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ScienceDirect Ribozymes that can be regulated by external stimuli Jennifer Frommer, Bettina Appel and Sabine Mu¨ller Ribozymes have been known for about 30 years, and nowadays are understood well enough to be turned into useful tools for a number of applications in vitro and in vivo. Allosteric ribozymes switch on and off their activity in response to a specific chemical (ligand) or physical (temperature, light) signal. The possibility of controlling ribozyme activity by external stimuli is of particular relevance for applications in different fields, such as environmental and medicinal diagnostics, molecular computing, control of gene expression and others. Herein, we review recent advances and describe selected examples of addressable ribozymes. Addresses Ernst Moritz Arndt University Greifswald, Institute for Biochemistry, Felix Hausdorff Str. 4, D-17487 Greifswald, Germany Corresponding author: Mu¨ller, Sabine ([email protected])
Current Opinion in Biotechnology 2015, 31:35–41 This review comes from a themed issue on Analytical biotechnology Edited by Hadley D Sikes and Nicola Zamboni
http://dx.doi.org/10.1016/j.copbio.2014.07.009 0958-1669/# 2014 Elsevier Ltd. All right reserved.
Introduction Since the discovery of catalytic RNA in 1982 [1], ribozymes have become important tools in molecular biology and medicine, in environmental diagnostics [2] and as nano materials [3]. Several catalytic RNAs were discovered in nature [4], and an even larger number of ribozymes were developed by in vitro selection and/or rational design [5,6]. Over the past 35 years, the structure and mechanism of ribozymes have been extensively studied, and by now RNA catalysis is understood to a level that allows engineering of custom-designed ribozymes for a defined purpose. As their protein counterparts, ribozymes can be activity regulated by external co-factors. In a pioneering work by Tang and Breaker, the first allosteric ribozyme was designed in the test tube [7]. The hammerhead ribozyme was connected to the ATP-binding aptamer, thus constituting an aptazyme, whose activity is dependent on the binding of ATP to the aptamer domain. An aptazyme consists of three functional components: an actuator comprising a ribozyme, a sensor, comprising an RNA aptamer and a communication module, comprising a sequence that transmits information between sensors www.sciencedirect.com
and actuator (Figure 1a). Nowadays, regulation of ribozyme activity by external effectors is a well-established principle that is used to temporarily and spatially regulate the activity of ribozymes, or to detect and sense defined analytes for diagnostic purposes. The general principle relies on a conformational change of the aptazyme triggered by an external stimulus and resulting in altered activity. In this scenario, the communication module linking the aptamer and the ribozyme is of utmost importance. The way of how the two components are linked determines the efficiency of aptazyme operation, and decides whether ligand binding induces or represses ribozyme activity. The modular nature of aptazymes allows for rational design, the aptamer, communication module and ribozyme domain being interchangeable among various aptazymes. However, usually for a defined system the communication module needs to be optimized to operate in a defined way. In addition to rational design and optimization, suitable linker sequences have been identified by in vitro selection (SELEX) [8] using randomized sequences that connect aptamer and ribozyme domain. The mechanism by which ligand binding is coupled to ribozyme activity is often not known in detail, but usually is associated with stabilization (inducer) or destabilization (repressor) of the catalytic domain. In addition to small organic molecules, also oligonucleotides, proteins or other biomolecules can act as ligands. Furthermore, temperature or light can be used as input to trigger the conformational change and to alter ribozyme activity. Over the past 15 years, excellent summaries of progress in the field of ribozyme activity regulation by external stimuli have appeared [9–13]. In this review, we focus on recent advances, and describe the design and the function mode of selected addressable ribozymes.
