9 research outputs found
Hachimoji DNA and RNA: A genetic system with eight building blocks
Reported here are DNA and RNA-like systems built from eight (hachi-) nucleotide letters (-moji) that form four orthogonal pairs. This synthetic genetic biopolymer meets the structural requirements needed to support Darwinism, including a polyelectrolyte backbone, predictable thermodynamic stability, and stereoregular building blocks that fit a Schrödinger aperiodic crystal. Measured thermodynamic parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to double the information density of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of molecular structures that might support life, including life throughout the cosmos
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RNA Catalysis: Structure, Function, and Evolution
Catalytic RNA or ribozymes present excellent systems to study the foundational principles of biological catalysis. In addition, they serve as models for investigating RNA structure and its relation to function. We report the first crystal structures of the Varkud satellite (VS) ribozyme, the largest among endonucleolytic ribozymes. The structures reveal a modular organization, in which independently folding domains are assembled into the functional conformation of the ribozyme by three-way junctions. The catalytic domain of the ribozyme recognizes and binds the substrate through tertiary interactions, and substrate docking is accompanied by remodeling of substrate structure that results in the formation of a catalytically-relevant active site. The catalytic strategies employed by the VS bear resemblance to that used by protein and DNAzyme ribonucleases, highlighting the robustness of the chemistry of catalytic RNA cleavage. In addition, the active site of the VS ribozyme is strikingly similar to that of the hairpin and hammerhead ribozymes, although the three endonucleolytic ribozymes have distinct sequences and structures. The presence of these architectural features in the context of what appears to be distinct mechanisms of catalysis underscores their functional importance and bolsters the case for convergent evolution.
However, our understanding of the possible mechanisms for the emergence of distinct endonucleolytic ribozyme function during evolution and the ease of access to distinct catalytic motifs involved in RNA cleavage is limited. We have explored the mutational connections between the VS, hairpin and hammerhead ribozymes and delineated plausible pathways by which these distinct ribonuclease motifs can be accessed via intersection of their neutral networks. Intersections between neutral networks are possible due to the existence of bifunctional sequences that exhibit catalytic functions corresponding to both networks. We have identified two such bifunctional sequences that can support hairpin and VS, and hammerhead and hairpin dual functions. Bifunctional sequences present plausible evolutionary nodes toward increasing complexity in functional RNA as illustrated by the hammerhead, hairpin and VS ribozymes in our study. Our results provide a framework to investigate the evolutionary origins of distinct catalytic function in RNA
Structural Basis for Substrate Helix Remodeling and Cleavage Loop Activation in the Varkud Satellite Ribozyme
The Varkud satellite (VS) ribozyme
catalyzes site-specific RNA
cleavage and ligation reactions. Recognition of the substrate involves
a kissing loop interaction between the substrate and the catalytic
domain of the ribozyme, resulting in a rearrangement of the substrate
helix register into a so-called “shifted” conformation
that is critical for substrate binding and activation. We report a
3.3 Å crystal structure of the complete ribozyme that reveals
the active, shifted conformation of the substrate, docked into the
catalytic domain of the ribozyme. Comparison to previous NMR structures
of isolated, inactive substrates provides a physical description of
substrate remodeling, and implicates roles for tertiary interactions
in catalytic activation of the cleavage loop. Similarities to the
hairpin ribozyme cleavage loop activation suggest general strategies
to enhance fidelity in RNA folding and ribozyme cleavage
In vitro selection of ribozyme ligases that use prebiotically plausible 2-aminoimidazole-activated substrates
The hypothesized central role of RNA in the origin of life suggests that RNA propagation predated the advent of complex protein enzymes. A critical step of RNA replication is the template-directed synthesis of a complementary strand. Two experimental approaches have been extensively explored in the pursuit of demonstrating protein-free RNA synthesis: template-directed nonenzymatic RNA polymerization using intrinsically reactive monomers and ribozyme-catalyzed polymerization using more stable substrates such as biological 5'-triphosphates. Despite significant progress in both approaches in recent years, the assembly and copying of functional RNA sequences under prebiotic conditions remains a challenge. Here, we explore an alternative approach to RNA-templated RNA copying that combines ribozyme catalysis with RNA substrates activated with a prebiotically plausible leaving group, 2-aminoimidazole (2AI). We applied in vitro selection to identify ligase ribozymes that catalyze phosphodiester bond formation between a template-bound primer and a phosphor-imidazolide-activated oligomer. Sequencing revealed the progressive enrichment of 10 abundant sequences from a random sequence pool. Ligase activity was detected in all 10 RNA sequences; all required activation of the ligator with 2AI and generated a 3'-5' phosphodiester bond. We propose that ribozyme catalysis of phosphodiester bond formation using intrinsically reactive RNA substrates, such as imidazolides, could have been an evolutionary step connecting purely nonenzymatic to ribozyme-catalyzed RNA template copying during the origin of life.11Ysciescopu
Frontiers in Prebiotic Chemistry and Early Earth Environments.
The Prebiotic Chemistry and Early Earth Environments (PCE3) Consortium is a community of researchers seeking to understand the origins of life on Earth and in the universe. PCE3 is one of five Research Coordination Networks (RCNs) within NASA's Astrobiology Program. Here we report on the inaugural PCE3 workshop, intended to cross-pollinate, transfer information, promote cooperation, break down disciplinary barriers, identify new directions, and foster collaborations. This workshop, entitled, "Building a New Foundation", was designed to propagate current knowledge, identify possibilities for multidisciplinary collaboration, and ultimately define paths for future collaborations. Presentations addressed the likely conditions on early Earth in ways that could be incorporated into prebiotic chemistry experiments and conceptual models to improve their plausibility and accuracy. Additionally, the discussions that followed among workshop participants helped to identify within each subdiscipline particularly impactful new research directions. At its core, the foundational knowledge base presented in this workshop should underpin future workshops and enable collaborations that bridge the many disciplines that are part of PCE3
Crystal structure of the Varkud satellite ribozyme
Varkud Satellite (VS) ribozyme mediates rolling circle replication of a plasmid found in the Neurospora mitochondria. We report crystal structures of this ribozyme at 3.1Å resolution, revealing an intertwined dimer formed by an exchange of substrate helices. Within each protomer, an arrangement of three-way helical junctions organizes seven helices into a global fold that creates a docking site for the substrate helix of the other protomer, resulting in the formation of two active sites in trans. This mode of RNA-RNA association resembles the process of domain swapping in proteins and has implications for RNA regulation and evolution. Within each active site, adenine and guanine nucleobases abut the scissile phosphate, poised to serve direct roles in catalysis. Similarities to the active sites of the hairpin and hammerhead ribozymes highlight the functional significance of active site features, underscore the ability of RNA to access functional architectures from distant regions of sequence space, and suggest convergent evolution