Structure and sequence at an RNA template 5' end influence insertion of transgenes by an R2 retrotransposon protein.

R2 non-long terminal repeat retrotransposons insert site-specifically into ribosomal RNA genes (rDNA) in a broad range of multicellular eukaryotes. R2-encoded proteins can be leveraged to mediate transgene insertion at 28S rDNA loci in cultured human cells. This strategy, Precise RNA-mediated INsertion of Transgenes (PRINT), relies on co-delivery of an mRNA encoding R2 protein and an RNA template encoding a transgene cassette of choice. Here we demonstrate that the PRINT RNA template 5' module, which as a complementary DNA 3' end will generate the transgene 5' junction with rDNA, influences the efficiency and mechanism of gene insertion. Iterative design and testing identified optimal 5' modules consisting of a hepatitis delta virus-like ribozyme fold with high thermodynamic stability, suggesting that RNA template degradation from its 5' end may limit transgene insertion efficiency. We also demonstrate that transgene 5' junction formation can be either precise, formed by annealing the 3' end of first-strand complementary DNA with the upstream target site, or imprecise, by end-joining, but this difference in junction formation mechanism is not a major determinant of insertion efficiency. Sequence characterization of imprecise end-joining events indicates surprisingly minimal reliance on microhomology. Our findings expand current understanding of the role of R2 retrotransposon transcript sequence and structure, and especially the 5' ribozyme fold, for retrotransposon mobility and RNA-templated gene synthesis in cells.

Ambiviricota, a novel ribovirian phylum for viruses with viroid-like properties.

Fungi harbor a vast diversity of mobile genetic elements (MGEs). Recently, novel fungal MGEs, tentatively referred to as 'ambiviruses,' were described. 'Ambiviruses' have single-stranded RNA genomes of about 4-5 kb in length that contain at least two open reading frames (ORFs) in non-overlapping ambisense orientation. Both ORFs are conserved among all currently known 'ambiviruses,' and one of them encodes a distinct viral RNA-directed RNA polymerase (RdRP), the hallmark gene of ribovirian kingdom Orthornavirae. However, 'ambivirus' genomes are circular and predicted to replicate via a rolling-circle mechanism. Their genomes are also predicted to form rod-like structures and contain ribozymes in various combinations in both sense and antisense orientations-features reminiscent of viroids, virusoids, ribozyvirian kolmiovirids, and yet-unclassified MGEs (such as 'epsilonviruses,' 'zetaviruses,' and some 'obelisks'). As a first step toward the formal classification of 'ambiviruses,' the International Committee on Taxonomy of Viruses (ICTV) recently approved the establishment of a novel ribovirian phylum, Ambiviricota, to accommodate an initial set of 20 members with well-annotated genome sequences.

In Vitro Amplification and Selection of Engineered RNase P Ribozyme for Gene Targeting Applications.

Ribozymes engineered from the RNase P catalytic RNA (M1 RNA) represent promising gene-targeting agents for clinical applications. We describe in this report an in vitro amplification and selection procedure for generating active RNase P ribozyme variants with improved catalytic efficiency. Using the amplification and selection procedure, we have previously generated ribozyme variants that were highly active in cleaving a herpes simplex virus 1-encoded mRNA in vitro and inhibiting its expression in virally infected human cells. In this chapter, we use an overlapping region of the mRNAs for the IE1 and IE2 proteins of human cytomegalovirus (HCMV) as a target substrate. We provide detailed protocols and include methods for establishing the procedure for the amplification and selection of active mRNA-cleaving RNase P ribozymes. The in vitro amplification and selection system represents an excellent approach for engineering highly active RNase P ribozymes that can be used in both basic research and clinical applications.

Rapid Ribonuclease P Kinetics Measured by Stopped-Flow Fluorescence and Fluorescence Anisotropy.

Stopped-flow fluorescence spectroscopy is a highly sensitive method for measuring rapid enzyme kinetics. A wide range of fluorophores can be employed, and fluorescence and fluorescence polarization can be measured. Thus, binding, conformational changes, and catalysis can, in principle, be measured, making it helpful in probing the entire kinetic landscape of a reaction. In this chapter, we use the bacterial RNA processing enzyme ribonuclease P (RNase P) as a model system to illustrate the determination of the kinetic constants for substrate binding and cleavage, thus allowing mechanistic questions regarding the effects of reaction conditions, mutations, or drug binding to be answered.