True allosteric ribozymes As mentioned above, the first small ligand that was used for regulation of ribozyme activity was ATP [7], and many others followed [reviewed in [9]. Initially, this type of allosteric regulation was intended to be used mainly for diagnostic purposes. In this case, the analyte of interest was required to act as ligand for the aptazyme. In addition to the hammerhead ribozyme [14] other nucleic acid enzymes as for example the L1 ligase ribozyme [15] or DNAzymes [16] have been employed as actuators in aptazymes. By coupling ligand dependent ribozyme activation or inactivation to a suitable readout, as for example a fluorescence reporter, convenient biosensor assays were established [13]. In most of these systems, the ligand induced activity enhancement is between 10-fold and 103-fold, and the sensitivity reaches the nanomolar range. Current Opinion in Biotechnology 2015, 31:35–41
36 Analytical biotechnology
Figure 1
(a) Allosteric regulation 5′
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Modes of regulation of ribozyme activity by external stimuli. Exemplary the hammerhead ribozyme activity is shown as catalytic unit in all constructs. Cleavage can occur in cis or trans. Owing to the modular design of the devices, the hammerhead ribozyme can be easily replaced by other catalytic units. Apart from (e), ON switches are shown in all examples. Dependent on the specific design, the devices can also operate as OFF switches. (a) Allosteric regulation. An aptazyme consists of three functional units: a sensor, comprising an RNA aptamer, an actuator comprising a ribozyme, and a communication module, comprising a sequence that transmits information between sensor and actuator. In the shown example, a specific ligand binds to the sensor. The binding event is transmitted via a structural change in the communication module to the actuator, and activity is switched ON. (b) Regulation by oligo effector. The ribozyme is trapped in an inactive conformation. Addition of an oligo effector restores the functional conformation, and activity is switched ON. (c) Regulation by light. Essential functional groups of the ribozyme are protected with photolabile groups preventing the required functional fold. Irradiation with light leads to removal of all labile groups and allows the ribozyme to fold into the required conformation for activity. (d) Ligand activation by light. A photolabile group protects the ligand and prevents binding to the aptamer. Irradiation with light removes the labile group rendering the ligand competent for binding and ribozyme activation. (e) Regulation by temperature. A thermosensitive hairpin is appended to the ribozyme. A temperature increase melts the hairpin stem. As a result activity is switched OFF.
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Ribozyme activity control Frommer, Appel and Mu¨ller 37
There is still much interest in developing aptazymes as biosensors for environmental monitoring [reviewed in [2]] or medicinal diagnostic [17]. However, more recently significant efforts have been directed towards the construction of ribozyme-based devices for modulation of gene expression. An integral part of these devices is that they are capable of responding to a defined chemical signal. The gene-regulatory activity depends on the relative partitioning of the RNA device between two functional conformations, one of them being a ribozyme-active state, the other a ribozyme-inactive state (Figure 2). Dependent on the specific design, ligand binding can support the ribozyme-active or the ribozyme-inactive conformation, thus deciding on downstream events. In many of those systems, initially theophylline acted as the chemical signal that triggers ribozyme activity. The theophylline aptamer was developed in 1994 [18], and short after the first theophylline responsive hammerhead ribozyme was developed by rational design [8]. For the construction of ribozymebased gene-regulatory devices, it was particularly important to use high-speed hammerhead ribozymes. Based on fast-cleaving hammerhead formats with stem I/II contacts [19], Breaker and colleagues developed, by in vitro selection, hammerhead ribozyme variants that are allosterically regulated by theophylline at physiological concentrations of magnesium ions [20]. In further studies, theophyllineresponsive hammerhead ribozyme variants were placed into the 50 -untranslated or 30 -untranslated region (UTR) of prokaryotic and eukaryotic mRNA to regulate translation by ligand-dependent mRNA cleavage [21]. The modularity of the approach enables easy reprogramming of ligand selectivity. Thus, in addition to theophylline, other ligands, as for example guanine, thiamine pyrophosphate or aminoglycosides were used with hammerhead ribozyme constructs, and post-transcriptional regulation of gene expression by control of mRNA [21,22,23–25], tRNA [26,27] or rRNA [28] functions was demonstrated. In addition to the hammerhead ribozyme other selfcleaving RNA structures were used. A recent example is a theophylline and guanine responsive hepatitis delta virus ribozyme that was shown to regulate cis-gene expression up to 29.5-fold in mammalian cell culture [29]. In addition to cis-acting allosteric ribozymes also trans-gene switches have been developed. Theophylline and guanine-dependent hammerhead ribozymes were designed in a way that activation by the ligand unmasks a microRNA precursor analog that silences a separately transcribed mRNA [25,30]. The ligand-dependent regulation of ribozyme activity also allows for screening purposes for example when intracellular activity of a certain enzyme is analyzed. A possible scenario is that the product of an enzyme reaction serves as ligand for the ribozyme, which for its part controls translation of a reporter protein [31]. Furthermore, ligand-dependent self-cleaving ribozymes have www.sciencedirect.com
