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ScienceDirect Riboswitch engineering — making the all-important second and third steps Christian Berens and Beatrix Suess Synthetic biology uses our understanding of biological systems to develop innovative solutions for challenges in fields as diverse as genetic control and logic devices, bioremediation, materials production or diagnostics and therapy in medicine by designing new biological components. RNA-based elements are key components of these engineered systems. Their structural and functional diversity is ideal for generating regulatory riboswitches that react with many different types of output to molecular and environmental signals. Recent advances have added new sensor and output domains to the existing toolbox, and demonstrated the portability of riboswitches to many different organisms. Improvements in riboswitch design and screens for selecting in vivo active switches provide the means to isolate riboswitches with regulatory properties more like their natural counterparts. Addresses Fachbereich Biologie, Technische Universita¨t Darmstadt, Schnittspahnstraße 10, 64287 Darmstadt, Germany Corresponding authors: Berens, Christian ([email protected]) and Suess, Beatrix ([email protected])
Current Opinion in Biotechnology 2014, 31:10–15 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.2014.07.014 0958-1669/# 2014 Published by Elsevier Ltd.
Introduction The thirty years following the discovery of catalytic RNA have seen a veritable radiative burst in our knowledge of the many levels at which RNA molecules operate within cells. Ribozymes, riboswitches, RNA thermometers, a plethora of small RNAs in eukaryotes and bacteria, as well as different types of long non-coding RNAs in eukaryotes demonstrate that RNA appears to be everywhere, and that there is still much to learn about how RNA influences cell physiology, differentiation and development [1]. It sometimes seems that the only limit to what RNA can do or be made to do lies solely within our imagination. RNA as a regulatory molecule has the distinct advantage that it can operate in a protein-independent manner, thereby allowing firstly faster regulatory Current Opinion in Biotechnology 2015, 31:10–15
responses, due to it already having been transcribed, secondly easier transfer of a single-step genetic control element to other organisms, and thirdly flexible combination with different downstream readout platforms for a maximum of regulatory outputs. This makes RNA an attractive target for the development of genetic control elements in gene functional studies, biotechnology and synthetic biology. Its attractiveness is even further enhanced by the addition of riboswitches — small structured RNA elements which regulate gene expression in response to a small-molecule ligand — to the RNA toolbox. Their use allows spatial, temporal and dosage control over gene expression. Riboswitches, discovered about a decade ago, have been the subject of intense scrutiny not only to understand their mechanisms of action [2–4], but also to put them to use in genetic engineering [5,6]. This is probably best exemplified by the more than 100 reviews on riboswitches that have been published so far, as well as by several special editions on ‘Artificial Riboswitches’ (Methods in Molecular Biology, 2014), ‘Riboswitches: new aspects of an old story’ (RNA Biology, 2010) or the two editions from Biochimica et Biophysica Acta — Gene Regulatory Mechanisms on ‘Structure and Function of Regulatory RNA Elements’ (2009) and ‘Riboswitches’ (2014). Here, we cover recent advances in the fields of riboswitch applicability and design, focusing on the time period following publication of the last review on engineered riboswitches in this journal in 2012 [7]. It clearly emphasized the major goal during early riboswitch engineering — to establish a robust foundation of ‘proof of principle’ applications delineating the spectrum of gene regulatory mechanisms that can be addressed with riboswitches (Table 1). Their functionality was mainly investigated in bacteria and yeast, popular model organisms for prokaryotes and eukaryotes. We are now coming to another phase of riboswitch applicability, in which the emphasis on the one hand will be on improving activity, robustness and reliability of riboswitch regulation and on the other hand continuing and intensifying the transfer of riboswitch regulatory systems to mammalian cell culture and maybe even to transgenic animals and plants.
Riboswitch regulation functions in Archaea, diverse bacteria and in viruses Most engineered riboswitches have been tested in the well-characterized model organisms Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae and in mammalian cell lines [2]. However, many other bacteria and organisms www.sciencedirect.com
Engineered riboswitches Berens and Suess 11
Table 1 Proof-of-principle in vivo applications in riboswitch engineering Expression platform
Aptamer domain Theophylline
Translational ON Translational OFF Transcriptional ON Transcriptional OFF Ribozyme Splicing Trans-acting switches
B-[46], B-[47] Y-[48], B-[49] B-[35], B-[36] B-[37] B-[51], Y-[47], C-[49]
Tetracycline Y-[50] B-[36] B-[37] Y-[52], C Y-[54]
a
Others: neomycin, ammeline, 5-aza-cytosine, PPDA B-[23] Y-[22] B-[36] B-[31], B-[37] Y-[53]
B-[55]
The table contains a combinatorial matrix of aptamer domains and expression platforms, which lists the publications implementing the different combinations in bacteria (B), yeast (Y) or in cell culture (C). Citations in bold are novel ‘proof-of-principle’ applications published after the last review on the topic in this Journal [7]. a Conditional control of mammalian gene expression by tetracycline-dependent hammerhead ribozymes: Kim Beilstein, Alexander Wittmann, Manuel Grez, Beatrix Suess, submitted for publication.
