The split intron-exon (SIE) system is introduced, which separates ribozymes from substrates to facilitate the efficient production of modified circular RNAs, optimizing production process.
Abstract
Ribozyme-based approaches have been extensively employed for in vitro circular RNA (circRNA) synthesis. However, several critical challenges are impeding the progress in this field, including the relatively low ribozyme utilization efficiency, limited synthetic flexibility, and complicated downstream purification procedures. Here, we introduce a split intron-exon (SIE) system for circRNA synthesis by physically separating the ribozyme from its substrates. This design ensures efficient circRNA synthesis by overcoming the inherent limitations of current ribozyme-based techniques, in which each ribozyme can only catalyse the synthesis of at most one circRNA molecule. Moreover, the SIE system enables the efficient synthesis of both unmodified and chemically modified circRNAs. Furthermore, the recyclability and immobilization of the ribozyme within the SIE system reduce reaction impurities and improves circRNA enrichment and yield. Overall, the SIE system holds great potential to advance the diversification and large-scale development of circRNA therapeutics. Current ribozyme-based methods enable efficient circular RNA synthesis but suffer from inflexibility. Here, the authors introduce the split intron-exon (SIE) system, which separates ribozymes from substrates to facilitate the efficient production of modified circular RNAs, optimizing production process.
The “RNA World” hypothesis posits a reasonable scenario for the origin of life. A question central to this scenario is: which RNA species may have emerged as the first ribozyme, thereby initiating the earliest stage of life? To date, experimental and computational studies have focused on ribozymes playing a constructive role in RNA synthesis—such as those facilitating template-directed copying or nucleotide synthesis. However, the answer remains unconvincing, primarily because their functional complexity likely demands lengthy sequences, making de novo emergence improbable. Here, we propose an alternative scenario: a destructive ribozyme, which is potentially quite short, may have emerged first. This ribozyme could cleave other RNA molecules to generate building blocks for its own replication. Notably, the dilemma for a destructive ribozyme is that it may degrade molecules of its own type, thus making it difficult to thrive as an RNA species. Using computational modeling, we demonstrate that an exonuclease ribozyme encoded within a circular RNA genome (thus evading self-attack) can spread within an RNA pool and may subsequently give rise to those constructive ribozymes. Our findings offer a new perspective on the initiation of the RNA world, thereby enriching both theoretical and experimental frameworks for understanding the origin of life.
A new chemically regulated circRNA translation system based on small ligand-induced stabilization of RNA-binding proteins (RBPs), engineered by incorporating RNA motifs into an internal ribosomal entry site and fusing RBPs with small ligand-stabilized conditional proteins is developed.
In vitro transcription using bacteriophage T7 RNA polymerase (T7 RNAP) is the gold-standard platform for RNA production in both research and therapeutic applications. Despite its high processivity and promoter specificity, T7 RNAP generates multiple RNA by-products, including double-stranded RNA, 3'-extended transcripts, abortive RNAs, and prematurely terminated products. These impurities reduce RNA yield, complicate downstream purification, and raise safety concerns for RNA-based therapeutics by activating adverse innate immune pathways. Although reaction optimization and downstream purification strategies can mitigate these issues, they typically involve trade-offs between RNA purity and yield. Enzyme engineering has therefore emerged as a powerful upstream strategy to suppress by-product formation at its molecular origin. Here, we synthesize current knowledge on the structural and mechanistic basis of T7 RNAP by-product formation and systematically review engineering strategies to improve RNA purity. T7 RNAP variants are classified according to their underlying mechanisms of action, including enhanced thermostability, reduced non-specific template binding, smoother initiation-to-elongation transition, reduced premature termination, and template-biased polymerase designs. This analysis identifies general principles governing the trade-off between specificity and processivity and highlights synergistic combinations of mutations that improve RNA purity without compromising transcriptional efficiency. We conclude by discussing the remaining challenges for engineering T7 RNAP to meet the stringent purity requirements of next-generation RNA therapeutics.
Pauline Hermans, Y.I. Serikova, José Castillo et al.· 0 citations
circDesign is presented, an algorithm that explicitly incorporates IRES structural deviation into circRNA sequence design while jointly optimizing codon adaptation and thermodynamic stability and establishes IRES structural preservation as a mechanistic design principle for circRNA engineering and position circDesign as a rational framework for therapeutic circRNA development.
Congcong Xu, F. Jiang, Yifan Jiang et al.· Angewandte Chemie· 0 citations
Ribozymes capable of self-replication from nucleotides would have been central to the hypothesized RNA World. The leading laboratory models for such molecules were converted from a class I ligase by in vitro evolution but then developed without 3D structures. Here, scaffolded cryo-EM of the substrate-free tC19Z RNA polymerase ribozyme at 3.1 Å resolution shows how this conversion was achieved. An accessory domain evolved from random sequence grips the ancestral ligase through a loop-loop contact, a seam of magnesium ions, and a six-base stack, and rebuilds the ligase’s substrate binding site from different residues of its own. A previously unrecognized pairing, present before substrate binds, sequesters the 5′ end that must otherwise pair with the template. Compensatory mutations to the ribozyme and template, designed to break this ectopic pairing, increase the extension rate. These results suggest that accelerating RNA structure determination may speed progress toward nucleotide-based self-replication.
Deni Szokoli, J. Hingey, V. Wu et al.· bioRxiv· 0 citations
Abstract Synthetic riboswitches are efficient tools to render gene expression inducible by small molecules. Here, we present a novel riboswitch platform based on RNA triplexes stabilizing non-polyadenylated transcripts while still allowing translation of the respective messenger RNA. By incorporating a tetracycline aptamer, we rendered triplex formation dependent upon the presence of the ligand. The new riboswitch platform was able to induce gene expression up to 25-fold as a standalone switch. Combination of the triplex system with an aptazyme placed into the 5′-UTR achieved a 42-fold induction of gene expression. Further, we were able to exchange the triplex, the aptamer, the maturation platform cleaving off the poly-A tail of the switch platform, and the regulated transgene, demonstrating a high degree of modularity. The new riboswitch platform is designed to function independently of nuclear processes such as transcription and RNA processing, potentially expanding the application of these synthetic riboswitches to strictly cytoplasmic therapeutic RNA vectors.
Pierre Gommeringer, Elisabeth Müller, Sıla Köse et al.· Nucleic Acids Research· 1 citation