also been customized for regulation of DNA and RNA viruses [32,33]. A theophylline-responsive hammerhead ribozyme was inserted into the adenoviral immediate early gene E1A, enabling small-molecule triggered dose-dependent inhibition of gene expression. As a result, adenoviral genome replication as well as infectious particle production and cytotoxicity/oncolysis were inhibited. Similarly, knockdown of a measles virus structural gene with the result of reduction of progeny infectivity and virus spread was achieved [33,34]. A series of allosteric ribozymes that respond to the bacterial second messenger cyclic diguanosyl-50 -monophosphate (c-di-GMP) were developed by Breaker and colleagues [35,36]. Initially, a group I self-splicing ribozyme responding to c-di-GMP was placed into the 50 UTR of the mRNA for a putative virulence gene in the pathogenic bacteria Clostridium difficile. Binding of c-diGMP induced conformational changes at splice site junctions and thus alternated further RNA processing [35]. In another approach, the hammerhead ribozyme was joined via a random-sequence bridge to an aptamer from the natural c-di-GMP riboswitch, and functional constructs were identified by in vitro selection. Representative candidates exhibited quite low EC50 (half-max. effective conc.) and IC50 (half-max. inhibitory conc.) values for cdi-GMP, which makes them potential tools for monitoring of c-di-GMP levels in bacteria or in other complex biological or chemical samples [36]. Another interesting example of ligand dependent ribozyme regulation is the application of an allosteric ribozyme in a self-sustained RNA replication system [37,38]. The theophylline aptamer was linked to an RNA enzyme that catalyzes the ligation of two RNA substrates producing another RNA enzyme that undergoes self-replication. The concentration of the ligand is critical for the time needed to reach a threshold concentration of replication products. By replacing the theophylline aptamer with other aptamers for various ligands, and a standardized plot of time to threshold versus ligand concentration can be used for quantitative detection of the ligand in an unknown sample [37]. Yet another area is the construction of aptazyme-based molecular devices and logic gates with potential applications in the fields of molecular computing and synthetic biology [39–41].
Regulation by oligonucleotides In addition to allosteric ligands described above, intermolecularly associating oligonucleotides have been used as effectors to modulate ribozyme activity (reviewed in [9] and [42]). Therein, the strategy often does not follow the classical concept of allosteric regulation, but rather employs the principle of noncompetitive inhibition. It involves incorporation of an ‘attenuator’ strand appended to the sequence of a defined ribozyme (Figure 1b). The attenuator is designed to bind to a critical sequence of the Current Opinion in Biotechnology 2015, 31:35–41
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5′ RBS
RBS
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Figure 2
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gene expression ON Current Opinion in Biotechnology
A ribozyme-based ON switch. The ribozyme-based device residing in the 50 -UTR adopts two distinct conformations. In the absence of a specific ligand, the ribosome binding site (RBS) is sequestered in a double-stranded conformation. Translation cannot take place, gene expression is switched off. Binding of the ligand to the aptamer induces a structural change that shifts the ribozyme conformation to the active state. Ribozyme-mediated cleavage sets the RBS free, resulting in translation initiation and expression of downstream genes.
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Ribozyme activity control Frommer, Appel and Mu¨ller 39
ribozyme’s catalytic core, thus inhibiting activity. Addition of an exogenous oligonucleotide effector that binds to the attenuator disrupts attenuator binding to the ribozyme and thus up-regulates activity [43,44]. In addition to inducible ribozymes, also variants that are repressed by an external oligonucleotide effector, were generated [44]. A related recent example is an engineered hammerhead ribozyme that allows sensing of small transacting RNAs (taRNAs) in E. coli by control of translation initiation [45]. The ribozyme resides in the 50 -untranslated region of a chosen reporter mRNA in a manner that the ribosome binding site is blocked. Autocatalytic cleavage of the ribozyme would uncover the ribosome binding site such that translation can occur. In the presence of a specific trans-acting RNA (taRNA) sequence, cleavage is inhibited due to binding of the taRNA to the taRNA responsive element within the ribozyme. As a result, the ribosome binding site remains block and translation is repressed. In addition to the hammerhead [43] and hairpin ribozyme [44], activity of the hepatitis delta virus ribozyme was modulated with a series of rationally designed aptamers and effector oligonucleotides in vitro and in mammalian cells [46]. A short hairpin structure was appended to the ribozyme, repressing ribozyme activity. An added oligonucleotide effector that binds to the hairpin structure was shown to restore ribozyme activity in a concentration-dependent manner. In general, oligonucleotide-controlled ribozymes offer the detection of specific DNA or RNA sequences and thus may be used for example for monitoring of cellular mRNA expression. Recently, a computational method for selection of allosteric ribozymes that sense a specific sequence of human telomerase reverse transcriptase mRNAs was described [47]. Experimental validation of one of the selected ribozymes revealed fast self-cleavage and high selectivity for the oligonucleotide effector. Importantly, the algorithm is universal, allowing the design of allosteric ribozymes to sense any specific RNA or DNA of interest.