also serve as model species for diverse physiological or developmental processes, as pathogens or as potential cell factories in biotechnology. Their characterization requires strictly controlled inducible expression systems to ensure that the products to be analyzed or synthesized are only produced when desired. Recognizing this need, the Gallivan lab initially developed a series of ligandinducible riboswitches, termed A–E, for control of gene expression in diverse species of Gram-negative and Gram-positive bacteria, like the model organisms E. coli, B. subtilis and Mycobacterium smegmatis or the plant and human pathogens Agrobacterium tumefaciens, Acinetobacter baumannii and Streptococcus pyogenes [8]. This organismal palette was then further extended by using theophyllinedependent riboswitches, mostly derived from the abovementioned series, but additionally including the constructs E* [8], F (clone 8.1; [9]) and G (clone D2; [10]), to achieve stringent and dose-dependent control of protein expression in the Cyanobacterium Synechococcus elongatus, a model organism for photosynthesis and a potential producer of biomaterials [11], in Streptomyces coelicolor, a major producer of antibiotics, enzymes, antiviral and anticancer drugs [12], as well as the important human pathogens Francisella tularensis [13] and Mycobacterium tuberculosis [14], but also in tobacco chloroplasts [15], which might be interesting for plant biotechnology, due to high transgene expression levels, and for biosafety reasons. Relevant parameters of riboregulation are shown in Table 2, but several points are still noteworthy. In all organisms tested, riboswitch-mediated regulation was efficient enough to establish a conditional knockout of selected endogenous genes, or even to inducibly control the circadian rhythm in S. elongatus [11]. The maximum regulatory factors obtained in M. smegmatis and S. elongatus were in the same range as those obtained with a transcriptional regulation system under control of the bacterial transcription factor TetR [14,16]. In the medically relevant pathogens F. tularensis and M. tuberculosis, gene expression is also controllable in a macrophage infection model [13,14]. Spraying the tobacco plants with theophylline www.sciencedirect.com
was sufficient to induce reporter gene expression in the plastids [15]. In contrast, a tetracycline-regulated riboswitch served as inducible control element in Methanosarcina acetivorans, an anaerobic model organism from the third superkingdom of life, the Archaea [17]. So far, naturally occurring riboswitches have not been validated experimentally in Archaea, although sequences for fluoride [18] and thiamine pyrophosphate (TPP) [19] controlled riboswitches have been found. It is therefore interesting to see if and how effective riboregulation might be in this organism. A tetracycline-regulated switch with low stability and partial inclusion of the ribosome-binding site (RBS) in the closing stem was the most active variant tested, demonstrating proof-of-principle for riboswitch functionality in Archaea [17]. Some clear tendencies can be derived from these publications. Theophylline is the preferred ligand of choice, as well as translational ON-switches. The former will hardly ever interfere with general host cell physiology, the latter appears more easy to introduce into the sequence of the respective target gene [11]. Nevertheless, the choice of riboswitch will still be affected by target gene and experimental requirements. For example, riboswitch ‘E’ displayed the highest activation factor and reporter gene expression level in F. tularensis, but the lower basal expression obtained with construct ‘F’ was needed to conditionally complement a growth-defective mutant [13]. Similarly, in S. coelicolor, riboswitch ‘A’ appears to be a good choice for tight repression, while riboswitch ‘E*’ would be used when high protein levels are needed [12]. But not only bacterial gene expression can be controlled by engineered riboswitches. Flanking insertions of theophylline-dependent aptazymes enabled ligand-triggered shutdown of immediate early gene expression in DNA viruses (adenovirus, adeno-associated virus, oncolytic adenovirus) and glycoprotein expression in an RNA virus (measles virus), leading to inhibition of viral replication Current Opinion in Biotechnology 2015, 31:10–15
12 Analytical biotechnology
Table 2 Riboswitches as genetic tools in model or potential cell factory organisms Organism Archaea Methanosarcina acetivorans Eubacteria Francisella novicida U112 Francisella tularensis ShuS4 Mycobacterium smegmatis mc2155 Mycobacterium tuberculosis H37Rv Streptomyces coelicolor M145 Synechococcus elongatus PCC7942 Organelle Chloroplast
Phylum
Process regulated
Switchtype
Euryarchaeota
RBS access
OFF
Proteobacteria (g)
Ligand
Regulatory factor
Reporter gene
Reference
Tetracycline (200 mM)
12; C a
b-Lactamase
[17]
RBS access (E, F) b ON
Theophylline (2 mM)
9.5; P a
b-Galactosidase
[13]
Proteobacteria (g)
RBS access (E) b
ON
Theophylline (2 mM)
5; P a
b-Galactosidase
[13]
Actinobacteria
RBS access (E*) b
ON
Theophylline (2–5 mM)
69; P a
GFP
[14]
Actinobacteria
RBS access (E*) b
ON
Theophylline (2–5 mM)
8.2; P a
b-Galactosidase
[14]
Actinobacteria
RBS access (E*) b
ON
Theophylline (4 mM) c
30–260d; C a b-Glucuronidase
[12]
Cyanobacteria
RBS access (E*) b
ON
Theophylline (2 mM) c
190; C a
Luciferase
[11]
RBS access
ON
Theophylline (2.5 mM) c Qualitative only; C a
GFP
[15]
The ligand concentration in brackets corresponds to the concentration required for maximum target protein expression. C: chromosomally encoded; P: plasmid-encoded. b Designation of synthetic riboswitches named and developed by the Gallivan group [8–10]. c Maximum dose applicable, due to toxicity issues. d The different factors were achieved with promoters of different expression strength. a
and infectivity and, ultimately, viral spread and cytotoxicity [20,21]. Taken together, theophylline-controlled and tetracycline-controlled riboswitches permit stringent, dosedependent, reversible and efficient control in DNA and RNA viruses, in Archaea, Bacteria and in organelles from widely diverse taxonomic origins. This regulatory approach, thus, appears to be sufficiently robust for portability to any organism of choice.
