Evolution of the substrate specificity of an RNA ligase ribozyme from phosphorimidazole to triphosphate activation.

The acquisition of new RNA functions through evolutionary processes was essential for the diversification of RNA-based primordial biology and its subsequent transition to modern biology. However, the mechanisms by which RNAs access new functions remain unclear. Do RNA enzymes need completely new folds to support new but related functions, or is reoptimization of the active site sufficient? What are the roles of neutral and adaptive mutations in evolutionary innovation? Here, we address these questions experimentally by focusing on the evolution of substrate specificity in RNA-catalyzed RNA assembly.

Synthetic antibodies for accelerated RNA crystallography.

RNA structure is crucial to a wide range of cellular processes. The intimate relationship between macromolecular structure and function necessitates the determination of high-resolution structures of functional RNA molecules. X-ray crystallography is the predominant technique used for macromolecular structure determination; however, solving RNA structures has been more challenging than their protein counterparts, as reflected in their poor representation in the Protein Data Bank (<1%).

Natural soda lakes provide compatible conditions for RNA and membrane function that could have enabled the origin of life.

The origin of life likely occurred within environments that concentrated cellular precursors and enabled their co-assembly into cells. Soda lakes (those dominated by Na ions and carbonate species) can concentrate precursors of RNA and membranes, such as phosphate, cyanide, and fatty acids. Subsequent assembly of RNA and membranes into cells is a long-standing problem because RNA function requires divalent cations, e.

Molecular Crowding Facilitates Ribozyme-Catalyzed RNA Assembly.

Catalytic RNAs or ribozymes are considered to be central to primordial biology. Most ribozymes require moderate to high concentrations of divalent cations such as Mg to fold into their catalytically competent structures and perform catalysis. However, undesirable effects of Mg such as hydrolysis of reactive RNA building blocks and degradation of RNA structures are likely to undermine its beneficial roles in ribozyme catalysis.

RNA-Catalyzed RNA Ligation within Prebiotically Plausible Model Protocells.

Demonstrating RNA catalysis within prebiotically relevant models of primordial cells (protocells) remains a challenge in origins of life research. Fatty acid vesicles encapsulating genomic and catalytic RNAs (ribozymes) are attractive models for protocells; however, RNA catalysis has largely been incompatible with fatty acid vesicles due to their instability in the presence of Mg at the concentrations required for ribozyme function. Here, we report a ribozyme that catalyzes template-directed RNA ligation at low Mg concentrations and thus remains active within stable vesicles.

Frontiers in Prebiotic Chemistry and Early Earth Environments.

The Prebiotic Chemistry and Early Earth Environments (PCE) Consortium is a community of researchers seeking to understand the origins of life on Earth and in the universe. PCE is one of five Research Coordination Networks (RCNs) within NASA's Astrobiology Program. Here we report on the inaugural PCE workshop, intended to cross-pollinate, transfer information, promote cooperation, break down disciplinary barriers, identify new directions, and foster collaborations.

REVERSE: a user-friendly web server for analyzing next-generation sequencing data from in vitro selection/evolution experiments.

Next-generation sequencing (NGS) enables the identification of functional nucleic acid sequences from in vitro selection/evolution experiments and illuminates the evolutionary process at single-nucleotide resolution. However, analyzing the vast output from NGS can be daunting, especially with limited programming skills. We developed REVERSE (Rapid EValuation of Experimental RNA Selection/Evolution) (https://www.

Nonenzymatic assembly of active chimeric ribozymes from aminoacylated RNA oligonucleotides.

Aminoacylated transfer RNAs, which harbor a covalent linkage between amino acids and RNA, are a universally conserved feature of life. Because they are essential substrates for ribosomal translation, aminoacylated oligonucleotides must have been present in the RNA world prior to the evolution of the ribosome. One possibility we are exploring is that the aminoacyl ester linkage served another function before being recruited for ribosomal protein synthesis.

Structural basis for substrate binding and catalysis by a self-alkylating ribozyme.