Regulation by light An elegant way of RNA activity control is masking the function of ribozymes by photolabile groups (Figure 1c). This has been applied in the past to impair catalysis of RNA or DNA enzymes that process nucleic acid substrates, and more recently also to the Diels-Alderase ribozyme [48]. (S)-1-(2-nitrophenyl)ethyl (NPE) groups were specifically incorporated at crucial positions within the RNA structure, thus inhibiting activity. Photolysis by irradiation at 355 nm removed the NPE groups and restored activity. In an alternative scenario, the binding of photoactive ligands that induce or repress activity of self-cleaving hammerhead ribozymes was controlled with light [49,50] (Figure 1d). For example, caged toyocamycin www.sciencedirect.com
was used in conjunction with a toyocamycin-repressible hammerhead ribozyme for light-induced gene expression. Photolysis of the caging groups generates toyocamycin that upon binding to the ribozyme inhibits selfcleavage and thus induces expression of an open reading frame [49]. In another example, an aptamer specific for binding one but not the other of two light induced isomers of a dihydropyrene derivative was joined with the hammerhead ribozyme, such that self-cleavage became controllable by irradiation with visible versus ultraviolet light [50]. A similar scenario was used with a flavine mononucleotide (FMN) responsive hairpin ribozyme, where ligand binding was controlled not by light, but by the FMN oxidation state [51].
Regulation by temperature A highly interesting example of a ribozyme that can be regulated by temperature (Figure 1e) is a hammerhead construct fused to a natural RNA thermometer from Salmonella [52]. When located in the 50 -UTR of a reporter gene, the fusion construct allows hammerhead ribozyme self-cleavage activity with the result of liberating the ribosome binding site and permitting gene expression. The thermometer element melts with rising temperature, thereby impairing ribozyme activity and switching off translation.
Conclusions Over the years, a variety of ribozymes that are regulated by external stimuli have been developed. Initial work was focused on proof-of principle, but soon turned into application directed studies. The combination of aptamers with ribozymes to aptazymes in a modular way has allowed the design of aptasensors to be applied in medical and environmental diagnostics. The modular design of aptazymes enables easy reprogramming of ligand selectivity, such that a variety of analytes can be sensed by the same basic platform. Furthermore, robust systems for control of gene expression in bacteria and eukaryotes have been developed by integrating aptazymes in the 50 -UTRs, or 30 -UTRs of mRNAs, or by appending them to functional RNA species. A critical parameter is the communication module linking the aptamer domain (sensor) with the catalytic domain (actuator), because this sequence is required to translate ligand binding into ribozyme activity. Meanwhile several communication modules are available from the literature, or alternatively, can be selected from aptazyme libraries with randomized linking sequences. Very importantly, bioinformatics has developed to a level that allows design of allosteric ribozymes to sense a specific ligand by universal algorithms [53]. The potential of switchable ribozymes for diagnostic and therapeutic application as well as for the design of complex circuits in the field of synthetic biology is obvious. There is much to be anticipated. Current Opinion in Biotechnology 2015, 31:35–41
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Acknowledgement S.M. acknowledges financial support by the Deutsche Forschungsgemeinschaft (Mu1396/9-11).
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53. Penchovsky R: Computational design of allosteric ribozymes as molecular biosensors. Biotechnol Adv 2014. [Ahead of print]. The paper describes diverse computational methods for designing ligandresponsive ribozymes that sense oligonucleotides or small molecules and discusses methods for synthesis and testing of allosteric ribozymes.