Improving riboswitch activity — concepts and their implementation The ‘proof-of-principle’ applications (Table 1 and [7]) and the high degree of functional portability to other organisms (Table 2) foretell a bright future for riboswitches in synthetic biology. However, to tap into the full potential of riboswitch regulation requires that we not only expand the toolbox of riboswitch components, but also improve the performance of the existing types of engineered riboswitches.
Expanding the toolbox — identifying new aptamer domains for riboswitches To date, most engineered riboswitches are equipped with theophylline or tetracycline aptamers (Table 1 and [5]), with only a few examples utilizing an in vivo screened neomycin aptamer [22] or heterocyclic ligands, like ammeline or 5-azacytosine, for a re-engineered Current Opinion in Biotechnology 2015, 31:10–15
adenine-responsive riboswitch [23]. There is therefore an acute need to identify new ligands and aptamer domains to increase the diversity of engineered riboswitches, especially if complex regulatory gates and networks are to be established [24–26]. Unfortunately, despite the demonstrated modularity of riboswitch aptamer domains and expression platforms [2] and despite the sophisticated advanced technology available for in vitro selecting aptamers [27], only a few sequence/ligand pairs have been used so far in vivo [5]. The in vivo active neomycin aptamer, which was highly underrepresented in the original in vitro selected aptamer pool [28], might shed some light on why this is the case. It displays a destabilized, open unbound state which undergoes extensive structural changes upon ligand binding. In contrast, in vivo inactive, but highly represented neomycin aptamers are already pre-formed in the ligand’s absence [29]. Thus, constraints on in vivo activity and in vitro functionality appear to differ considerably, so we have to find in vitro conditions that more closely mimic the in vivo situation. The optimal ligand for an engineered riboswitch should be cheap and easy to produce, be non-toxic and applicable in both prokaryotes and eukaryotes, should not interfere with cell physiology, have good solubility and cell permeability and allow the facile isolation of highly specific aptamers. So far, such a ligand/aptamer combination has not been presented. A new aptamer for an in vivo active www.sciencedirect.com
Engineered riboswitches Berens and Suess 13
riboswitch was presented by Davidson et al. [30]. They isolated a 2,4-dinitrotoluene (DNT) responsive riboswitch in E. coli by placing a 30 nucleotide-long randomized sequence between a DNT aptamer and an RBS, and by screening in vivo for active expression platforms in the presence of DNT. A ten-fold increase in GFP fluorescence was observed with the best candidate [30]. The Micklefield group used a different approach. They mutagenized a natural riboswitch to not respond to its native ligand, adenine, anymore and screened the mutant pool for binding to synthetic, non-natural heterocyclic ligands [23]. This screen yielded a ligand, PPDA (pyrimido[4,5d]pyrimidine-2,4-diamine), that formed a pair with the M600 aptamer with superior regulatory properties [31]. Such re-engineering of natural riboswitches might turn out to be an attractive approach if it is also transferable to other natural riboswitches, especially because many have been and are being subjected to intense drug discovery screens [32–34].
Expanding the toolbox — establishing new expression platforms Inspection of Table 1 reveals quite a few gaps in the matrix. An eye-catching one is the lack of any engineered transcriptionally regulated control elements. This gap has been filled in the meantime. Wachsmuth et al. used an in silico selection process involving multiple constraints and combined it with in vitro and in vivo experiments to design and isolate theophylline-dependent transcriptional ON switches [35]. Half of their constructs regulated reporter gene expression. Further sequence optimization led to a maximum activation of six-fold, which is similar to a naturally occurring transcriptional ON switch [35]. An important observation made was that intermediate terminator stability was important for riboswitch functionality. The Batey group instead preferred a modular ‘mix-and-match’ approach to construct both transcriptional ON [36] and OFF [37] switches. It uses a set of specific expression platforms, which can be uncoupled structurally from their aptamer domain and grafts arbitrary aptamer domains to these. Optimization of the regulatory properties required some sequence adjustments to the expression platforms. An important advantage of this technique is that it does not require a ‘communication module’ to transfer the signal from the aptamer domain to the expression platform, eliminating the time-consuming and laborious mutagenesis and selection experiments previously needed. Robinson et al. used a similar approach to generate a transcriptional OFF switch with their re-engineered M600 adenine aptamer domain [31]. Less than tenfold repression was obtained, due to the fact that a plateau of reporter activity had still not been reached at the maximum PPDA concentration applicable to the cells. A second gap in Table 1 was filled by Ogawa [38]. He brought a rationally modified synthetic internal ribosome entry site (IRES), based on the Plautia stali intestine virus IRES, under the control of aptamers www.sciencedirect.com
against theophylline, tetracycline, FMN and sulforhodamine B, thereby obtaining ON/OFF ratios between five and thirty in vitro [38]. The in vivo activity of these switches remains to be tested.
