Abstract:An allosteric ribozyme has been designed using the hammerhead ribozyme as the active site and aflavin-specific RNA aptamer as a regulatory site. We constructed six variants with a series of base pairs in the linker region (stem II). Under single turnover conditions, kinetic studies were carried out in the absence and presence of flavin mononucleotide (FMN). Interestingly, FMN addition did not influence the cleavage rate of constructs with a 5-6 bp linker but stimulated the catalytic activity of those bearing a… Show more
“…The reward for acquiring this capability is substantial, considering that many applications in medicine, industry, and biotechnology demand high-speed enzymes with precisely tailored catalytic functions. ''Modular rational design'' has proven to be an effective means for conferring additional chemical and kinetic complexity on existing protein (1)(2)(3)(4) and RNA enzymes (5)(6)(7)(8)(9). This engineering strategy takes advantage of the modular nature of many protein (10) and RNA subdomains (11)(12)(13), which can be judiciously integrated to form new multifunctional constructs.…”
Ligand-specific molecular switches composed of RNA were created by coupling preexisting catalytic and receptor domains via structural bridges. Binding of ligand to the receptor triggers a conformational change within the bridge, and this structural reorganization dictates the activity of the adjoining ribozyme. The modular nature of these tripartite constructs makes possible the rapid construction of precision RNA molecular switches that trigger only in the presence of their corresponding ligand. By using similar enzyme engineering strategies, new RNA switches can be made to operate as designer molecular sensors or as a new class of genetic control elements.Mastery of the molecular forces that dictate biopolymer folding and function would allow molecular engineers to participate in the design of enzymes-a task that to date has been managed largely by the random processes of evolution. The reward for acquiring this capability is substantial, considering that many applications in medicine, industry, and biotechnology demand high-speed enzymes with precisely tailored catalytic functions. ''Modular rational design'' has proven to be an effective means for conferring additional chemical and kinetic complexity on existing protein (1-4) and RNA enzymes (5-9). This engineering strategy takes advantage of the modular nature of many protein (10) and RNA subdomains (11-13), which can be judiciously integrated to form new multifunctional constructs. The recent discoveries of new catalytic RNA motifs (14, 15) and new ligand-binding motifs (16, 17) have considerably expanded the opportunities for ribozyme engineering.We have used modular rational design to create several artificial ribozymes that are activated or deactivated by the binding of specific small organic molecules such as ATP (5, 8) and flavin mononucleotide (FMN) (unpublished data). Each of these allosteric † ribozymes is composed of two independent structural domains: one an RNA-cleaving ribozyme and the other a receptor (or ''aptamer'') for a specific ligand. The conformational changes that occur within an aptamer domain on introduction of the ligand, termed ''adaptive binding '' (22-25), can trigger kinetic modulation of the adjoining catalytic domain by several different mechanisms that ultimately influence ribozyme folding (8,9). In this report, we describe the combined application of modular rational design and in vitro selection techniques that provide an effective strategy for the rapid generation of precision molecular switches made of RNA.
MATERIALS AND METHODSOligonucleotides. Synthetic DNA and the 14-nt substrate RNA were prepared by standard solid phase methods (Keck Biotechnology Resource Laboratory, Yale University) and purified by denaturing (8 M urea) PAGE as described (5).RNA substrate was 5Ј 32 P-labeled with T4 polynucleotide kinase and [␥-32 P]ATP, and repurified by PAGE. Doublestranded DNA templates for in vitro transcription using T7 RNA polymerase were generated by extension of primer A (5Ј-TAATACGACTCACTATAGGGCGACCCTGATGAG) on a D...
“…The reward for acquiring this capability is substantial, considering that many applications in medicine, industry, and biotechnology demand high-speed enzymes with precisely tailored catalytic functions. ''Modular rational design'' has proven to be an effective means for conferring additional chemical and kinetic complexity on existing protein (1)(2)(3)(4) and RNA enzymes (5)(6)(7)(8)(9). This engineering strategy takes advantage of the modular nature of many protein (10) and RNA subdomains (11)(12)(13), which can be judiciously integrated to form new multifunctional constructs.…”
Ligand-specific molecular switches composed of RNA were created by coupling preexisting catalytic and receptor domains via structural bridges. Binding of ligand to the receptor triggers a conformational change within the bridge, and this structural reorganization dictates the activity of the adjoining ribozyme. The modular nature of these tripartite constructs makes possible the rapid construction of precision RNA molecular switches that trigger only in the presence of their corresponding ligand. By using similar enzyme engineering strategies, new RNA switches can be made to operate as designer molecular sensors or as a new class of genetic control elements.Mastery of the molecular forces that dictate biopolymer folding and function would allow molecular engineers to participate in the design of enzymes-a task that to date has been managed largely by the random processes of evolution. The reward for acquiring this capability is substantial, considering that many applications in medicine, industry, and biotechnology demand high-speed enzymes with precisely tailored catalytic functions. ''Modular rational design'' has proven to be an effective means for conferring additional chemical and kinetic complexity on existing protein (1-4) and RNA enzymes (5-9). This engineering strategy takes advantage of the modular nature of many protein (10) and RNA subdomains (11-13), which can be judiciously integrated to form new multifunctional constructs. The recent discoveries of new catalytic RNA motifs (14, 15) and new ligand-binding motifs (16, 17) have considerably expanded the opportunities for ribozyme engineering.We have used modular rational design to create several artificial ribozymes that are activated or deactivated by the binding of specific small organic molecules such as ATP (5, 8) and flavin mononucleotide (FMN) (unpublished data). Each of these allosteric † ribozymes is composed of two independent structural domains: one an RNA-cleaving ribozyme and the other a receptor (or ''aptamer'') for a specific ligand. The conformational changes that occur within an aptamer domain on introduction of the ligand, termed ''adaptive binding '' (22-25), can trigger kinetic modulation of the adjoining catalytic domain by several different mechanisms that ultimately influence ribozyme folding (8,9). In this report, we describe the combined application of modular rational design and in vitro selection techniques that provide an effective strategy for the rapid generation of precision molecular switches made of RNA.
