An RNA aptamer that shifts the reduction potential of metabolic cofactors.

The discovery of ribozymes has inspired exploration of RNA's potential to serve as primordial catalysts in a hypothesized RNA world. Modern oxidoreductase enzymes employ differential binding between reduced and oxidized forms of redox cofactors to alter cofactor reduction potential and enhance the enzyme's catalytic capabilities. The utility of differential affinity has been underexplored as a chemical strategy for RNA.

RNA sequence and structure control assembly and function of RNA condensates.

Intracellular condensates formed through liquid-liquid phase separation (LLPS) primarily contain proteins and RNA. Recent evidence points to major contributions of RNA self-assembly in the formation of intracellular condensates. As the majority of previous studies on LLPS have focused on protein biochemistry, effects of biological RNAs on LLPS remain largely unexplored.

Long Tracts of Guanines Drive Aggregation of RNA G-Quadruplexes in the Presence of Spermine.

G-Quadruplexes (GQs) are compact, stable structures in DNA and RNA comprised of two or more tiers of quartets whose G-rich motif of tracts of two or more G's occurs commonly within genomes and transcriptomes. While thermodynamically stable , these structures remain difficult to study . One approach to understanding GQ behavior is to test whether conditions and molecules found in cells facilitate their folding.

Measuring the activity and structure of functional RNAs inside compartments formed by liquid-liquid phase separation.

Liquid-liquid phase separation (LLPS) has been known to drive formation of biomolecular compartments, which can encapsulate RNA and proteins among other cosolutes. Such compartments, which lack a lipid membrane, have been implicated in origins of life scenarios as they can easily uptake and concentrate biomolecules, similar to intracellular condensates. Indeed, chemical interactions that drive LLPS in vitro have also been shown to lead to similar sub-cellular compartments in vivo.

Polyanion-Assisted Ribozyme Catalysis Inside Complex Coacervates.

Owing to their ability to encapsulate biomolecules, complex coacervates formed by associative phase separation of oppositely charged polyelectrolytes have been postulated as prebiotic nonmembranous compartments (NMCs). Recent studies show that NMCs sequester RNA and enhance ribozyme reactions, a critical tenet of the RNA World Hypothesis. As RNA is negatively charged, it is expected to interact with polycationic coacervate components.

Template-directed RNA polymerization and enhanced ribozyme catalysis inside membraneless compartments formed by coacervates.

Membraneless compartments, such as complex coacervates, have been hypothesized as plausible prebiotic micro-compartments due to their ability to sequester RNA; however, their compatibility with essential RNA World chemistries is unclear. We show that such compartments can enhance key prebiotically-relevant RNA chemistries. We demonstrate that template-directed RNA polymerization is sensitive to polycation identity, with polydiallyldimethylammonium chloride (PDAC) outperforming poly(allylamine), poly(lysine), and poly(arginine) in polycation/RNA coacervates.

Physical Principles and Extant Biology Reveal Roles for RNA-Containing Membraneless Compartments in Origins of Life Chemistry.

This Perspective focuses on RNA in biological and nonbiological compartments resulting from liquid-liquid phase separation (LLPS), with an emphasis on origins of life. In extant cells, intracellular liquid condensates, many of which are rich in RNAs and intrinsically disordered proteins, provide spatial regulation of biomolecular interactions that can result in altered gene expression. Given the diversity of biogenic and abiogenic molecules that undergo LLPS, such membraneless compartments may have also played key roles in prebiotic chemistries relevant to the origins of life.

Nucleobase modification by an RNA enzyme.

Ribozymes can catalyze phosphoryl or nucleotidyl transfer onto ribose hydroxyls of RNA chains. We report a single ribozyme that performs both reactions, with a nucleobase serving as initial acceptor moiety. This unprecedented combined reaction was revealed while investigating potential contributions of ribose hydroxyls to catalysis by kinase ribozyme K28.

Selective Inactivation of Functional RNAs by Ribozyme-Catalyzed Covalent Modification.

The diverse functions of RNA provide numerous opportunities for programming biological circuits. We describe a new strategy that uses ribozyme K28min to covalently tag a specific nucleobase within an RNA or DNA target strand to regulate and selectively inactivate those nucleic acids. K28min variants with appropriately reprogrammed internal guide sequences efficiently tagged multiple sites from an mRNA and from aptamer and ribozyme targets.

Preparation of ribosomes for smFRET studies: A simplified approach.

During the past decade, single-molecule studies of the ribosome have significantly advanced our understanding of protein synthesis. The broadest application of these methods has been towards the investigation of ribosome conformational dynamics using single-molecule Förster resonance energy transfer (smFRET). The recent advances in fluorescently labeled ribosomes and translation components have resulted in success of smFRET experiments.

Lewis acid catalysis of phosphoryl transfer from a copper(II)-NTP complex in a kinase ribozyme.

The chemical strategies used by ribozymes to enhance reaction rates are revealed in part from their metal ion and pH requirements. We find that kinase ribozyme K28(1-77)C, in contrast with previously characterized kinase ribozymes, requires Cu(2+) for optimal catalysis of thiophosphoryl transfer from GTPγS. Phosphoryl transfer from GTP is greatly reduced in the absence of Cu(2+), indicating a specific catalytic role independent of any potential interactions with the GTPγS thiophosphoryl group.