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Ligand‐dependent ribozymes

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The discovery of catalytic RNA (ribozymes) more than 30 years ago significantly widened the horizon of RNA‐based functions in natural systems. Similarly to the activity of protein enzymes that are often modulated by the presence of an interaction partner, some examples of naturally occurring ribozymes are influenced by ligands that can either act as cofactors or allosteric modulators. Recent discoveries of new and widespread ribozyme motifs in many different genetic contexts point toward the existence of further ligand‐dependent RNA catalysts. In addition to the presence of ligand‐dependent ribozymes in nature, researchers have engineered ligand dependency into natural and artificial ribozymes. Because RNA functions can often be assembled in a truly modular way, many different systems have been obtained utilizing different ligand‐sensing domains and ribozyme activities in diverse applications. We summarize the occurrence of ligand‐dependent ribozymes in nature and the many examples realized by researchers that engineered ligand‐dependent catalytic RNA motifs. We will also highlight methods for obtaining ligand dependency as well as discuss the many interesting applications of ligand‐controlled catalytic RNAs. WIREs RNA 2017, 8:e1395. doi: 10.1002/wrna.1395 This article is categorized under: RNA-Based Catalysis > RNA–Mediated Cleavage RNA-Based Catalysis > Miscellaneous RNA–Catalyzed Reactions Regulatory RNAs/RNAi/Riboswitches > Riboswitches
Naturally occurring ligand‐dependent ribozymes. (a) Group I intron ribozymes promote self‐splicing activity using guanosine nucleotides as cofactors. The consensus secondary structure of the group I introns consists of 10 paired regions (P1–P10). The guanosine cofactor (G) binds to the G site located in helix P7 (in blue). The processing of the group I introns starts with the nucleophilic attack of the 3′‐OH of the exogenous G cofactor to the phosphodiester bond at the 5′ splice site (5′SS) in P1. The terminal G (ωG) of the intron replaces the G cofactor in the G site. Afterward a second trans‐esterification takes place with the 3′‐OH of the upstream exon attacking the phosphodiester bond in the 3′ splice site (3′SS), leading to the ligation of the two exons. (b) Mechanism of gene regulation by the c‐di‐GMP‐dependent group I intron of Clostridium difficile. In the absence of c‐di‐GMP (top) the GTP cofactor attacks the 3′SS generating an mRNA that lacks a ribosome‐binding site (RBS). In the presence of c‐di‐GMP (bottom) the ribozyme selects alternative splice sites (5′SS and 3′SS). The splicing results in the production of an mRNA containing a strong RBS and start codon (UUG). (c) The glmS ribozyme exhibits self‐cleavage in the presence of glucosamine‐6‐phosphate (GlcN6P), which is used as a cofactor. The cleavage of the mRNA generates a free 5′ hydroxyl group that allows mRNA degradation by RNase J.
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Strategies for aptazyme‐mediated gene regulation in eukaryotes. Allosteric control of gene expression can be achieved by inserting an aptazyme either (a) into the 3′‐UTR or (b) into the 5′‐UTR of the gene of interest. In the first strategy the regulated cleavage reaction results in removal of the poly(A)‐tail, whereas in the 5′‐UTR the 5′‐cap is removed. In both cases mRNA is rapidly degraded upon ribozyme cleavage. (c) In an alternative strategy the ligand‐induced cleavage releases a pri‐miRNA species that is sequentially processed by Drosha and Dicer leading to RNA interference and conditional knockdown of gene expression.
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Strategies for aptazyme‐mediated gene regulation in bacteria. (a) In the first approach a ligand‐dependent group I intron is inserted into an interrupted gene. The addition of the ligand results in correct splicing and in the formation of a functional gene. (b) In an alternative strategy, the ribozyme motif sequesters the ribosome‐binding site (RBS), enabling control of translation initiation. (c) The ligand‐dependent cleavage within the 3′‐UTR using an aptazyme can be used to control gene expression in bacteria. This is probably due to the fact that secondary structures in the 3′‐UTR of bacterial mRNAs were shown to have a protective function against RNase degradation. (d) Aptazyme‐mediated control can be performed with tRNAs. When an amber stop codon is placed into the gene of interest, gene expression can be controlled via ligand‐dependent activation of the amber suppressor tRNA. (e) Aptazymes can control in a ligand‐dependent way the integrity of an orthogonalized 16S rRNA.
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Common designs for oligonucleotide‐dependent ribozymes. (a) In the inhibitor control strategy the effector oligonucleotide binds to the sensor domain disrupting an inhibitory structure and increasing the activity of the ribozyme. (b) In the repair strategy a repair RNA strand displaces a nonfunctional intramolecular strand, thereby activating the ribozyme. (c) An alternative strategy consists in using a sensor strand that is not directly interacting with the catalytic domain, allowing the system to be adaptable to different target–effector sequences. (d) In the expansive regulation model, the activation of the catalytic activity is due to an enhancement of the stability of the enzyme–substrate complex exerted by the effector oligonucleotide.
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In vitro applications of aptazymes and readout systems. (a) Ligand binding induces the cleavage of a radioactively labeled RNA. The detection of the cleavage products is performed by polyacrylamide gel electrophoresis (PAGE) (bottom left) or on array (bottom right). The use of arrays allows the parallel analysis of different analytes in a mixture. (b) The detection of cleavage or ligation products can be performed by quartz crystal microbalance (QCM). (c) Amplification of the signal can be obtained using in trans constructs. An amplification system in which two synthetic ligand‐dependent ligases catalyze each other's synthesis was developed using a total of four RNA strands. (d) Fluorescence resonance energy transfer (FRET) can be used to monitor changes in the cleavage activity of an aptazyme by fluorescence using donor–acceptor dye pairs. (e) Aptazymes can regulate the state of aggregation of gold nanoparticles, thus inducing a visible color shift of the solution upon ligand binding. (f) Using a ligand‐dependent Diels–Alderase aptazyme, the presence of the ligand can be detected monitoring the conversion of the fluorescent substrate anthracene into the nonfluorescent cycloaddition product.
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Tripartite organization of aptazymes. The actuator domain, here a hammerhead ribozyme, is represented in black. The cleavage site is indicated with a gray arrowhead. The sensor domain and the effector ligand are represented in light and dark blue, respectively. The communication module, which functionally connects the actuator and the sensor domain, is displayed in gray.
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Regulatory RNAs/RNAi/Riboswitches > Riboswitches
RNA-Based Catalysis > Miscellaneous RNA-Catalyzed Reactions
RNA-Based Catalysis > RNA-Mediated Cleavage

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