MATERIALS AND METHODSOligonucleotides. Synthetic DNA and the 14-nt substrate RNA were prepared by standard solid phase methods (Keck Biotechnology Resource Laboratory, Yale University) and purified by denaturing (8 M urea) PAGE as described (5).RNA substrate was 5Ј 32 P-labeled with T4 polynucleotide kinase and [␥-32 P]ATP, and repurified by PAGE. Doublestranded DNA templates for in vitro transcription using T7 RNA polymerase were generated by extension of primer A (5Ј-TAATACGACTCACTATAGGGCGACCCTGATGAG) on a D...
“…Allosteric regulation has been imposed onto the hammerhead (Kertsburg and Soukup 2002), HDV (Kertsburg and Soukup 2002), hairpin , Tetrahymena group I intron (Kertsburg and Soukup 2002), and X-motif ribozymes (Kertsburg and Soukup 2002). The activity-modulating ligands include ATP (Tang and Breaker 1997;Robertson and Ellington 2000), theophylline (Soukup and Breaker 1999a;Robertson and Ellington 2000;Soukup et al 2000), flavin mononucleotide (FMN; Araki et al 1998Araki et al , 2001Breaker 1999a, 1999b;Robertson and Ellington 2000), cyclic nucleotide monophosphates (cNMPs; Koizumi et al 1999), doxycycline (Piganeau et al 2000), 3-methylxanthine (Soukup et al 2000), and pefloxacin (Piganeau et al 2001), as well as various metal ions (Seetharaman et al 2001), oligonucleotides (Porta and Lizardi 1995;Kuwabara et al 1998;Robertson and Ellington 1999;Komatsu et al 2000), and several proteins Vaish et al 2002). Allosteric regulation has also been extended to DNA enzymes that are activated by ATP (Levy and Ellington 2002).…”
Section: Allosteric Ribozyme Sensors (Aptazymes) and General "Communimentioning
Switches and sensors play important roles in our everyday lives. The chemical properties of RNA make it amenable for use as a switch or sensor, both artificially and in nature. This review focuses on recent advances in artificial RNA switches and sensors. Researchers have been applying classical biochemical principles such as allostery in elegant ways that are influencing the development of biosensors and other applications. Particular attention is given here to allosteric ribozymes (aptazymes) that are regulated by small organic molecules, by proteins, or by oligonucleotides. Also discussed are ribozymes whose activities are controlled by various nonallosteric strategies.
“…These latter artificial regulators are called allosteric ribozymes, or aptazymes. To date, several aptazymes have been constructed using hammerhead ribozyme for various types of ligands, including ATP (Tang and Breaker 1997), FMN (Araki et al 1998;Soukup and Breaker 1999a), theophylline (Soukup and Breaker 1999a;Wieland and Hartig 2008), cyclic nucleotide monophosphates (Koizumi et al 1999), and thiamine pyrophosphate (TPP) (Wieland et al 2009). These aptazymes have been used for a wide range of applications, such as 5 artificial gene regulation in vivo (Kumar et al 2009;Carothers et al 2011) and as biosensors in vitro (Breaker 2002;Hesselberth et al 2003;Ogawa and Maeda 2007).…”
Aptazymes are useful as RNA-based switches of gene expression responsive to several types of compounds. One of the most important properties of the switching ability is the signal/noise (S/N) ratio, i.e., the ratio of gene expression in the presence of ligand to that in the absence of ligand. The present study was performed to gain a quantitative understanding of how the aptazyme S/N ratio is determined by factors involved in gene expression, such as transcription, RNA self-cleavage, RNA degradation, protein translation, and their ligand dependencies. We performed switching of gene expression using two onswitch aptazymes with different properties in a cell-free translation system, and constructed a kinetic model that quantitatively describes the dynamics of RNA and protein species involved in switching. Both theoretical and experimental analyses consistently demonstrated that factors determining both the absolute value and the dynamics of the S/N ratio are highly dependent on the routes of translation in the absence of ligand: translation from the ligand-independently cleaved RNA or leaky translation from the noncleaved RNA. The model obtained here is useful to assess the factors that restrict the S/N ratio and to improve aptazymes more efficiently.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.