Improving riboswitch performance Many in vivo active engineered riboswitches suffer from low dynamic regulatory ranges, high background activity in the absence of their cognate ligand and high effector concentrations required for switching. Consequently, the following two questions are among the most important hurdles synthetic riboswitch research has to overcome at the moment: how does ligand binding to the aptamer drive riboswitch function, and how can we better improve the process of aptamer/riboswitch selection? Better understanding the former will aid us in designing more effective aptamer domain/expression platform interfaces, while improving the latter will increase our rather dismal success rate in converting in vitro selected aptamers to in vivo active riboswitches. The most reliable approach to identify in vivo active riboswitches with new and untested aptamer domains is still to perform a genetic in vivo screen of a pool of mutagenized sequence variants of the newly combined riboswitch in the desired or a closely related host organism. This was first impressively demonstrated for in vitro selected neomycin aptamers in the construction of an in vivo active neomycin riboswitch [22] and, more recently, for the DNT riboswitch [30]. A genetic screen was also used to transform a TPP-OFF switch into a TPP-ON switch [39], again highlighting the power of in vivo selection systems. Fusing an aptamer domain to an expression platform will in most cases create a suboptimal interface between the two elements which might be the cause for impaired performance by the resulting riboswitch. Natural sequence variants of riboswitches display a high degree of functional diversity, which is believed to represent ‘tuning’ to the required regulatory response [40]. Stoddard et al. [41] and Weigand et al. [42], accordingly, were able to modulate the regulatory parameters of riboswitches by mutagenizing residues not directly involved in ligand binding. This indicates a high degree of structural and functional plasticity in any given riboswitch, which may have to be optimized to obtain the desired regulatory results. It is tempting to speculate that if we combine the mutagenesis results from aptamer specificity switches [31] with those of riboswitch tuning [41,42], it might even be possible that mutagenesis of more nucleotides in or even entire subsections of a natural riboswitch would yield riboswitches with completely different ligand specificities. Natural riboswitches could, then, serve as established regulatory ‘base camps’ from which new unexpected and unexplored ligand ‘peaks’ might be scaled. Taken together, it appears that a combination of in silico structural analysis and in vivo genetic tuning will be necessary if we want to obtain well-functioning and Current Opinion in Biotechnology 2015, 31:10–15
14 Analytical biotechnology
highly active riboswitches as opposed to just functional riboswitches.
Conclusion Small-molecule controlled regulation of gene expression in synthetic RNA biology has come a long way since its inception in vitro with allosteric ribozymes [43] and in vivo with RNA aptamers against Hoechst33258 [44] approximately fifteen years ago. Numerous ‘proof-of-principle’ applications have been published since then, demonstrating the great diversity of aptamer domains and expression platforms in establishing functional regulatory modules [5]. Currently, remaining gaps in this riboswitch toolbox are being filled with new aptamer domains, new expression modules and combinations thereof. In addition, advanced in vitro and in vivo screening systems and improved in silico and structure-based design approaches have added to our riboswitch engineering principles and methods, so that the riboswitches we generate should more closely resemble their natural counterparts. Distilling the knowledge gained in all of these and in future experiments will create positive feedback and ultimately permit the design of even better tools that should finally allow standardized ‘off the shelf’ components to be utilized in ‘plug-and-play’ approaches [45] for generating synthetic genetic circuits of adjustable complexity, sophisticated biosensors, ‘intelligent’ responsive metabolic pathways and optimized diagnostic and therapeutic tools.
Acknowledgements The authors thank Dr. Julia E. Weigand for helpful suggestions and critical comments. This work was funded by the Deutsche Forschungsgemeinschaft (SFB902 A2) and Lowe CGT.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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41. Stoddard CD, Widmann J, Trausch JJ, Marcano-Vela´zquez JG, Knight R, Batey RT: Nucleotides adjacent to the ligand-binding pocket are linked to activity tuning in the purine riboswitch. J Mol Biol 2013, 425:1596-1611. Integrated bioinformatic, structural, in vitro and in vivo approach to demonstrate that modest sequence alterations in a structurally critical region have a dramatic impact on biological activity. 42. Weigand JE, Gottstein-Schmidtke SR, Demolli S, Groher F, Duchardt-Ferner E, Wo¨hnert J, Suess B: Sequence elements distal to the ligand binding pocket modulate the efficiency of a synthetic riboswitch. ChemBioChem 2014, 15:1627-1637. Structure-based analysis of neomycin riboswitch mutants in a terminal loop that highlights the importance of structure-affecting, non-ligandbinding nucleotides for biological activity of a riboswitch. 43. Soukup GA, Breaker RR: Engineering precision RNA molecular switches. Proc Natl Acad Sci U S A 1999, 96:3584-3589. 44. Werstuck G, Green MR: Controlling gene expression in living cells through small molecule–RNA interactions. Science 1998, 282:296-298. 45. Agapakis CM: Designing synthetic biology. ACS Synth Biol 2014, 3:121-128. 46. Suess B, Fink B, Berens C, Stentz R, Hillen W: A theophylline responsive riboswitch based on helix slipping controls gene expression in vivo. Nucleic Acids Res 2004, 32:1610-1614. 47. Win MN, Smolke CD: A modular and extensible RNA-based gene-regulatory platform for engineering cellular function. Proc Natl Acad Sci U S A 2007, 104:14283-14288. 