Current Opinion in Biotechnology 2015, 31:35–41
ScienceDirect Ribozymes that can be regulated by external stimuli Jennifer Frommer, Bettina Appel and Sabine Mu¨ller Ribozymes have been known for about 30 years, and nowadays are understood well enough to be turned into useful tools for a number of applications in vitro and in vivo. Allosteric ribozymes switch on and off their activity in response to a specific chemical (ligand) or physical (temperature, light) signal. The possibility of controlling ribozyme activity by external stimuli is of particular relevance for applications in different fields, such as environmental and medicinal diagnostics, molecular computing, control of gene expression and others. Herein, we review recent advances and describe selected examples of addressable ribozymes. Addresses Ernst Moritz Arndt University Greifswald, Institute for Biochemistry, Felix Hausdorff Str. 4, D-17487 Greifswald, Germany Corresponding author: Mu¨ller, Sabine ([email protected])
Current Opinion in Biotechnology 2015, 31:35–41 This review comes from a themed issue on Analytical biotechnology Edited by Hadley D Sikes and Nicola Zamboni
http://dx.doi.org/10.1016/j.copbio.2014.07.009 0958-1669/# 2014 Elsevier Ltd. All right reserved.
Introduction Since the discovery of catalytic RNA in 1982 [1], ribozymes have become important tools in molecular biology and medicine, in environmental diagnostics [2] and as nano materials [3]. Several catalytic RNAs were discovered in nature [4], and an even larger number of ribozymes were developed by in vitro selection and/or rational design [5,6]. Over the past 35 years, the structure and mechanism of ribozymes have been extensively studied, and by now RNA catalysis is understood to a level that allows engineering of custom-designed ribozymes for a defined purpose. As their protein counterparts, ribozymes can be activity regulated by external co-factors. In a pioneering work by Tang and Breaker, the first allosteric ribozyme was designed in the test tube [7]. The hammerhead ribozyme was connected to the ATP-binding aptamer, thus constituting an aptazyme, whose activity is dependent on the binding of ATP to the aptamer domain. An aptazyme consists of three functional components: an actuator comprising a ribozyme, a sensor, comprising an RNA aptamer and a communication module, comprising a sequence that transmits information between sensors www.sciencedirect.com
and actuator (Figure 1a). Nowadays, regulation of ribozyme activity by external effectors is a well-established principle that is used to temporarily and spatially regulate the activity of ribozymes, or to detect and sense defined analytes for diagnostic purposes. The general principle relies on a conformational change of the aptazyme triggered by an external stimulus and resulting in altered activity. In this scenario, the communication module linking the aptamer and the ribozyme is of utmost importance. The way of how the two components are linked determines the efficiency of aptazyme operation, and decides whether ligand binding induces or represses ribozyme activity. The modular nature of aptazymes allows for rational design, the aptamer, communication module and ribozyme domain being interchangeable among various aptazymes. However, usually for a defined system the communication module needs to be optimized to operate in a defined way. In addition to rational design and optimization, suitable linker sequences have been identified by in vitro selection (SELEX) [8] using randomized sequences that connect aptamer and ribozyme domain. The mechanism by which ligand binding is coupled to ribozyme activity is often not known in detail, but usually is associated with stabilization (inducer) or destabilization (repressor) of the catalytic domain. In addition to small organic molecules, also oligonucleotides, proteins or other biomolecules can act as ligands. Furthermore, temperature or light can be used as input to trigger the conformational change and to alter ribozyme activity. Over the past 15 years, excellent summaries of progress in the field of ribozyme activity regulation by external stimuli have appeared [9–13]. In this review, we focus on recent advances, and describe the design and the function mode of selected addressable ribozymes.