Two RNA Folds from One Sequence: A Ribozyme with Versatile Substrate Processing Abilities.

We report the design of a single RNA sequence capable of adopting one of two ribozyme folds and catalyzing the cleavage and/or ligation of the respective substrates. The RNA is able to change its conformation in response to its environment, hence it is called chameleon ribozyme (CHR). Efficient RNA cleavage of two different substrates as well as RNA ligation by CHR is demonstrated in separate experiments and in a one pot reaction. Our study shows that sequence variants of the hairpin ribozyme intersect with the hammerhead ribozyme and that rather short RNA molecules can have comprehensive conformational flexibility, which is an important feature for the emergence of new functional folds in early evolution.

Computational Design of Allosteric Ribozymes via Genetic Algorithms.

In vitro selection of allosteric ribozymes has many challenges, such as complex and time-consuming experimental procedures, uncertain results, and the unwanted functionality of the enriched sequences. The precise computational design of allosteric ribozymes is achievable using RNA secondary structure folding principles. The computational design of allosteric ribozymes is based on experimentally validated EAs, random search algorithms, and a partition function for RNA folding. The in silico design achieves an accuracy exceeding 90%. Various algorithms with different logic gates have been automated via computer programs that can quickly create many allosteric sequences. This can eliminate the need for in vitro selection of allosteric ribozymes, thus vastly reducing the time and cost required.

Cell-Free Biosensors Based on Modular Eukaryotic Riboswitches That Function in One Pot at Ambient Temperature.

Artificial riboswitches responsive to user-defined analytes can be constructed by successfully inserting in vitro selected aptamers, which bind to the analytes, into untranslated regions of mRNA. Among them, eukaryotic riboswitches are more promising as biosensors than bacterial ones because they function well at ambient temperature. In addition, cell-free expression systems allow the broader use of these riboswitches as cell-free biosensors in an environmentally friendly manner without cellular limitations. The current best cell-free eukaryotic riboswitch regulates eukaryotic canonical translation initiation through self-cleavage mediated by an implanted analyte-responsive ribozyme (i.e., an aptazyme, an aptamer-ribozyme fusion). However, it has critical flaws as a sensor: due to the less-active ribozyme used, self-cleavage and translation reactions must be conducted separately and sequentially, and a different aptazyme has to be selected to change the analyte specificity, even if an aptamer for the next analyte is available. We here stepwise engineered novel types of cell-free eukaryotic riboswitches that harness highly active self-cleavage and thus require no reaction partitioning. Despite the single-step and one-pot reaction, these riboswitches showed higher analyte dose dependency and sensitivities than the current best cell-free eukaryotic riboswitch requiring multistep reactions. In addition, the analyte specificity can be changed in an extremely facile way, simply by aptamer substitution (and the subsequent simple fine-tuning for giant aptamers). Given that cell-free systems can be lyophilized for storage and transport, the present one-pot and thus easy-to-handle cell-free biosensors utilizing eukaryotic riboswitches are expected to be widely used for on-the-spot sensing of analytes at ambient temperature.

Internal initiation of reverse transcription in a Penelope-like retrotransposon.

Eukaryotic retroelements are generally divided into two classes: long terminal repeat (LTR) retrotransposons and non-LTR retrotransposons. A third class of eukaryotic retroelement, the Penelope-like elements (PLEs), has been well-characterized bioinformatically, but relatively little is known about the transposition mechanism of these elements. PLEs share some features with the R2 retrotransposon from Bombyx mori, which uses a target-primed reverse transcription (TPRT) mechanism, but their distinct phylogeny suggests PLEs may utilize a novel mechanism of mobilization. Using protein purified from E. coli, we report unique in vitro properties of a PLE from the green anole (Anolis carolinensis), revealing mechanistic aspects not shared by other retrotransposons. We found that reverse transcription is initiated at two adjacent sites within the transposon RNA that is not homologous to the cleaved DNA, a feature that is reflected in the genomic "tail" signature shared between and unique to PLEs. Our results for the first active PLE in vitro provide a starting point for understanding PLE mobilization and biology.