48. Harvey I, Garneau P, Pelletier J: Inhibition of translation by RNA– small molecule interactions. RNA 2002, 8:452-463. 49. Ausla¨nder S, Ketzer P, Hartig JS: A ligand-dependent hammerhead ribozyme switch for controlling mammalian gene expression. Mol Biosyst 2010, 6:807-814. 50. Suess B, Hanson S, Berens C, Fink B, Schroeder R, Hillen W: Conditional gene expression by controlling translation with tetracycline-binding aptamers. Nucleic Acids Res 2003, 31:1853-1858. 51. Wieland M, Hartig JS: Artificial riboswitches: synthetic mRNAbased regulators of gene expression. ChemBioChem 2008, 9:1873-1878. 52. Wittmann A, Suess B: Selection of tetracycline inducible selfcleaving ribozymes as synthetic devices for gene regulation in yeast. Mol Biosyst 2011, 7:2419-2427. 53. Klauser B, Atanasov J, Siewert LK, Hartig JS: Ribozyme-based aminoglycoside switches of gene expression engineered by genetic selection in S. cerevisiae. ACS Synth Biol 2014. Clever combination of in vivo genetic screening and rational design to isolate aminoglycoside-responsive riboswitches that belong to the bestperforming RNA-based genetic regulators reported so far. 54. Weigand JE, Suess B: Tetracycline aptamer-controlled regulation of pre-mRNA splicing in yeast. Nucleic Acids Res 2007, 35:4179-4185. 55. Rodrigo G, Landrain TE, Majer E, Daros JA, Jaramillo A: Full design automation of multi-state RNA devices to program gene expression using energy-based optimization. PLoS Comput Biol 2013, 9:e1003172. A novel and innovative computational approach to design highly effective trans-acting sRNA riboregulators for directed gene targeting.
Current Opinion in Biotechnology 2015, 31:10–15
ScienceDirect Riboswitch engineering — making the all-important second and third steps Christian Berens and Beatrix Suess Synthetic biology uses our understanding of biological systems to develop innovative solutions for challenges in fields as diverse as genetic control and logic devices, bioremediation, materials production or diagnostics and therapy in medicine by designing new biological components. RNA-based elements are key components of these engineered systems. Their structural and functional diversity is ideal for generating regulatory riboswitches that react with many different types of output to molecular and environmental signals. Recent advances have added new sensor and output domains to the existing toolbox, and demonstrated the portability of riboswitches to many different organisms. Improvements in riboswitch design and screens for selecting in vivo active switches provide the means to isolate riboswitches with regulatory properties more like their natural counterparts. Addresses Fachbereich Biologie, Technische Universita¨t Darmstadt, Schnittspahnstraße 10, 64287 Darmstadt, Germany Corresponding authors: Berens, Christian ([email protected]) and Suess, Beatrix ([email protected])
Current Opinion in Biotechnology 2014, 31:10–15 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.2014.07.014 0958-1669/# 2014 Published by Elsevier Ltd.
Introduction The thirty years following the discovery of catalytic RNA have seen a veritable radiative burst in our knowledge of the many levels at which RNA molecules operate within cells. Ribozymes, riboswitches, RNA thermometers, a plethora of small RNAs in eukaryotes and bacteria, as well as different types of long non-coding RNAs in eukaryotes demonstrate that RNA appears to be everywhere, and that there is still much to learn about how RNA influences cell physiology, differentiation and development [1]. It sometimes seems that the only limit to what RNA can do or be made to do lies solely within our imagination. RNA as a regulatory molecule has the distinct advantage that it can operate in a protein-independent manner, thereby allowing firstly faster regulatory Current Opinion in Biotechnology 2015, 31:10–15
responses, due to it already having been transcribed, secondly easier transfer of a single-step genetic control element to other organisms, and thirdly flexible combination with different downstream readout platforms for a maximum of regulatory outputs. This makes RNA an attractive target for the development of genetic control elements in gene functional studies, biotechnology and synthetic biology. Its attractiveness is even further enhanced by the addition of riboswitches — small structured RNA elements which regulate gene expression in response to a small-molecule ligand — to the RNA toolbox. Their use allows spatial, temporal and dosage control over gene expression. Riboswitches, discovered about a decade ago, have been the subject of intense scrutiny not only to understand their mechanisms of action [2–4], but also to put them to use in genetic engineering [5,6]. This is probably best exemplified by the more than 100 reviews on riboswitches that have been published so far, as well as by several special editions on ‘Artificial Riboswitches’ (Methods in Molecular Biology, 2014), ‘Riboswitches: new aspects of an old story’ (RNA Biology, 2010) or the two editions from Biochimica et Biophysica Acta — Gene Regulatory Mechanisms on ‘Structure and Function of Regulatory RNA Elements’ (2009) and ‘Riboswitches’ (2014). Here, we cover recent advances in the fields of riboswitch applicability and design, focusing on the time period following publication of the last review on engineered riboswitches in this journal in 2012 [7]. It clearly emphasized the major goal during early riboswitch engineering — to establish a robust foundation of ‘proof of principle’ applications delineating the spectrum of gene regulatory mechanisms that can be addressed with riboswitches (Table 1). Their functionality was mainly investigated in bacteria and yeast, popular model organisms for prokaryotes and eukaryotes. We are now coming to another phase of riboswitch applicability, in which the emphasis on the one hand will be on improving activity, robustness and reliability of riboswitch regulation and on the other hand continuing and intensifying the transfer of riboswitch regulatory systems to mammalian cell culture and maybe even to transgenic animals and plants.
