first occurred in the DNA-RNA-protein world but the divergence of present-day lineages in eukaryotes occurred after the ancestral eukaryote has acquired the mitochondria as the endosymbionts of O2-respiratory eubacteria. These results strongly suggest that the divergence of unicellular organisms is closely related with the drastic changes in cell contents such as the formation of ribosome, the generation of RNA genes, the conversion from the RNA genes to DNA genes, gene duplication or the endosymbiosis. In the present paper, such drastic changes in evolution of unicellular organisms are theoretically formulated on the basis of the concept of biological activity which consists of the acquired energy, stored energy and systematization. Upon the drastic change in cell contents, the biological activity of an organism is first lowered by the increase in stored energy, but it is gradually recovered as the systematization advances to increase the acquired energy overcoming the increased stored energy and negative entropy due to the systematization itself. Throughout this process, the divergence of new style organisms occurred utilizing new material and energy sources. Such evolutionary process will be explained in detail at the main divergence points in the phylogenetic tree. The relation of such drastic changes in evolution with Darwinian evolution is also clarified. 

Nonenzymatic RNA replication in a mixture of 'spent' nucleotides.

Nonenzymatic template-directed replication would have been affected by co-solutes in a heterogeneous prebiotic soup due to lack of enzymatic machinery. Unlike in contemporary biology, these reactions use chemically activated nucleotides, which undergo rapid hydrolysis forming nucleoside monophosphates ('spent' monomers). These co-solutes cannot extend the primer but continue to base pair with the template, thereby interfering with replication. We, therefore, aimed to understand how a mixture of 'spent' ribonucleotides would affect nonenzymatic replication. We observed the inhibition of replication in the mixture, wherein the predominant contribution came from the cognate Watson-Crick monomer, showing potential sequence dependence. Our study highlights how nonenzymatic RNA replication would have been directly affected by co-solutes, with ramifications for the emergence of functional polymers in an RNA World.

The RNA world of fungal pathogens.

The RNA world of fungal pathogens.

From the RNA World to the Clinic: RNA Therapeutics for Substance Use Disorders

RNA terapötikleri, hastalıkları tedavi etmeyi veya önlemeyi amaçlayan ve hızla genişleyen bir ilaç kategorisidir. Madde bağımlılığının mekanizması tam olarak aydınlatılamadığından, önlenmesi ve tedavisinde halen büyük zorluklar yaşanmaktadır. Bağımlılık yapan maddelerin uzun süreli alımının gen ekspresyon profilini modüle ettiği, ödül mekanizması ve hafıza ile ilgili beyin bölgelerinde patolojik nöroadaptasyona yol açtığı bilinmektedir. Kodlamayan RNA’ların, kompulsif madde kullanımını yönlendiren uyumsuz nöroadaptasyonları düzenlediği gösterilmiş olsa da madde kullanım bozukluğu üzerindeki etkisi henüz klinik olarak test edilmemiştir. Bu gözden geçirme çalışması, madde kullanım bozukluğunun tedavisine yönelik potansiyel bir yaklaşım olan, kodlamayan RNA bazlı terapötiklere odaklanmayı amaçlamaktadır. Bu perspektifte, madde kullanım bozukluğunda yer alan kodlamayan RNA süreçleri gözden geçirilmiştir. Kodlamayan RNA'ları hedeflemeye yönelik son terapötik yaklaşımlar tartışılarak, madde kullanım bozukluğu için kodlamayan RNA'yı hedefleyen terapötiklerin potansiyel fırsatları ve zorlukları vurgulanmıştır. Sonuç olarak, RNA modülasyonunun çeşitli hastalıkların tedavisindeki etki mekanizmalarının umut verici bir strateji haline gelmesi, kodlamayan RNA'ların madde kullanım bozukluğunun tedavisinde de uygulanabilir terapötik hedefler olarak keşfedilmesi gerektiği kanaati oluşturmaktadır.