Robust and heritable knockdown of gene expression using a self-cleaving ribozyme in Drosophila.

The current toolkit for genetic manipulation in the model animal Drosophila melanogaster is extensive and versatile but not without its limitations. Here, we report a powerful and heritable method to knockdown gene expression in D. melanogaster using the self-cleaving N79 hammerhead ribozyme, a modification of a naturally occurring ribozyme found in the parasite Schistosoma mansoni. A 111 bp ribozyme cassette, consisting of the N79 ribozyme surrounded by insulating spacer sequences, was inserted into four independent long noncoding RNA genes as well as the male-specific splice variant of doublesex using scarless CRISPR/Cas9-mediated genome editing. Ribozyme-induced RNA cleavage resulted in robust destruction of 3' fragments typically exceeding 90%. Single molecule RNA fluorescence in situ hybridization results suggest that cleavage and destruction can even occur for nascent transcribing RNAs. Knockdown was highly specific to the targeted RNA, with no adverse effects observed in neighboring genes or the other splice variants. To control for potential effects produced by the simple insertion of 111 nucleotides into genes, we tested multiple catalytically inactive ribozyme variants and found that a variant with scrambled N79 sequence best recapitulated natural RNA levels. Thus, self-cleaving ribozymes offer a novel approach for powerful gene knockdown in Drosophila, with potential applications for the study of noncoding RNAs, nuclear-localized RNAs, and specific splice variants of protein-coding genes.

Design and Synthesis of Circular RNA Expression Vectors.

The recent success of the synthetic mRNA-based anti-COVID-19 vaccines has demonstrated the broad potential of the mRNA platform for applications in medicine, thanks to the combined efforts of a small community that has vastly improved key determinants such as design and formulation of synthetic mRNA during the past three decades. However, the cost of production and sensitivity to enzymatic degradation are still limiting the broader application of synthetic mRNA for therapeutic applications. The increased interest in mRNA-based technologies has spurred a renaissance for circular RNA (circRNA), as the lack of free 5' and 3' ends substantially increases resistance against enzymatic degradation in biological systems and does not require expensive cap analogs, as translation is controlled by an Internal Ribosome Entry Site (IRES) sequence. Thus, it can be expected that circRNA will play an important role for future mRNA therapeutics. Here we provide a detailed guide to the production of synthetic circRNA.

The RNA-DNA world and the emergence of DNA-encoded heritable traits.

The RNA world hypothesis confers a central role to RNA molecules in information encoding and catalysis. Even though evidence in support of this hypothesis has accumulated from both experiments and computational modelling, the transition from an RNA world to a world where heritable genetic information is encoded in DNA remains an open question. Recent experiments show that both RNA and DNA templates can extend complementary primers using free RNA/DNA nucleotides, either non-enzymatically or in the presence of a replicase ribozyme. Guided by these experiments, we analyse protocellular evolution with an expanded set of reaction pathways made possible through the presence of DNA nucleotides. By encapsulating these reactions inside three different types of protocellular compartments, each subject to distinct modes of selection, we show how protocells containing DNA-encoded replicases in low copy numbers and replicases in high copy numbers can dominate the population. This is facilitated by a reaction that leads to auto-catalytic synthesis of replicase ribozymes from DNA templates encoding the replicase after the chance emergence of a replicase through non-enzymatic reactions. Our work unveils a pathway for the transition from an RNA world to a mixed RNA-DNA world characterized by Darwinian evolution, where DNA sequences encode heritable phenotypes.

Development of Allosteric Ribozymes for ATP and l-Histidine Based on the R3C Ligase Ribozyme.

During the evolution of the RNA, short RNAs are thought to have joined together to form long RNAs, enhancing their function as ribozymes. Previously, the artificial R3C ligase ribozyme (73 nucleotides) was successfully reduced to 46 nucleotides; however, its activity decreased significantly. Therefore, we aimed to develop allosteric ribozymes, whose activities could be regulated by effector compounds, based on the reduced R3C ligase ribozyme (R3C-A). Among the variants prepared by fusing an ATP-binding aptamer RNA with R3C-A, one mutant showed increased ligation activity in an ATP-dependent manner. Melting temperature measurements of the two RNA mutants suggested that the region around the aptamer site was stabilized by the addition of ATP. This resulted in a suitable conformation for the reaction at the ligation site. Another ribozyme was prepared by fusing R3C-A with a l-histidine-binding aptamer RNA, and the ligase activity increased with increasing l-histidine concentrations. Both ATP and l-histidine play prominent roles in current molecular biology and the interaction of RNAs and these molecules could be a key step in the evolution of the world of RNAs. Our results suggest promise in the development of general allosteric ribozymes that are independent of the type of effector molecule and provide important clues to the evolution of the RNA world.