Riboswitch regulation functions in Archaea, diverse bacteria and in viruses Most engineered riboswitches have been tested in the well-characterized model organisms Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae and in mammalian cell lines [2]. However, many other bacteria and organisms www.sciencedirect.com
Engineered riboswitches Berens and Suess 11
Table 1 Proof-of-principle in vivo applications in riboswitch engineering Expression platform
Aptamer domain Theophylline
Translational ON Translational OFF Transcriptional ON Transcriptional OFF Ribozyme Splicing Trans-acting switches
B-[46], B-[47] Y-[48], B-[49] B-[35], B-[36] B-[37] B-[51], Y-[47], C-[49]
Tetracycline Y-[50] B-[36] B-[37] Y-[52], C Y-[54]
a
Others: neomycin, ammeline, 5-aza-cytosine, PPDA B-[23] Y-[22] B-[36] B-[31], B-[37] Y-[53]
B-[55]
The table contains a combinatorial matrix of aptamer domains and expression platforms, which lists the publications implementing the different combinations in bacteria (B), yeast (Y) or in cell culture (C). Citations in bold are novel ‘proof-of-principle’ applications published after the last review on the topic in this Journal [7]. a Conditional control of mammalian gene expression by tetracycline-dependent hammerhead ribozymes: Kim Beilstein, Alexander Wittmann, Manuel Grez, Beatrix Suess, submitted for publication.
also serve as model species for diverse physiological or developmental processes, as pathogens or as potential cell factories in biotechnology. Their characterization requires strictly controlled inducible expression systems to ensure that the products to be analyzed or synthesized are only produced when desired. Recognizing this need, the Gallivan lab initially developed a series of ligandinducible riboswitches, termed A–E, for control of gene expression in diverse species of Gram-negative and Gram-positive bacteria, like the model organisms E. coli, B. subtilis and Mycobacterium smegmatis or the plant and human pathogens Agrobacterium tumefaciens, Acinetobacter baumannii and Streptococcus pyogenes [8]. This organismal palette was then further extended by using theophyllinedependent riboswitches, mostly derived from the abovementioned series, but additionally including the constructs E* [8], F (clone 8.1; [9]) and G (clone D2; [10]), to achieve stringent and dose-dependent control of protein expression in the Cyanobacterium Synechococcus elongatus, a model organism for photosynthesis and a potential producer of biomaterials [11], in Streptomyces coelicolor, a major producer of antibiotics, enzymes, antiviral and anticancer drugs [12], as well as the important human pathogens Francisella tularensis [13] and Mycobacterium tuberculosis [14], but also in tobacco chloroplasts [15], which might be interesting for plant biotechnology, due to high transgene expression levels, and for biosafety reasons. Relevant parameters of riboregulation are shown in Table 2, but several points are still noteworthy. In all organisms tested, riboswitch-mediated regulation was efficient enough to establish a conditional knockout of selected endogenous genes, or even to inducibly control the circadian rhythm in S. elongatus [11]. The maximum regulatory factors obtained in M. smegmatis and S. elongatus were in the same range as those obtained with a transcriptional regulation system under control of the bacterial transcription factor TetR [14,16]. In the medically relevant pathogens F. tularensis and M. tuberculosis, gene expression is also controllable in a macrophage infection model [13,14]. Spraying the tobacco plants with theophylline www.sciencedirect.com
was sufficient to induce reporter gene expression in the plastids [15]. In contrast, a tetracycline-regulated riboswitch served as inducible control element in Methanosarcina acetivorans, an anaerobic model organism from the third superkingdom of life, the Archaea [17]. So far, naturally occurring riboswitches have not been validated experimentally in Archaea, although sequences for fluoride [18] and thiamine pyrophosphate (TPP) [19] controlled riboswitches have been found. It is therefore interesting to see if and how effective riboregulation might be in this organism. A tetracycline-regulated switch with low stability and partial inclusion of the ribosome-binding site (RBS) in the closing stem was the most active variant tested, demonstrating proof-of-principle for riboswitch functionality in Archaea [17]. Some clear tendencies can be derived from these publications. Theophylline is the preferred ligand of choice, as well as translational ON-switches. The former will hardly ever interfere with general host cell physiology, the latter appears more easy to introduce into the sequence of the respective target gene [11]. Nevertheless, the choice of riboswitch will still be affected by target gene and experimental requirements. For example, riboswitch ‘E’ displayed the highest activation factor and reporter gene expression level in F. tularensis, but the lower basal expression obtained with construct ‘F’ was needed to conditionally complement a growth-defective mutant [13]. Similarly, in S. coelicolor, riboswitch ‘A’ appears to be a good choice for tight repression, while riboswitch ‘E*’ would be used when high protein levels are needed [12]. But not only bacterial gene expression can be controlled by engineered riboswitches. Flanking insertions of theophylline-dependent aptazymes enabled ligand-triggered shutdown of immediate early gene expression in DNA viruses (adenovirus, adeno-associated virus, oncolytic adenovirus) and glycoprotein expression in an RNA virus (measles virus), leading to inhibition of viral replication Current Opinion in Biotechnology 2015, 31:10–15