Ribozymes that react with small-molecule probes have important applications in transcriptomics and chemical biology, such as RNA labeling and imaging. Understanding the structural basis for these RNA-modifying reactions will enable the development of better tools for studying RNA. Nevertheless, high-resolution structures and underlying catalytic mechanisms for members of this ribozyme class remain elusive.

The hammerhead self-cleaving motif as a precursor to complex endonucleolytic ribozymes.

Connections between distinct catalytic RNA motifs through networks of mutations that retain catalytic function (neutral networks) were likely central to the evolution of biocatalysis. Despite suggestions that functional RNAs collectively form an interconnected web of neutral networks, little evidence has emerged to demonstrate the existence of such intersecting networks in naturally occurring RNAs. Here we show that neutral networks of two naturally occurring, seemingly unrelated endonucleolytic ribozymes, the hammerhead (HH) and hairpin (HP), intersect.

The Varkud Satellite Ribozyme: A Thirty-Year Journey through Biochemistry, Crystallography, and Computation.

The discovery of catalytic RNAs or ribozymes introduced a new class of enzymes to biology. In addition to their increasingly important roles in modern life, ribozymes are key players in the RNA World hypothesis, which posits that life started or flourished with RNA supporting both genetic and enzymatic functions. Therefore, investigations into the mechanisms of ribozyme function provide an exciting opportunity to examine the foundational principles of biological catalysis.

Molecular crowding and RNA catalysis.

RNA enzymes or ribozymes catalyze some of the most important reactions in biology and are thought to have played a central role in the origin and evolution of life on earth. Catalytic function in RNA has evolved in crowded cellular environments that are different from dilute solutions in which most in vitro assays are performed. The presence of molecules such as amino acids, polypeptides, alcohols, and sugars in the cell introduces forces that modify the kinetics and thermodynamics of ribozyme-catalyzed reactions.

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.

Hachimoji DNA and RNA: A genetic system with eight building blocks.

We report DNA- and RNA-like systems built from eight nucleotide "letters" (hence the name "hachimoji") that form four orthogonal pairs. These synthetic systems meet the structural requirements needed to support Darwinian evolution, 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 increase the information density of natural terran DNA.

Affinity maturation of a portable Fab-RNA module for chaperone-assisted RNA crystallography.

Antibody fragments such as Fabs possess properties that can enhance protein and RNA crystallization and therefore can facilitate macromolecular structure determination. In particular, Fab BL3-6 binds to an AAACA RNA pentaloop closed by a GC pair with ∼100 nM affinity. The Fab and hairpin have served as a portable module for RNA crystallization.

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.

Laboratory evolution of artificially expanded DNA gives redesignable aptamers that target the toxic form of anthrax protective antigen.

Reported here is a laboratory in vitro evolution (LIVE) experiment based on an artificially expanded genetic information system (AEGIS). This experiment delivers the first example of an AEGIS aptamer that binds to an isolated protein target, the first whose structural contact with its target has been outlined and the first to inhibit biologically important activities of its target, the protective antigen from Bacillus anthracis We show how rational design based on secondary structure predictions can also direct the use of AEGIS to improve the stability and binding of the aptamer to its target. The final aptamer has a dissociation constant of ∼35 nM.

Crystal structure of the Varkud satellite ribozyme.

The Varkud satellite (VS) ribozyme mediates rolling-circle replication of a plasmid found in the Neurospora mitochondrion. We report crystal structures of this ribozyme from Neurospora intermedia at 3.1 Å resolution, which revealed an intertwined dimer formed by an exchange of substrate helices.

Spinach RNA aptamer detects lead(II) with high selectivity.

Spinach RNA aptamer contains a G-quadruplex motif that serves as a platform for binding and fluorescence activation of a GFP-like fluorophore. Here we show that Pb(2+) induces formation of Spinach's G-quadruplex and activates fluorescence with high selectivity and sensitivity. This device establishes the first example of an RNA-based sensor that provides a simple and inexpensive tool for Pb(2+) detection.