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.g. Mg2+, but Mg2+ disrupts fatty acid membranes. The low solubility of Mg-containing carbonates limits soda lakes to moderate Mg2+ concentrations (∼1 mM), so we investigated whether both RNAs and membranes function within these lakes. We collected water from Last Chance Lake and Goodenough Lake in Canada. Because we sampled after seasonal evaporation, the lake water contained ∼1 M Na+ and ∼1 mM Mg2+ near pH 10. In the laboratory, nonenzymatic, RNA-templated polymerization of 2-aminoimidazole-activated ribonucleotides occurred at comparable rates in lake water and standard laboratory conditions (50 mM MgCl2, pH 8). Additionally, we found that a ligase ribozyme that uses oligonucleotide substrates activated with 2-aminoimidazole was active in lake water after adjusting pH from ∼10 to 9. We also observed that decanoic acid and decanol assembled into vesicles in a dilute solution that resembled lake water after seasonal rains, and that those vesicles retained encapsulated solutes despite salt-induced flocculation when the external solution was replaced with dry-season lake water. By identifying compatible conditions for nonenzymatic and ribozyme-catalyzed RNA assembly, and for encapsulation by membranes, our results suggest that soda lakes could have enabled cellular life to emerge on Earth, and perhaps elsewhere.

Emergence of catalytic function in prebiotic information-coding polymers.

Life as we know it relies on the interplay between catalytic activity and information processing carried out by biological polymers. Here we present a plausible pathway by which a pool of prebiotic information-coding oligomers could acquire an early catalytic function, namely sequence-specific cleavage activity. Starting with a system capable of non-enzymatic templated replication, we demonstrate that even non-catalyzed spontaneous cleavage would promote proliferation by generating short fragments that act as primers. Furthermore, we show that catalytic cleavage function can naturally emerge and proliferate in this system. Specifically, a cooperative catalytic network with four subpopulations of oligomers is selected by the evolution in competition with chains lacking catalytic activity. The cooperative system emerges through the functional differentiation of oligomers into catalysts and their substrates. The model is inspired by the structure of the hammerhead RNA enzyme as well as other DNA- and RNA-based enzymes with cleavage activity that readily emerge through natural or artificial selection. We identify the conditions necessary for the emergence of the cooperative catalytic network. In particular, we show that it requires the catalytic rate enhancement over the spontaneous cleavage rate to be at least 102-103, a factor consistent with the existing experiments. The evolutionary pressure leads to a further increase in catalytic efficiency. The presented mechanism provides an escape route from a relatively simple pairwise replication of oligomers toward a more complex behavior involving catalytic function. This provides a bridge between the information-first origin of life scenarios and the paradigm of autocatalytic sets and hypercycles, albeit based on cleavage rather than synthesis of reactants.

Deciphering the Role of Post-Translational Modifications and Cellular Location of Hepatitis Delta Virus (HDV) Antigens in HDV-Mediated Liver Damage in Mice.

Hepatitis D virus (HDV) infection represents the most severe form of chronic viral hepatitis. We have shown that the delivery of HDV replication-competent genomes to the hepatocytes using adeno-associated virus (AAV-HDV) as gene delivery vehicles offers a unique platform to investigate the molecular aspects of HDV and associated liver damage. For the purpose of this study, we generated HDV genomes modified by site-directed mutagenesis aimed to (i) prevent some post-translational modifications of HDV antigens (HDAgs) such as large-HDAg (L-HDAg) isoprenylation or short-HDAg (S-HDAg) phosphorylation; (ii) alter the localization of HDAgs within the subcellular compartments; and (iii) inhibit the right conformation of the delta ribozyme. First, the different HDV mutants were tested in vitro using plasmid-transfected Huh-7 cells and then in vivo in C57BL/6 mice using AAV vectors. We found that Ser177 phosphorylation and ribozymal activity are essential for HDV replication and HDAg expression. Mutations of the isoprenylation domain prevented the formation of infectious particles and increased cellular toxicity and liver damage. Furthermore, altering HDAg intracellular localization notably decreased viral replication, though liver damage remained unchanged versus normal HDAg distribution. In addition, a mutation in the nuclear export signal impaired the formation of infectious viral particles. These findings contribute valuable insights into the intricate mechanisms of HDV biology and have implications for therapeutic considerations.