12 Analytical biotechnology
Table 2 Riboswitches as genetic tools in model or potential cell factory organisms Organism Archaea Methanosarcina acetivorans Eubacteria Francisella novicida U112 Francisella tularensis ShuS4 Mycobacterium smegmatis mc2155 Mycobacterium tuberculosis H37Rv Streptomyces coelicolor M145 Synechococcus elongatus PCC7942 Organelle Chloroplast
Phylum
Process regulated
Switchtype
Euryarchaeota
RBS access
OFF
Proteobacteria (g)
Ligand
Regulatory factor
Reporter gene
Reference
Tetracycline (200 mM)
12; C a
b-Lactamase
[17]
RBS access (E, F) b ON
Theophylline (2 mM)
9.5; P a
b-Galactosidase
[13]
Proteobacteria (g)
RBS access (E) b
ON
Theophylline (2 mM)
5; P a
b-Galactosidase
[13]
Actinobacteria
RBS access (E*) b
ON
Theophylline (2–5 mM)
69; P a
GFP
[14]
Actinobacteria
RBS access (E*) b
ON
Theophylline (2–5 mM)
8.2; P a
b-Galactosidase
[14]
Actinobacteria
RBS access (E*) b
ON
Theophylline (4 mM) c
30–260d; C a b-Glucuronidase
[12]
Cyanobacteria
RBS access (E*) b
ON
Theophylline (2 mM) c
190; C a
Luciferase
[11]
RBS access
ON
Theophylline (2.5 mM) c Qualitative only; C a
GFP
[15]
The ligand concentration in brackets corresponds to the concentration required for maximum target protein expression. C: chromosomally encoded; P: plasmid-encoded. b Designation of synthetic riboswitches named and developed by the Gallivan group [8–10]. c Maximum dose applicable, due to toxicity issues. d The different factors were achieved with promoters of different expression strength. a
and infectivity and, ultimately, viral spread and cytotoxicity [20,21]. Taken together, theophylline-controlled and tetracycline-controlled riboswitches permit stringent, dosedependent, reversible and efficient control in DNA and RNA viruses, in Archaea, Bacteria and in organelles from widely diverse taxonomic origins. This regulatory approach, thus, appears to be sufficiently robust for portability to any organism of choice.
Improving riboswitch activity — concepts and their implementation The ‘proof-of-principle’ applications (Table 1 and [7]) and the high degree of functional portability to other organisms (Table 2) foretell a bright future for riboswitches in synthetic biology. However, to tap into the full potential of riboswitch regulation requires that we not only expand the toolbox of riboswitch components, but also improve the performance of the existing types of engineered riboswitches.
Expanding the toolbox — identifying new aptamer domains for riboswitches To date, most engineered riboswitches are equipped with theophylline or tetracycline aptamers (Table 1 and [5]), with only a few examples utilizing an in vivo screened neomycin aptamer [22] or heterocyclic ligands, like ammeline or 5-azacytosine, for a re-engineered Current Opinion in Biotechnology 2015, 31:10–15
adenine-responsive riboswitch [23]. There is therefore an acute need to identify new ligands and aptamer domains to increase the diversity of engineered riboswitches, especially if complex regulatory gates and networks are to be established [24–26]. Unfortunately, despite the demonstrated modularity of riboswitch aptamer domains and expression platforms [2] and despite the sophisticated advanced technology available for in vitro selecting aptamers [27], only a few sequence/ligand pairs have been used so far in vivo [5]. The in vivo active neomycin aptamer, which was highly underrepresented in the original in vitro selected aptamer pool [28], might shed some light on why this is the case. It displays a destabilized, open unbound state which undergoes extensive structural changes upon ligand binding. In contrast, in vivo inactive, but highly represented neomycin aptamers are already pre-formed in the ligand’s absence [29]. Thus, constraints on in vivo activity and in vitro functionality appear to differ considerably, so we have to find in vitro conditions that more closely mimic the in vivo situation. The optimal ligand for an engineered riboswitch should be cheap and easy to produce, be non-toxic and applicable in both prokaryotes and eukaryotes, should not interfere with cell physiology, have good solubility and cell permeability and allow the facile isolation of highly specific aptamers. So far, such a ligand/aptamer combination has not been presented. A new aptamer for an in vivo active www.sciencedirect.com
Engineered riboswitches Berens and Suess 13
riboswitch was presented by Davidson et al. [30]. They isolated a 2,4-dinitrotoluene (DNT) responsive riboswitch in E. coli by placing a 30 nucleotide-long randomized sequence between a DNT aptamer and an RBS, and by screening in vivo for active expression platforms in the presence of DNT. A ten-fold increase in GFP fluorescence was observed with the best candidate [30]. The Micklefield group used a different approach. They mutagenized a natural riboswitch to not respond to its native ligand, adenine, anymore and screened the mutant pool for binding to synthetic, non-natural heterocyclic ligands [23]. This screen yielded a ligand, PPDA (pyrimido[4,5d]pyrimidine-2,4-diamine), that formed a pair with the M600 aptamer with superior regulatory properties [31]. Such re-engineering of natural riboswitches might turn out to be an attractive approach if it is also transferable to other natural riboswitches, especially because many have been and are being subjected to intense drug discovery screens [32–34].
