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The acquisition of functions via the elongation of nucleotides is an important factor in the development of the RNA world. In our previous study, we found that the introduction of complementary seven-membered kissing loops into inactive R3C ligase ribozymes revived their ligation activity. In this study, we applied the kissing complex formation-induced rearrangement of RNAs to two nonfunctional RNAs by introducing complementary seven-membered loops into each of them. By combining these two forms of RNAs, the ligase activity (derived from the R3C ligase ribozyme) as well as cleavage activity (derived from the hammerhead ribozyme) was obtained. Thus, effective RNA evolution toward the formation of a life system may require the achievement of “multiple” functions via kissing-loop interactions, as indicated in this study. Our results point toward the versatility of kissing-loop interactions in the evolution of RNA, i.e., two small nonfunctional RNAs can gain dual functions via a kissing-loop interaction.

Unlike in vivo conditions, group II intron ribozymes are known to require high magnesium(II) concentrations ([Mg2+]) and high temperatures (42 °C) for folding and catalysis in vitro. A possible explanation for this difference is the highly crowded cellular environment, which can be mimicked in vitro by macromolecular crowding agents. Here, we combined bulk activity assays and single-molecule Förster Resonance Energy Transfer (smFRET) to study the influence of polyethylene glycol (PEG) on catalysis and folding of the ribozyme. Our activity studies reveal that PEG reduces the [Mg2+] required, and we found an “optimum” [PEG] that yields maximum activity. smFRET experiments show that the most compact state population, the putative active state, increases with increasing [PEG]. Dynamic transitions between folded states also increase. Therefore, this study shows that optimal molecular crowding concentrations help the ribozyme not only to reach the native fold but also to increase its in vitro activity to approach that in physiological conditions.

RNAs fold into 3D structures that range from simple helical elements to complex tertiary structures and quaternary ribonucleoprotein assemblies. The functions of many regulatory RNAs depend on how their 3D structure changes in response to a diverse array of cellular conditions. In this Review, we examine how the structural characterization of RNA as dynamic ensembles of conformations, which form with different probabilities and at different timescales, is improving our understanding of RNA function in cells. We discuss the mechanisms of gene regulation by microRNAs, riboswitches, ribozymes, post-transcriptional RNA modifications and RNA-binding proteins, and how the cellular environment and processes such as liquid–liquid phase separation may affect RNA folding and activity. The emerging RNA-ensemble–function paradigm is changing our perspective and understanding of RNA regulation, from in vitro to in vivo and from descriptive to predictive.The functions of many regulatory RNAs depend on how their 3D structure changes in response to cellular conditions. Recent studies have revealed that RNA exists as a dynamic ensemble of conformations, which form with different probabilities in different cellular conditions and thus modulate RNA function.

Aim. Identification of native and structurally-engineered variants of LCRs with faster processing kinetics and increased lariat capping, and their use for mRNA translation in cells. Conclusions. The capless translation system constructed from functionally-coupled lariat capping ribozyme and viral IRES significantly increased overall protein production compared to IRES-only configuration, while still inferior to co-translational capping with ARCA.Structural engineering of DiLCR stems by modulation of their thermodynamic stability, allowed us to control lariat cap/cleavage products ratio and design variants with near-quantitative capping achievable in vitro, leading to increased protein accumulation in the cell-based translation assay.Alternative LCR-IRES reporter combinations demonstrate high dependence of functional activity on sequence context possibly due to the mutual folding/interaction environment interference.

Biocatalysis is fundamental to biological processes and sustainable applications. Over time, the understanding of biocatalysis has evolved considerably. Initially, protein enzymes were recognized as the primary biocatalysts due to their high catalytic efficiency under mild conditions. The discovery of ribozymes expanded the scope of biocatalysts to include nucleic acids and the development of synthetic or semisynthetic artificial enzymes sought to overcome the limitations of natural enzymes. The emergence of nanozymes, nanomaterials with intrinsic biocatalytic activity, has further broadened this field. Nanozymes possess abundant active sites, multiple active phases, and nanostructures that maintain stability even under extreme conditions, along with unique physicochemical properties. These attributes enable nanozymes to perform efficient biocatalysis in diverse forms and under a wide range of conditions. The discovery of natural biogenic nanozymes, such as magnetosomes, ferritin iron cores, and amyloid protein assemblies, underscores their potential physiological functions and roles in disease pathogenesis. This review explores the distinct properties and catalytic mechanisms of nanozymes, elucidates their structure-activity relationships, and discusses their transformative impact on biocatalysis, highlighting their potential to reshape fundamental concepts and practical applications in the field. Biocatalysis is fundamental to biological processes and sustainable applications, and the emergence of nanozymes, nanomaterials with intrinsic biocatalytic activity, has broadened the field of biocatalysis. This review explores the fundamental definition and distinctive characteristics of nanozymes, and highlights the potential of nanozymes as biocatalytic materials in biomedical applications

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 Mg 2+ 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. Membraneless compartments have been theorized to be prebiotic micro-compartments as they spontaneously encapsulate RNA and proteins. Here, the authors report membraneless compartments can enhance RNA chemistries, affecting template directed RNA polymerization and stimulating nucleic acid enzymes.

