RNA aptamers as genetic control devices: The potential of...

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Biotechnol. J. 2015, 10, 246–257 DOI 10.1002/biot.201300498

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1 Introduction

Working with RNA can be like opening a “goodie bag” –for one, you never know what you might find until youlook, but once in a while you will discover somethingexciting and unknown you never had thought aboutbefore. Similarly, our knowledge of what RNA can do in a

cell has changed dramatically since the “central dogma ofmolecular biology”, in which RNA served primarily as aninformation carrier, was proposed about 45 years ago [1].And fortunately, our perception of what RNA might becapable of doing in a cell or in a test-tube is still expand-ing, since new regulatory functions of RNA are continu-ously being discovered [2]. Circular exonic RNA mole-cules, for example, are an abundant and differentiallyexpressed RNA species that have recently been proposedto serve as molecular “sinks” for trans-acting short RNAregulators, as mRNA traps in regulating protein expres-sion, or as interaction partners of RNA-binding proteins[3]. Another newly identified and apparently ubiquitousRNA species is long noncoding RNAs. This quite hetero-geneous population of RNA molecules participates inmany different molecular and cellular processes involvedin regulating nuclear organization, development, viability,immunity, and disease [4]. CRISPR/Cas systems are usedby various bacteria and archaea as an adaptive immune

Review

RNA aptamers as genetic control devices: The potential of riboswitches as synthetic elements for regulating gene expression

Christian Berens1, Florian Groher2 and Beatrix Suess2

1 Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Jena, Germany2 Synthetic Biology and Genetics, Department of Biology, Technical University Darmstadt, Germany

RNA utilizes many different mechanisms to control gene expression. Among the regulatory ele-ments that respond to external stimuli, riboswitches are a prominent and elegant example. Theyconsist solely of RNA and couple binding of a small molecule ligand to the so-called “aptamerdomain” with a conformational change in the downstream “expression platform” which thendetermines system output. The modular organization of riboswitches and the relative ease withwhich ligand-binding RNA aptamers can be selected in vitro against almost any molecule have ledto the rapid and widespread adoption of engineered riboswitches as artificial genetic controldevices in biotechnology and synthetic biology over the past decade. This review highlights proof-of-principle applications to demonstrate the versatility and robustness of engineered riboswitch-es in regulating gene expression in pro- and eukaryotes. It then focuses on strategies and param-eters to identify aptamers that can be integrated into synthetic riboswitches that are functional invivo, before finishing with a reflection on how to improve the regulatory properties of engineeredriboswitches, so that we can not only further expand riboswitch applicability, but also finally fullyexploit their potential as control elements in regulating gene expression.

Keywords: Engineered riboswitch · Regulatory circuits · RNA aptamer · RNA aptazyme · SELEX

Correspondence: Prof. Dr. Beatrix Suess, Synthetic Biology and Genetics,Department of Biology, Technical University Darmstadt, Schnittspahnstr. 10, 64287 Darmstadt, Germany.E-mail: [email protected]

Abbreviations: CRISPR, clustered regularly interspaced short palindromicrepeats; GFP, green fluorescent protein; miRNA, micro RNA; mRNA, mes-senger RNA; PPDA, pyrimido[4,5-d]pyrimidine-2,4-diamine; SD, Shine-Dal-garno; SELEX, systematic evolution of ligands by exponential enrichment;shRNA, short hairpin RNA; siRNA, short interfering RNA; sRNA, smallRNA; UTR, untranslated leader region


Received 14 OCT 2014Revised 23 DEC 2014Accepted 15 JAN 2015

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BiotechnologyJournal Biotechnol. J. 2015, 10, 246–257

system to mediate defence against bacteriophages andother foreign nucleic acids. They use a “guide RNA” withcomplementarity to a specific DNA target site to direct aCas9 nuclease which then initiates a double-strand breakresulting in degradation of the target DNA molecule [5].

Many of these types of RNA-mediated regulation ofgene expression have proven to be very attractive forbiotechnological and even therapeutic applications. RNAinterference [6], using miRNA, shRNA and siRNA, isemployed routinely in the lab for loss-of-function studiesin eukaryotes [7] and also shows potential as therapeuticmolecule in disease treatment [8]. Circular RNAs havebiotechnological potential due to their exceptional intra-cellular stability and their ability to titrate endogenousRNA species or to support internal ribosome entry site-mediated translation [3]. Finally, CRISPR/Cas9 systemsare very successful at precisely manipulating thegenomes of both pro- and eukaryotes [9, 10].