Expanding the toolbox — establishing new expression platforms Inspection of Table 1 reveals quite a few gaps in the matrix. An eye-catching one is the lack of any engineered transcriptionally regulated control elements. This gap has been filled in the meantime. Wachsmuth et al. used an in silico selection process involving multiple constraints and combined it with in vitro and in vivo experiments to design and isolate theophylline-dependent transcriptional ON switches [35]. Half of their constructs regulated reporter gene expression. Further sequence optimization led to a maximum activation of six-fold, which is similar to a naturally occurring transcriptional ON switch [35]. An important observation made was that intermediate terminator stability was important for riboswitch functionality. The Batey group instead preferred a modular ‘mix-and-match’ approach to construct both transcriptional ON [36] and OFF [37] switches. It uses a set of specific expression platforms, which can be uncoupled structurally from their aptamer domain and grafts arbitrary aptamer domains to these. Optimization of the regulatory properties required some sequence adjustments to the expression platforms. An important advantage of this technique is that it does not require a ‘communication module’ to transfer the signal from the aptamer domain to the expression platform, eliminating the time-consuming and laborious mutagenesis and selection experiments previously needed. Robinson et al. used a similar approach to generate a transcriptional OFF switch with their re-engineered M600 adenine aptamer domain [31]. Less than tenfold repression was obtained, due to the fact that a plateau of reporter activity had still not been reached at the maximum PPDA concentration applicable to the cells. A second gap in Table 1 was filled by Ogawa [38]. He brought a rationally modified synthetic internal ribosome entry site (IRES), based on the Plautia stali intestine virus IRES, under the control of aptamers www.sciencedirect.com
against theophylline, tetracycline, FMN and sulforhodamine B, thereby obtaining ON/OFF ratios between five and thirty in vitro [38]. The in vivo activity of these switches remains to be tested.
Improving riboswitch performance Many in vivo active engineered riboswitches suffer from low dynamic regulatory ranges, high background activity in the absence of their cognate ligand and high effector concentrations required for switching. Consequently, the following two questions are among the most important hurdles synthetic riboswitch research has to overcome at the moment: how does ligand binding to the aptamer drive riboswitch function, and how can we better improve the process of aptamer/riboswitch selection? Better understanding the former will aid us in designing more effective aptamer domain/expression platform interfaces, while improving the latter will increase our rather dismal success rate in converting in vitro selected aptamers to in vivo active riboswitches. The most reliable approach to identify in vivo active riboswitches with new and untested aptamer domains is still to perform a genetic in vivo screen of a pool of mutagenized sequence variants of the newly combined riboswitch in the desired or a closely related host organism. This was first impressively demonstrated for in vitro selected neomycin aptamers in the construction of an in vivo active neomycin riboswitch [22] and, more recently, for the DNT riboswitch [30]. A genetic screen was also used to transform a TPP-OFF switch into a TPP-ON switch [39], again highlighting the power of in vivo selection systems. Fusing an aptamer domain to an expression platform will in most cases create a suboptimal interface between the two elements which might be the cause for impaired performance by the resulting riboswitch. Natural sequence variants of riboswitches display a high degree of functional diversity, which is believed to represent ‘tuning’ to the required regulatory response [40]. Stoddard et al. [41] and Weigand et al. [42], accordingly, were able to modulate the regulatory parameters of riboswitches by mutagenizing residues not directly involved in ligand binding. This indicates a high degree of structural and functional plasticity in any given riboswitch, which may have to be optimized to obtain the desired regulatory results. It is tempting to speculate that if we combine the mutagenesis results from aptamer specificity switches [31] with those of riboswitch tuning [41,42], it might even be possible that mutagenesis of more nucleotides in or even entire subsections of a natural riboswitch would yield riboswitches with completely different ligand specificities. Natural riboswitches could, then, serve as established regulatory ‘base camps’ from which new unexpected and unexplored ligand ‘peaks’ might be scaled. Taken together, it appears that a combination of in silico structural analysis and in vivo genetic tuning will be necessary if we want to obtain well-functioning and Current Opinion in Biotechnology 2015, 31:10–15
14 Analytical biotechnology
highly active riboswitches as opposed to just functional riboswitches.
Conclusion Small-molecule controlled regulation of gene expression in synthetic RNA biology has come a long way since its inception in vitro with allosteric ribozymes [43] and in vivo with RNA aptamers against Hoechst33258 [44] approximately fifteen years ago. Numerous ‘proof-of-principle’ applications have been published since then, demonstrating the great diversity of aptamer domains and expression platforms in establishing functional regulatory modules [5]. Currently, remaining gaps in this riboswitch toolbox are being filled with new aptamer domains, new expression modules and combinations thereof. In addition, advanced in vitro and in vivo screening systems and improved in silico and structure-based design approaches have added to our riboswitch engineering principles and methods, so that the riboswitches we generate should more closely resemble their natural counterparts. Distilling the knowledge gained in all of these and in future experiments will create positive feedback and ultimately permit the design of even better tools that should finally allow standardized ‘off the shelf’ components to be utilized in ‘plug-and-play’ approaches [45] for generating synthetic genetic circuits of adjustable complexity, sophisticated biosensors, ‘intelligent’ responsive metabolic pathways and optimized diagnostic and therapeutic tools.
Acknowledgements The authors thank Dr. Julia E. Weigand for helpful suggestions and critical comments. This work was funded by the Deutsche Forschungsgemeinschaft (SFB902 A2) and Lowe CGT.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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