True allosteric ribozymes As mentioned above, the first small ligand that was used for regulation of ribozyme activity was ATP [7], and many others followed [reviewed in [9]. Initially, this type of allosteric regulation was intended to be used mainly for diagnostic purposes. In this case, the analyte of interest was required to act as ligand for the aptazyme. In addition to the hammerhead ribozyme [14] other nucleic acid enzymes as for example the L1 ligase ribozyme [15] or DNAzymes [16] have been employed as actuators in aptazymes. By coupling ligand dependent ribozyme activation or inactivation to a suitable readout, as for example a fluorescence reporter, convenient biosensor assays were established [13]. In most of these systems, the ligand induced activity enhancement is between 10-fold and 103-fold, and the sensitivity reaches the nanomolar range. Current Opinion in Biotechnology 2015, 31:35–41
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Figure 1
(a) Allosteric regulation 5′
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Modes of regulation of ribozyme activity by external stimuli. Exemplary the hammerhead ribozyme activity is shown as catalytic unit in all constructs. Cleavage can occur in cis or trans. Owing to the modular design of the devices, the hammerhead ribozyme can be easily replaced by other catalytic units. Apart from (e), ON switches are shown in all examples. Dependent on the specific design, the devices can also operate as OFF switches. (a) Allosteric regulation. An aptazyme consists of three functional units: a sensor, comprising an RNA aptamer, an actuator comprising a ribozyme, and a communication module, comprising a sequence that transmits information between sensor and actuator. In the shown example, a specific ligand binds to the sensor. The binding event is transmitted via a structural change in the communication module to the actuator, and activity is switched ON. (b) Regulation by oligo effector. The ribozyme is trapped in an inactive conformation. Addition of an oligo effector restores the functional conformation, and activity is switched ON. (c) Regulation by light. Essential functional groups of the ribozyme are protected with photolabile groups preventing the required functional fold. Irradiation with light leads to removal of all labile groups and allows the ribozyme to fold into the required conformation for activity. (d) Ligand activation by light. A photolabile group protects the ligand and prevents binding to the aptamer. Irradiation with light removes the labile group rendering the ligand competent for binding and ribozyme activation. (e) Regulation by temperature. A thermosensitive hairpin is appended to the ribozyme. A temperature increase melts the hairpin stem. As a result activity is switched OFF.
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Ribozyme activity control Frommer, Appel and Mu¨ller 37
There is still much interest in developing aptazymes as biosensors for environmental monitoring [reviewed in [2]] or medicinal diagnostic [17]. However, more recently significant efforts have been directed towards the construction of ribozyme-based devices for modulation of gene expression. An integral part of these devices is that they are capable of responding to a defined chemical signal. The gene-regulatory activity depends on the relative partitioning of the RNA device between two functional conformations, one of them being a ribozyme-active state, the other a ribozyme-inactive state (Figure 2). Dependent on the specific design, ligand binding can support the ribozyme-active or the ribozyme-inactive conformation, thus deciding on downstream events. In many of those systems, initially theophylline acted as the chemical signal that triggers ribozyme activity. The theophylline aptamer was developed in 1994 [18], and short after the first theophylline responsive hammerhead ribozyme was developed by rational design [8]. For the construction of ribozymebased gene-regulatory devices, it was particularly important to use high-speed hammerhead ribozymes. Based on fast-cleaving hammerhead formats with stem I/II contacts [19], Breaker and colleagues developed, by in vitro selection, hammerhead ribozyme variants that are allosterically regulated by theophylline at physiological concentrations of magnesium ions [20]. In further studies, theophyllineresponsive hammerhead ribozyme variants were placed into the 50 -untranslated or 30 -untranslated region (UTR) of prokaryotic and eukaryotic mRNA to regulate translation by ligand-dependent mRNA cleavage [21]. The modularity of the approach enables easy reprogramming of ligand selectivity. Thus, in addition to theophylline, other ligands, as for example guanine, thiamine pyrophosphate or aminoglycosides were used with hammerhead ribozyme constructs, and