Sophisticated molecular systems that sense externalstimuli and respond to these by modulating the output ofa cellular process are another important application inbiotechnology. Genetically, this problem can be tackledby several different approaches of which one is based onallosteric RNA elements termed riboswitches. Typically,riboswitches are genetic control elements that consist oftwo domains – an aptamer domain that binds a small mol-ecule ligand and an expression platform that converts lig-and binding into a change in gene expression by adopt-ing an alternative RNA structure [11]. Interestingly, suchallosteric aptamer-containing devices were designed andshown to be active in vitro [12, 13] and in vivo [14] aboutfive years before the first naturally occurring riboswitch-es were discovered [15–17]. Since then, a plethora of dif-ferent riboswitches have been identified in all threedomains of life and have been reviewed comprehensively[18, 19]; see also the recent special edition on “Ribo -switches” in Biochemica et Biophysica Acta – Gene Reg-ulatory Mechanisms (2014) [133].

There are several reasons why riboswitches are anattractive tool in biotechnology. Most importantly, theyconsist solely of RNA. So far, protein components have notbeen found to be necessary for their activity. Therefore,they are easy to implement, because they involve thetransfer of only a single genetic control element into anorganism. They are modular, so that different aptamerdomains can be combined with different expression plat-forms [20]. Finally, their use allows spatial, temporal anddosage control over target gene expression. It is thereforenot astonishing that engineered riboswitches were intro-duced early to the fields of genetic engineering and syn-thetic biology [14, 21] and have seen widespread applica-tion since then [22–26].

This review is not intended to serve as a comprehen-sive overview of all aspects of riboswitch engineering. Forexample, we do not cover the wide fields of diagnosticand therapeutic aptamers and riboswitches, which have

been addressed elsewhere [23, 27–30]; see also the Sep-tember 2014 special issue on Aptamers published inMolecular Therapy – Nucleic Acids. Our aim is rather topresent three topics we consider to be of prime impor-tance for riboswitch biotechnology. We not only demon-strate how far the field has progressed since the early daysof artificial in vitro “riboregulators” [12], but also presentstrategies we believe are important for isolating in vivoactive riboswitches, as well as challenges that will befaced in designing more versatile and effective ribo -switches. We hope this format will stimulate discussionsthat will lead to new and improved approaches and selection systems and, ultimately and hopefully, to thedevelopment of new and more efficient synthetic ribo -switches.

2 Engineered riboswitches: An overview of the status quo

2.1 The toolbox of engineered riboswitchesrecapitulates the functionality of naturalriboswitches

Natural riboswitches control translation, transcription,ribozyme activity, and splicing [15–17, 31, 32]. In themajority of these cases, they act in cis as part of the mRNAtranscript. But they can also regulate in trans either as aprocessed, terminated riboswitch that functions like ansRNA [33] or as a full-length transcript containing a bind-ing site for sequestering an activated RNA-binding pro-tein [34, 35] or by acting as an antisense RNA [36]. In syn-thetic biology, creating complex and more realistic genet-ic circuits requires that we eventually identify and design“BioBricks” that not only reflect the entire spectrum ofgenetic regulatory elements, but that can also be tailoredand fine-tuned to obtain the desired output level of thetarget gene. The first step towards achieving this goal isthe construction of proof-of-principle applications todemonstrate that we are able to reproduce these geneticelements using artificial and/or heterologous compo-nents. The following section will provide an overview ofsuch archetype constructs. More detailed descriptionscan be found in several other reviews [25, 26].

In bacteria, engineered riboswitches most often targettranslation initiation, either by controlling access to theribosomal binding site through helix slippage [37] or bysequestering this Shine-Dalgarno (SD) element (Fig. 1A)[38]. Regulation of gene expression by a theophylline-con-trolled translational switch was efficient enough to allowthe control of chemotaxis in Escherichia coli [39]. In con-trast, transcriptionally-controlled synthetic ribo switcheshave only recently been introduced to the toolbox by theMörl [40], Batey [41, 42], and Micklefield [43] groups. Here,a transcriptional terminator is formed depending on thebinding state of the aptamer (Fig. 1B). It will be interest-



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ing to see how these devices perform with respect to reg-ulatory efficiency, basal activity, noise, and kinetic param-eters, in comparison to analogous, but translationally con-trolled riboswitches in similar experimental settings.