RNA aptamers and RNA aptamer-based devices can be genetically encoded and expressed in cells to probe and manipulate cellular function. However, their usefulness in the mammalian cell is limited by low expression and rapid degradation. Here we describe the Tornado (Twister-optimized RNA for durable overexpression) expression system for achieving rapid RNA circularization, resulting in RNA aptamers with high stability and expression levels. Tornado-expressed transcripts contain an RNA of interest flanked by Twister ribozymes. The ribozymes rapidly undergo autocatalytic cleavage, leaving termini that are ligated by the ubiquitous endogenous RNA ligase RtcB. Using this approach, protein-binding aptamers that otherwise have minimal effects in cells become potent inhibitors of cellular signaling. Additionally, an RNA-based fluorescent metabolite biosensor for S-adenosyl methionine (SAM) that is expressed at low levels when expressed as a linear RNA achieves levels sufficient for detection of intracellular SAM dynamics when expressed as a circular RNA. The Tornado expression system thus markedly enhances the utility of RNA-based approaches in the mammalian cell.A ribozyme-based circularization strategy improves the stability of exogenously expressed RNA aptamers.

Functional biomolecules, such as RNA, encapsulated inside a protocellular membrane are believed to have comprised a very early, critical stage in the evolution of life, since membrane vesicles allow selective permeability and create a unit of selection enabling cooperative phenotypes. The biophysical environment inside a protocell would differ fundamentally from bulk solution due to the microscopic confinement. However, the effect of the encapsulated environment on ribozyme evolution has not been previously studied experimentally. Here, we examine the effect of encapsulation inside model protocells on the self-aminoacylation activity of tens of thousands of RNA sequences using a high-throughput sequencing assay. We find that encapsulation of these ribozymes generally increases their activity, giving encapsulated sequences an advantage over nonencapsulated sequences in an amphiphile-rich environment. In addition, highly active ribozymes benefit disproportionately more from encapsulation. The asymmetry in fitness gain broadens the distribution of fitness in the system. Consistent with Fisher’s fundamental theorem of natural selection, encapsulation therefore leads to faster adaptation when the RNAs are encapsulated inside a protocell during in vitro selection. Thus, protocells would not only provide a compartmentalization function but also promote activity and evolutionary adaptation during the origin of life.

Multivalent polyions can undergo complex coacervation, producing membraneless compartments that accumulate ribozymes and enhance catalysis, and offering a mechanism for functional prebiotic compartmentalization in the origins of life. Here, we evaluate the impact of lower, more prebiotically-relevant, polyion multivalency on the functional performance of coacervates as compartments. Positively and negatively charged homopeptides with 1–100 residues and adenosine mono-, di-, and triphosphate nucleotides are used as model polyions. Polycation/polyanion pairs are tested for coacervation, and resulting membraneless compartments are analyzed for salt resistance, ability to provide a distinct internal microenvironment (apparent local pH, RNA partitioning), and effect on RNA structure formation. We find that coacervates formed by phase separation of the shorter polyions more effectively generated distinct pH microenvironments, accumulated RNA, and preserved duplexes than those formed by longer polyions. Hence, coacervates formed by reduced multivalency polyions are not only viable as functional compartments for prebiotic chemistries, they can outperform higher molecular weight analogues. Short cationic peptides and nucleotides can form complex coacervates, but the influence of reduced multivalency on coacervate functionality was not investigated. Here, the authors report that coacervates formed from short polyions generate distinct pH microenvironments, accumulate RNA and preserve nucleic acid duplexes more efficiently than their longer counterparts.

It has long been proposed that phase-separated compartments can provide a basis for the formation of cellular precursors in prebiotic environments. However, we know very little about the properties of coacervates formed from simple peptides, their compatibility with ribozymes or their functional significance. Here we assess the conditions under which functional ribozymes form coacervates with simple peptides. We find coacervation to be most robust when transitioning from long homopeptides to shorter, more pre-biologically plausible heteropeptides. We mechanistically show that these RNA–peptide coacervates display peptide-dependent material properties and cofactor concentrations. We find that the interspacing of cationic and neutral amino acids increases RNA mobility, and we use isothermal calorimetry to reveal sequence-dependent Mg 2+ partitioning, two critical factors that together enable ribozyme activity. Our results establish how peptides of limited length, homogeneity and charge density facilitate the compartmentalization of active ribozymes into non-gelating, magnesium-rich coacervates, a scenario that could be applicable to cellular precursors with peptide-dependent functional phenotypes. Phase-separated compartments have long been proposed as precursors to cellular life. Now, it has been shown that RNA–peptide protocells are more robust when formed using shorter (rather than longer) peptides, and that peptide sequence determines the functional materials properties of these compartments.