Highly Reactive Group I Introns Ubiquitous in Pathogenic Fungi.

Systemic fungal infections are a growing public health threat, and yet viable antifungal drug targets are limited as fungi share a similar proteome with humans. However, features of RNA metabolism and the noncoding transcriptomes in fungi are distinctive. For example, fungi harbor highly structured RNA elements that humans lack, such as self-splicing introns within key housekeeping genes in the mitochondria. However, the location and function of these mitochondrial riboregulatory elements has largely eluded characterization. Here we used an RNA-structure-based bioinformatics pipeline to identify the group I introns interrupting key mitochondrial genes in medically relevant fungi, revealing their fixation within a handful of genetic hotspots and their ubiquitous presence across divergent phylogenies of fungi, including all highest priority pathogens such as Candida albicans, Candida auris, Aspergillus fumigatus and Cryptococcus neoformans. We then biochemically characterized two representative introns from C. albicans and C. auris, demonstrating their exceptionally efficient splicing catalysis relative to previously-characterized group I introns. Indeed, the C. albicans mitochondrial intron displays extremely rapid catalytic turnover, even at ambient temperatures and physiological magnesium ion concentrations. Our results unmask a significant new set of players in the RNA metabolism of pathogenic fungi, suggesting a promising new type of antifungal drug target.

RNA-catalyzed evolution of catalytic RNA.

An RNA polymerase ribozyme that was obtained by directed evolution can propagate a functional RNA through repeated rounds of replication and selection, thereby enabling Darwinian evolution. Earlier versions of the polymerase did not have sufficient copying fidelity to propagate functional information, but a new variant with improved fidelity can replicate the hammerhead ribozyme through reciprocal synthesis of both the hammerhead and its complement, with the products then being selected for RNA-cleavage activity. Two evolutionary lineages were carried out in parallel, using either the prior low-fidelity or the newer high-fidelity polymerase. The former lineage quickly lost hammerhead functionality as the population diverged toward random sequences, whereas the latter evolved new hammerhead variants with improved fitness compared to the starting RNA. The increase in fitness was attributable to specific mutations that improved the replicability of the hammerhead, counterbalanced by a small decrease in hammerhead activity. Deep sequencing analysis was used to follow the course of evolution, revealing the emergence of a succession of variants that progressively diverged from the starting hammerhead as fitness increased. This study demonstrates the critical importance of replication fidelity for maintaining heritable information in an RNA-based evolving system, such as is thought to have existed during the early history of life on Earth. Attempts to recreate RNA-based life in the laboratory must achieve further improvements in replication fidelity to enable the fully autonomous Darwinian evolution of RNA enzymes as complex as the polymerase itself.

Inhibition of Cpeb3 ribozyme elevates CPEB3 protein expression and polyadenylation of its target mRNAs and enhances object location memory.

A self-cleaving ribozyme that maps to an intron of the cytoplasmic polyadenylation element-binding protein 3 (Cpeb3) gene is thought to play a role in human episodic memory, but the underlying mechanisms mediating this effect are not known. We tested the activity of the murine sequence and found that the ribozyme's self-scission half-life matches the time it takes an RNA polymerase to reach the immediate downstream exon, suggesting that the ribozyme-dependent intron cleavage is tuned to co-transcriptional splicing of the Cpeb3 mRNA. Our studies also reveal that the murine ribozyme modulates maturation of its harboring mRNA in both cultured cortical neurons and the hippocampus: inhibition of the ribozyme using an antisense oligonucleotide leads to increased CPEB3 protein expression, which enhances polyadenylation and translation of localized plasticity-related target mRNAs, and subsequently strengthens hippocampal-dependent long-term memory. These findings reveal a previously unknown role for self-cleaving ribozyme activity in regulating experience-induced co-transcriptional and local translational processes required for learning and memory.