The cleavage activity of small ribozymes, like the ham-merhead ribozyme, was made ligand-dependent byinserting an aptamer domain into one of its three stem-loops [44]. Luckily, all three stem-loops of the hammer-head ribozyme are amenable to aptamer insertion [45–48],greatly facilitating the applicability of such “aptazymes”,because the “best fit” between ribozyme and targetsequence can now be exploited. In bacteria, such apta -zymes were used to liberate the ribosomal binding siteafter ligand-dependent ribozyme cleavage (Fig. 1C). Withrespect to the thermally-controlled riboswitches men-tioned below in Section 2.3 [49], it will be interesting tosee if the recently introduced “thermozymes” [50] can alsobe modified to include an additional aptamer control ele-ment. The examples listed here indicate that in bacteriaengineered riboswitches function in regulatory settingsthat are also found for natural riboswitches.

In eukaryotes, translational regulation by engineeredriboswitches functions differently. Here, insertion of the


riboswitch into the 5’-untranslated leader region leads toinhibition of translation initiation, either by preventingbinding of the small ribosomal subunit to the mRNA capstructure or by interfering with ribosomal subunit scan-ning for the AUG start codon (Fig. 2A). Here, aptamerswhich specifically recognize a Hoechst dye [14], mala-chite green [51], tetracycline [21, 52], neomycin [53],biotin or theophylline [54] (Fig. 2A) have been engineeredto serve as regulatory elements. The riboswitches wereactive in yeast, Xenopus oocytes and mammalian cell cul-ture [55]. Cap-independent translation initiation by aninternal ribosome entry site has also been controlled byengineered riboswitches. However, up to now this hasonly been demonstrated in vitro [56].

The only naturally occurring eukaryotic riboswitchesidentified so far have been discovered in filamentous fun-gi, green algae and higher plants where they control geneexpression via regulation of mRNA splicing [57–60]. Anal-ogously, theophylline- or tetracycline-binding aptamershave been used to control constitutive and alternativesplicing by placing them close to either the 5’-splice site,the 3’-splice site or the intron internal branch point (Fig. 2B) [61–63].

Figure 1. Common mechanisms of engineered riboswitches in bacteria. (A) Regulation of translation initiation:In the absence of a ligand, a stem-loopstructure is formed between the aptamerdomain (depicted in green) and asequence element complementary to theShine-Dalgarno (SD) sequence (magen-ta). Thus, the SD sequence (orange) isaccessible for 30S binding and transla-tion initiation occurs. As a consequenceof ligand binding (blue circle) and fold-ing of the aptamer domain, an alterna-tive stem-loop is formed whichsequesters the SD sequence, blockingthe binding of the 30S ribosomal sub-unit. (B) Regulation of transcription ter-mination: The aptamer domain is fusedto a short spacer region (grey), followedby a sequence complementary to the 3’ part of the aptamer (magenta) and aU stretch. In the absence of ligand, thecomplementary 3’ part is base-paired withthe aptamer forming a terminator struc-ture; thus, RNA polymerase (RNAP) dis-sociates and transcription is terminated.Upon ligand binding, terminator struc-ture formation is inhibited and transcrip-tion can proceed, resulting in expressionof the reporter gene. (C) Control of geneexpression with catalytic riboswitches: A ligand-dependent aptazyme is insertedinto the 5’-UTR of an mRNA in such away that ligand-induced self-cleavage liberates a sequestered SD sequence to induce translation initiation.




Aptazyme-mediated control of gene expression inyeast and in mammalian cell lines using hammerheadribozymes (Fig. 2C) has been demonstrated by severalgroups [46, 48, 64–66]. Here, ligand-dependent ribozymecleavage leads to mRNA degradation. Besides the ham-merhead ribozyme, the hepatitis delta virus ribozyme hasalso been successfully applied to RNA engineering [67].

