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. Here we show an RNA aptamer that preferentially binds oxidized forms of flavin over reduced forms and markedly shifts flavin reduction potential by -40 mV, similar to shifts for oxidoreductases. Nuclear magnetic resonance structural analysis revealed π-π and donor atom-π interactions between the aptamer and flavin that cause unfavorable contacts with the electron-rich reduced form, suggesting a mechanism by which the local environment of the RNA-binding pocket drives the observed shift in cofactor reduction potential. It seems likely that primordial RNAs could have used similar strategies in RNA world metabolisms.

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. In this study, we investigate the effects of crowding, metal ions, and RNA structure on formation of RNA condensates lacking proteins. Using bacterial riboswitches as a model system, we first demonstrate that LLPS of RNA is promoted by molecular crowding, as evidenced by formation of RNA droplets in the presence of polyethylene glycol (PEG 8K). Crowders are not essential for LLPS, however. Elevated Mg2+ concentrations promote LLPS of specific riboswitches without PEG. Calculations identify key RNA structural and sequence elements that potentiate the formation of PEG-free condensates; these calculations are corroborated by key wet-bench experiments. Based on this, we implement structure-guided design to generate condensates with novel functions including ligand binding. Finally, we show that RNA condensates help protect their RNA components from degradation by nucleases, suggesting potential biological roles for such higher-order RNA assemblies in controlling gene expression through RNA stability. By utilizing both natural and artificial RNAs, our study provides mechanistic insight into the contributions of intrinsic RNA properties and extrinsic environmental conditions to the formation and regulation of condensates comprised of RNAs.

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 in vitro, these structures remain difficult to study in vivo. One approach to understanding GQ in vivo behavior is to test whether conditions and molecules found in cells facilitate their folding. Polyamines are biogenic polycations that interact with RNA. Among common polyamines, spermine contains the highest charge and is found in eukaryotes, making it a good candidate for association with high-charge density nucleic acid structures like GQs. Using a variety of techniques, including ultraviolet-detected thermal denaturation, circular dichroism, size exclusion chromatography, and confocal microscopy, on an array of quadruplex sequence variants, we find that eukaryotic biological concentrations of spermine induce microaggregation of three-tiered G-rich sequences, but not of purely two-tiered structures, although higher spermine concentrations induce aggregation of even these. The formation of microaggregates can also be induced by addition of as little as a single G to a two-tiered structure; moreover, they form at biological temperatures, are sensitive to salt, and can form in the presence of at least some flanking sequence. Notably, GQ aggregation is not observed under prokaryotic-like conditions of no spermine and higher NaCl concentrations. The sequence, polyamine, and salt specificity of microaggregation reported herein have implications for the formation and stability of G-rich nucleic acid aggregates in vivo and for functional roles for understudied GQ sequences with only two quadruplex tiers.

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. Here we describe methods to prepare compartments formed by complex coacervates, which are driven by LLPS of oppositely-charged polyions, and to probe the structures and functions of RNAs in them. These methods can be adapted to study RNA biochemistry in compartments formed by diverse artificial and biological macromolecules.

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. The molecular basis for how identity and concentration of polyanionic components of complex coacervates affect ribozyme catalysis remains unexplored. We report here a general mechanism wherein diverse polyanions enhance ribozyme catalysis in complex coacervates. By competing for unproductive RNA-polycation interactions, polyanions enhance ribozyme reaction more than 12-fold. The generality of our findings is supported by similar behavior in three polyanions-polycarboxylates, polysulfates, and polysulfates/carboxylates-as well as two different ribozymes, the hammerhead and hairpin. These results reveal potential roles for polyanions in prebiotic chemistry and extant biology.

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. Differences in RNA diffusion rates between PDAC/RNA and oligoarginine/RNA coacervates imply distinct biophysical environments. Template-directed RNA polymerization is relatively insensitive to Mg2+ concentration when performed in PDAC/RNA coacervates as compared to buffer, even enabling partial rescue of the reaction in the absence of magnesium. Finally, we show enhanced activities of multiple nucleic acid enzymes including two ribozymes and a deoxyribozyme, underscoring the generality of this approach, in which functional nucleic acids like aptamers and ribozymes, and in some cases key cosolutes localize within the coacervate microenvironments.