Ling-Ling Chen: RNA has its own features; don’t study it as a protein

Abstract Non-coding RNA (ncRNA) has been a very active research area over the past 30 years. From small ncRNA – the discovery of RNA interference won the lead researchers the Nobel Prize, to long ncRNA (lncRNA), which has attracted much attention in recent years, various ncRNAs participate in all kinds of biological processes and show a variety of biomedical application prospects. Recently, National Science Review (NSR) interviewed Ling-Ling Chen, a professor at the Center for Excellence in Molecular Cell Science (CEMCS) of the Chinese Academy of Sciences (CAS), deputy director of the State Key Laboratory of Molecular Biology and director of the CAS Key Laboratory of RNA Science and Engineering, to talk about the magic world of RNA. Ling-Ling Chen and her team have been studying ncRNA for more than 10 years, and have witnessed and promoted the development of this field. They discovered unconventional lncRNAs without polyadenylated (polyA) tails or N7-methylguanosine (m7G) caps, including sno-lncRNAs (small nucleolar lncRNAs), SPAs (5' snoRNA capped and 3' polyadenylated RNAs) and circRNAs (circular RNAs), and have made remarkable progress clarifying the biogenetic mechanisms and functions of these RNAs and exploring their biomedical application. Chen said: ‘I am 45 years old. I hope I will not stagnate here, but make new discoveries.’

One-Pot Formation of Pairing Proto-RNA Nucleotides and Their Supramolecular Assemblies.

Most contemporary theories for the chemical origins of life include the prebiotic synthesis of informational polymers, including strong interpretations of the RNA World hypothesis. Existing challenges to the prebiotic emergence of RNA have encouraged exploration of the possibility that RNA was preceded by an ancestral informational polymer, or proto-RNA, that formed more easily on the early Earth. We have proposed that the proto-nucleobases of proto-RNA would have readily formed glycosides with ribose and that these proto-nucleosides would have formed base pairs as monomers in aqueous solution, two properties not exhibited by the extant nucleosides or nucleotides. Here we demonstrate that putative proto-nucleotides of the model proto-nucleobases barbituric acid and melamine can be formed in the same one-pot reaction with ribose-5-phosphate. Additionally, the proto-nucleotides formed in these reactions spontaneously form assemblies that are consistent with the presence of Watson-Crick-like base pairs. Together, these results provide further support for the possibility that heterocycles closely related to the extant bases of RNA facilitated the prebiotic emergence of RNA-like molecules, which were eventually replaced by RNA over the course of chemical and biological evolution.

Effect of montmorillonite K10 clay on RNA structure and function

One of the earliest living systems was likely based on RNA (“the RNA world”). Mineral surfaces have been postulated to be an important environment for the prebiotic chemistry of RNA. In addition to adsorbing RNA and thus potentially reducing the chance of parasitic takeover through limited diffusion, minerals have been shown to promote a range of processes related to the emergence of life, including RNA polymerization, peptide bond formation, and self-assembly of vesicles. In addition, self-cleaving ribozymes have been shown to retain activity when adsorbed to the clay mineral montmorillonite. However, simulation studies suggest that adsorption to minerals is likely to interfere with RNA folding and, thus, function. To further evaluate the plausibility of a mineral-adsorbed RNA world, here we studied the effect of the synthetic clay montmorillonite K10 on the malachite green RNA aptamer, including binding of the clay to malachite green and RNA, as well as on the formation of secondary structures in model RNA and DNA oligonucleotides. We evaluated the fluorescence of the aptamer complex, adsorption to the mineral, melting curves, Förster resonance energy transfer interactions, and 1H-NMR signals to study the folding and functionality of these nucleic acids. Our results indicate that while some base pairings are unperturbed, the overall folding and binding of the malachite green aptamer are substantially disrupted by montmorillonite. These findings suggest that minerals would constrain the structures, and possibly the functions, available to an adsorbed RNA world.

Equilibrium and Non‐equilibrium Reaction Schemes for Prebiotic Polymerization of Ribonucleotides

AbstractThe RNA World theory for the origin of life requires polymers to be generated initially by abiotic reactions. Experiments have studied polymerization of 5′‐monophosphates, 2′,3′‐cyclic phosphates, and 5′‐triphosphates. We consider theoretical models of polymerization in solution illustrating the differences between these cases. We consider (i) a basic model where all monomers undergo reversible joining and breaking; (ii) a model where 2′,3′‐cyclic phosphates can join, and breaking regenerates the cyclic phosphate; (iii) a model where 5′‐triphosphates can join irreversibly, in addition to the joining and breaking of 2′,3′‐cyclic phosphates. In cases (i) and (ii) there is an equilibrium steady state with balance between making and breaking bonds. In case (iii) there is a circular reaction flux in which monomers are activated by an external phosphate source, activated monomers form polymers, and polymers break to release non‐activated monomers. The mean length can be calculated as a function of concentration. In case (iii), the mean length switches from a low‐concentration regime controlled by the 5′‐triphosphates to a high‐concentration regime controlled by the 2′,3′‐cyclic phosphates. The circular reaction flux is reminiscent of a metabolism. If formation of 5’‐triphosphates was already in place for RNA synthesis, ATP could subsequently been coopted for metabolism.