post-transcriptional regulation of gene expression by control of mRNA [21,22,23–25], tRNA [26,27] or rRNA [28] functions was demonstrated. In addition to the hammerhead ribozyme other selfcleaving RNA structures were used. A recent example is a theophylline and guanine responsive hepatitis delta virus ribozyme that was shown to regulate cis-gene expression up to 29.5-fold in mammalian cell culture [29]. In addition to cis-acting allosteric ribozymes also trans-gene switches have been developed. Theophylline and guanine-dependent hammerhead ribozymes were designed in a way that activation by the ligand unmasks a microRNA precursor analog that silences a separately transcribed mRNA [25,30]. The ligand-dependent regulation of ribozyme activity also allows for screening purposes for example when intracellular activity of a certain enzyme is analyzed. A possible scenario is that the product of an enzyme reaction serves as ligand for the ribozyme, which for its part controls translation of a reporter protein [31]. Furthermore, ligand-dependent self-cleaving ribozymes have www.sciencedirect.com
also been customized for regulation of DNA and RNA viruses [32,33]. A theophylline-responsive hammerhead ribozyme was inserted into the adenoviral immediate early gene E1A, enabling small-molecule triggered dose-dependent inhibition of gene expression. As a result, adenoviral genome replication as well as infectious particle production and cytotoxicity/oncolysis were inhibited. Similarly, knockdown of a measles virus structural gene with the result of reduction of progeny infectivity and virus spread was achieved [33,34]. A series of allosteric ribozymes that respond to the bacterial second messenger cyclic diguanosyl-50 -monophosphate (c-di-GMP) were developed by Breaker and colleagues [35,36]. Initially, a group I self-splicing ribozyme responding to c-di-GMP was placed into the 50 UTR of the mRNA for a putative virulence gene in the pathogenic bacteria Clostridium difficile. Binding of c-diGMP induced conformational changes at splice site junctions and thus alternated further RNA processing [35]. In another approach, the hammerhead ribozyme was joined via a random-sequence bridge to an aptamer from the natural c-di-GMP riboswitch, and functional constructs were identified by in vitro selection. Representative candidates exhibited quite low EC50 (half-max. effective conc.) and IC50 (half-max. inhibitory conc.) values for cdi-GMP, which makes them potential tools for monitoring of c-di-GMP levels in bacteria or in other complex biological or chemical samples [36]. Another interesting example of ligand dependent ribozyme regulation is the application of an allosteric ribozyme in a self-sustained RNA replication system [37,38]. The theophylline aptamer was linked to an RNA enzyme that catalyzes the ligation of two RNA substrates producing another RNA enzyme that undergoes self-replication. The concentration of the ligand is critical for the time needed to reach a threshold concentration of replication products. By replacing the theophylline aptamer with other aptamers for various ligands, and a standardized plot of time to threshold versus ligand concentration can be used for quantitative detection of the ligand in an unknown sample [37]. Yet another area is the construction of aptazyme-based molecular devices and logic gates with potential applications in the fields of molecular computing and synthetic biology [39–41].
Regulation by oligonucleotides In addition to allosteric ligands described above, intermolecularly associating oligonucleotides have been used as effectors to modulate ribozyme activity (reviewed in [9] and [42]). Therein, the strategy often does not follow the classical concept of allosteric regulation, but rather employs the principle of noncompetitive inhibition. It involves incorporation of an ‘attenuator’ strand appended to the sequence of a defined ribozyme (Figure 1b). The attenuator is designed to bind to a critical sequence of the Current Opinion in Biotechnology 2015, 31:35–41
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5′ RBS
RBS
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Figure 2
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gene expression ON Current Opinion in Biotechnology
A ribozyme-based ON switch. The ribozyme-based device residing in the 50 -UTR adopts two distinct conformations. In the absence of a specific ligand, the ribosome binding site (RBS) is sequestered in a double-stranded conformation. Translation cannot take place, gene expression is switched off. Binding of the ligand to the aptamer induces a structural change that shifts the ribozyme conformation to the active state. Ribozyme-mediated cleavage sets the RBS free, resulting in translation initiation and expression of downstream genes.