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. The RNA World hypothesis posits that RNA may have served as both a genetic information carrier and a catalyst during the origin of life. Because of its polyanionic backbone, RNA can undergo LLPS by complex coacervation in the presence of polycations. Phase separation could provide a mechanism for concentrating monomers for RNA synthesis and selectively partition longer RNAs with enzymatic functions, thus driving prebiotic evolution. We introduce several types of LLPS that could lead to compartmentalization and discuss potential roles in template-mediated non-enzymatic polymerization of RNA and other related biomolecules, functions of ribozymes and aptamers, and benefits or penalties imparted by liquid demixing. We conclude that tiny liquid droplets may have concentrated precious biomolecules and acted as bioreactors in the RNA World.

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. Upon covalent modification by the corresponding K28min variant, an ATP-binding aptamer lost all affinity for ATP, and the fluorogenic Mango aptamer lost its ability to activate fluorescence of its dye ligand. Modifying a hammerhead ribozyme near the catalytic core led to loss of almost all of its substrate-cleaving activity, but modifying the same hammerhead ribozyme within a tertiary stabilizing element that reduces magnesium dependence only impaired substrate cleavage at low magnesium concentration. Thus, ribozyme-mediated covalent modification can be used both to selectively inactivate and to fine-tune the activities of targeted functional RNAs, analogous to the effects of post-translational modifications of proteins. Ribozyme-catalyzed covalent modification could therefore be developed to regulate nucleic acids components of synthetic and natural circuits.

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. For a 58nt, cis-acting form of K28, each nucleotide could be replaced with the corresponding 2΄F analog without loss of activity, indicating that no particular 2΄OH is specifically required. Reactivities of two-stranded K28 variants with oligodeoxynucleotide acceptor strands devoid of any 2΄OH moieties implicate modification on an internal guanosine N-2, rather than a ribose hydroxyl. Product mass suggests formation of a GDP(S) adduct along with a second thiophosphorylation, implying that the ribozyme catalyzes both phosphoryl and nucleotidyl transfers. This is further supported by transfer of radiolabels into product from both α and γ phosphates of donor molecules. Furthermore, periodate reactivity of the final product signifies acquisition of a ribose sugar with an intact 2΄-3΄ vicinal diol. Neither nucleobase modification nor nucleotidyl transfer has previously been reported for a kinase ribozyme, making this a first-in-class ribozyme. Base-modifying ribozymes may have played important roles in early RNA world evolution by enhancing nucleic acid functions.

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. Various methods have been employed to target fluorescent dyes to specific locations within the ribosome. Primarily, these methods have involved additional steps including subunit dissociation and/or full reconstitution, which could result in ribosomes of reduced activity and translation efficiency. In addition, substantial time and effort are required to produce limited quantities of material. To enable rapid and large-scale production of highly active, fluorescently labeled ribosomes, we have developed a procedure that combines partial reconstitution with His-tag purification. This allows for a homogeneous single-step purification of mutant ribosomes and subsequent integration of labeled proteins. Ribosomes produced with this method are shown to be as active as ribosomes purified using classical methods. While we have focused on two labeling sites in this report, the method is generalizable and can in principle be extended to any non-essential ribosomal protein.

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. In-line probing and ATPγS competition both argue against direct Cu(2+) binding by RNA; rather, these data establish that Cu(2+) enters the active site within a Cu(2+)•GTPγS or Cu(2+)•GTP chelation complex, and that Cu(2+)•nucleobase interactions further enforce Cu(2+) selectivity and position the metal ion for Lewis acid catalysis. Replacing Mg(2+) with [Co(NH3)6](3+) significantly reduced product yield, but not kobs, indicating that the role of inner-sphere Mg(2+) coordination is structural rather than catalytic. Replacing Mg(2+) with alkaline earths of increasing ionic radii (Ca(2+), Sr(2+) and Ba(2+)) gave lower yields and approximately linear rates of product accumulation. Finally, we observe that reaction rates increased with pH in log-linear fashion with an apparent pKa = 8.0 ± 0.1, indicating deprotonation in the rate-limiting step.