Stored within DNA are the instructions cells need to make proteins. In order for proteins to get made, the region of DNA that codes for the desired protein (known as the gene) must first be copied into a molecule called messenger RNA (or mRNA for short). Once transcribed, the mRNA undergoes further modifications, including removing redundant segments known as introns. It then travels to molecular machines that translate its genetic sequence into the building blocks of the protein. Following transcription, some RNAs can fold into catalytic segments known as self-cleaving ribozymes which promote the scission of their own genetic sequence. One such ribozyme resides in the intron of a gene for CPEB3, a protein which adds a poly(A) tail to various mRNAs, including some involved in learning and memory. Although this ribozyme is found in most mammals, its biological role is poorly understood. Previous studies suggested that the ribozyme cleaves itself at the same time as the mRNA for CPEB3 is transcribed. This led Chen et al. to hypothesize that the rate at which these two events occur impacts the amount of CPEB3 produced, resulting in changes in memory and learning. If the ribozyme cleaves quickly, the intron is disrupted and may not be properly removed, leading to less CPEB3 being made. However, if the ribozyme is inhibited, the intron remains intact and is efficiently excised, resulting in higher levels of CPEB3 protein. To test how the ribozyme impacts CPEB3 production, Chen et al. inhibited the enzyme from cutting itself with antisense oligonucleotides (ASOs). The ASOs were applied to in vitro transcription systems, neurons cultured in the laboratory and the brains of living mice in an area called the hippocampus. The in vitro and cell culture experiments led to higher levels of CPEB3 protein and the addition of more poly(A) tails to mRNAs involved in neuron communication. Injection of the ASOs into the brains of mice had the same effect, and also improved their memory and learning. The findings of Chen et al. show a new mechanism for controlling protein production, and suggest that ASOs could be used to increase the levels of CPEB3 and modulate neuronal activity. This is the first time a biological role for a self-cleaving ribozyme in mammals has been identified, and the approach used could be applied to investigate the function of two other self-cleaving ribozymes located in introns in humans.

Genetic disruption of the bacterial raiA motif noncoding RNA causes defects in sporulation and aggregation.

Several structured noncoding RNAs in bacteria are essential contributors to fundamental cellular processes. Thus, discoveries of additional ncRNA classes provide opportunities to uncover and explore biochemical mechanisms relevant to other major and potentially ancient processes. A candidate structured ncRNA named the "raiA motif" has been found via bioinformatic analyses in over 2,500 bacterial species. The gene coding for the RNA typically resides between the raiA and comFC genes of many species of Bacillota and Actinomycetota. Structural probing of the raiA motif RNA from the Gram-positive anaerobe Clostridium acetobutylicum confirms key features of its sophisticated secondary structure model. Expression analysis of raiA motif RNA reveals that the RNA is constitutively produced but reaches peak abundance during the transition from exponential growth to stationary phase. The raiA motif RNA becomes the fourth most abundant RNA in C. acetobutylicum, excluding ribosomal RNAs and transfer RNAs. Genetic disruption of the raiA motif RNA causes cells to exhibit substantially decreased spore formation and diminished ability to aggregate. Restoration of normal cellular function in this knock-out strain is achieved by expression of a raiA motif gene from a plasmid. These results demonstrate that raiA motif RNAs normally participate in major cell differentiation processes by operating as a trans-acting factor.

Directed Circularization of a Short RNA.

Basic research and functional analyses of circular RNA (circRNA) have been limited by challenges in circRNA formation of desired length and sequence in adequate yields. Nowadays, circular RNA can be obtained using enzymatic, "ribozymatic," or modulated splice events. However, there are few records for the directed circularization of RNA. Here, we present a proof of principle for an affordable and efficient RNA-based system for the controlled synthesis of circRNA with a physiological 3',5'-phosphodiester conjunction. The engineered hairpin ribozyme variant circular ribozyme 3 (CRZ-3) performs self-cleavage poorly. We designed an activator-polyamine complex to complete cleavage as a prerequisite for subsequent circularization. The developed protocol allows synthesizing circRNA without external enzymatic assistance and adds a controllable way of circularization to the existing methods.