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Ribozyme activity control Frommer, Appel and Mu¨ller 39
ribozyme’s catalytic core, thus inhibiting activity. Addition of an exogenous oligonucleotide effector that binds to the attenuator disrupts attenuator binding to the ribozyme and thus up-regulates activity [43,44]. In addition to inducible ribozymes, also variants that are repressed by an external oligonucleotide effector, were generated [44]. A related recent example is an engineered hammerhead ribozyme that allows sensing of small transacting RNAs (taRNAs) in E. coli by control of translation initiation [45]. The ribozyme resides in the 50 -untranslated region of a chosen reporter mRNA in a manner that the ribosome binding site is blocked. Autocatalytic cleavage of the ribozyme would uncover the ribosome binding site such that translation can occur. In the presence of a specific trans-acting RNA (taRNA) sequence, cleavage is inhibited due to binding of the taRNA to the taRNA responsive element within the ribozyme. As a result, the ribosome binding site remains block and translation is repressed. In addition to the hammerhead [43] and hairpin ribozyme [44], activity of the hepatitis delta virus ribozyme was modulated with a series of rationally designed aptamers and effector oligonucleotides in vitro and in mammalian cells [46]. A short hairpin structure was appended to the ribozyme, repressing ribozyme activity. An added oligonucleotide effector that binds to the hairpin structure was shown to restore ribozyme activity in a concentration-dependent manner. In general, oligonucleotide-controlled ribozymes offer the detection of specific DNA or RNA sequences and thus may be used for example for monitoring of cellular mRNA expression. Recently, a computational method for selection of allosteric ribozymes that sense a specific sequence of human telomerase reverse transcriptase mRNAs was described [47]. Experimental validation of one of the selected ribozymes revealed fast self-cleavage and high selectivity for the oligonucleotide effector. Importantly, the algorithm is universal, allowing the design of allosteric ribozymes to sense any specific RNA or DNA of interest.
Regulation by light An elegant way of RNA activity control is masking the function of ribozymes by photolabile groups (Figure 1c). This has been applied in the past to impair catalysis of RNA or DNA enzymes that process nucleic acid substrates, and more recently also to the Diels-Alderase ribozyme [48]. (S)-1-(2-nitrophenyl)ethyl (NPE) groups were specifically incorporated at crucial positions within the RNA structure, thus inhibiting activity. Photolysis by irradiation at 355 nm removed the NPE groups and restored activity. In an alternative scenario, the binding of photoactive ligands that induce or repress activity of self-cleaving hammerhead ribozymes was controlled with light [49,50] (Figure 1d). For example, caged toyocamycin www.sciencedirect.com
was used in conjunction with a toyocamycin-repressible hammerhead ribozyme for light-induced gene expression. Photolysis of the caging groups generates toyocamycin that upon binding to the ribozyme inhibits selfcleavage and thus induces expression of an open reading frame [49]. In another example, an aptamer specific for binding one but not the other of two light induced isomers of a dihydropyrene derivative was joined with the hammerhead ribozyme, such that self-cleavage became controllable by irradiation with visible versus ultraviolet light [50]. A similar scenario was used with a flavine mononucleotide (FMN) responsive hairpin ribozyme, where ligand binding was controlled not by light, but by the FMN oxidation state [51].
Regulation by temperature A highly interesting example of a ribozyme that can be regulated by temperature (Figure 1e) is a hammerhead construct fused to a natural RNA thermometer from Salmonella [52]. When located in the 50 -UTR of a reporter gene, the fusion construct allows hammerhead ribozyme self-cleavage activity with the result of liberating the ribosome binding site and permitting gene expression. The thermometer element melts with rising temperature, thereby impairing ribozyme activity and switching off translation.
Conclusions Over the years, a variety of ribozymes that are regulated by external stimuli have been developed. Initial work was focused on proof-of principle, but soon turned into application directed studies. The combination of aptamers with ribozymes to aptazymes in a modular way has allowed the design of aptasensors to be applied in medical and environmental diagnostics. The modular design of aptazymes enables easy reprogramming of ligand selectivity, such that a variety of analytes can be sensed by the same basic platform. Furthermore, robust systems for control of gene expression in bacteria and eukaryotes have been developed by integrating aptazymes in the 50 -UTRs, or 30 -UTRs of mRNAs, or by appending them to functional RNA species. A critical parameter is the communication module linking the aptamer domain (sensor) with the catalytic domain (actuator), because this sequence is required to translate ligand binding into ribozyme activity. Meanwhile several communication modules are available from the literature, or alternatively, can be selected from aptazyme libraries with randomized linking sequences. Very importantly, bioinformatics has developed to a level that allows design of allosteric ribozymes to sense a specific ligand by universal algorithms [53]. The potential of switchable ribozymes for diagnostic and therapeutic application as well as for the design of complex circuits in the field of synthetic biology is obvious. There is much to be anticipated. Current Opinion in Biotechnology 2015, 31:35–41
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Acknowledgement S.M. acknowledges financial support by the Deutsche Forschungsgemeinschaft (Mu1396/9-11).
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