RNA interference is an important and widely usedmechanism in eukaryotes to repress gene expression by

base-pairing to mRNA molecules. RNA interference con-trols a broad range of developmental and physiologicalprocesses [68–70]. It is also a standard method in molec-ular biology with a possible therapeutic option which isbeing tested in clinical trials [71]. Consequently, attemptsto control the biogenesis pathway of RNA interferencewere made by introducing theophylline-dependentaptamers [72, 73] or aptazymes [74] at sites of siRNA orshRNA molecules where their presence can interfere with


Figure 2. Common mechanisms of engineered riboswitches in eukaryotes.(A) Regulation of ribosome scanning:After insertion of the aptamer (depictedin green) into the 5’UTR of a mRNA, lig-and binding (blue circle) to the aptamerprevents scanning of the 40S ribosomalsubunit and, consequently, translationinitiation. (B) Regulation of mRNA splic-ing: An aptamer is integrated into anintron of a eukaryotic mRNA to controlthe accessibility of the 5’ splice site (SS).Ligand binding inhibits splicing. (C)Control of gene expression with catalyticriboswitches: A ligand-dependentaptazyme is inserted into the 5’-UTR of aeukaryotic mRNA. Ligand-induced self-cleavage triggers RNA degradation(magenta pacman). (D) Regulation ofRNA interference: An integrated aptamerdomain (green) interferes in its ligand-bound state with the enzymatic activityof Dicer.




processing by either Drosha or Dicer (Fig. 2D). So far,extremely high ligand concentrations are needed for effi-cient regulation. Optimization of the experimental strate-gies is clearly needed and it will be challenging to see ifendogenous miRNA molecules can also become subjectto riboswitch control.

2.2 Riboswitch regulation is becoming more robustand versatile

While proof-of-principle applications are important fordemonstrating the functionality of any kind of experi-mental approach, they do not, per se, demonstrate theirgeneral transferability to other systems, organisms oreven across the kingdoms of life. Undoubtedly, the gener-al applicability and the ease with which system function-ality can be achieved is an important parameter for deter-mining success and acceptance of an application as a toolby the respective community. So far, most of the regula-tory strategies presented in the previous section weretested in the well-characterized model organisms E. colifor Gram-negative bacteria, Bacillus subtilis for Gram-positive bacteria, Saccharomyces cerevisiae for lowereukaryotes and mammalian cell lines for higher eukary-otes [19]. In developmental biology, biotechnology and inmedicine, other organisms are frequently employed asphysiological, developmental and disease models or ascell factories for the production of materials [75–80]. Sinceendogenous riboswitches have been identified in thegenomes of many different organisms [81], it seem rea-sonable to assume that engineered riboswitches, basedon similar regulatory principles, should also be active inthese species.

In bacteria, the Gallivan lab, consequently, designed aset of theophylline-inducible translationally controlledriboswitches, initially termed A-E, which vary in thestrength of their ribosomal binding sites and the stabilityof their secondary structures [38]. Later, riboswitches des-ignated E* (supplemental material in [38]), F (clone 8.1from [82]), and G (clone D2 from [83]) were added to theset. These riboswitches were then tested for activity indiverse bacterial species, including the developmentalmodel organism and major producer of antibiotics, anti-cancer and antiviral drugs or enzymes, Streptomycescoelicolor [84], cyanobacteria, model organisms for photo-synthesis and potential material-producing cell factories[85, 86] or tobacco chloroplasts, which are of interest inplant biotechnology [87]. The riboswitch set was alsoevaluated in important pathogens like Agrobacteriumtumefaciens, Acinetobacter baumannii, Streptococcuspyogenes [38], Mycobacterium [88] and Francisella [89].The tetracycline aptamer was shown to be functional inthe anaerobic model organism Methanosarcina acetivo-rans, an archaeon [90]. In all organisms tested, at least oneriboswitch was active enough to establish a conditionalknockout of an endogenous gene. In some cases, the reg-

ulatory factors obtained were similar to those obtainedwith transcriptional regulation. Effector molecule pene-tration across several membranes was also effectiveenough to permit target gene expression control in chloro-plasts and in intracellular infections models.

Theophylline was the preferred ligand of choice. It canbe added at high concentrations (2–4 mM) to the mediumwithout causing obvious toxicity. In contrast, tetracyclineand neomycin are antibiotics in bacteria and the concen-trations needed for riboswitch activity can be close to orabove the respective minimal inhibitory concentrations.Unfortunately, anhydrotetracycline, a less toxic derivativeof tetracycline, does not bind to the tetracycline aptamer[91]. Translational ON-switches are the most frequentlyused type of expression platform. They appear the easiestto integrate into the sequence of the target gene. Andfinally, there is no single “optimal” riboswitch. Dependingon the species and the specific target gene, differentriboswitches displayed the best regulatory properties. Sofar, several constructs have to be tested to find the rightcombination for a given application.

Regulation of gene expression by synthetic ribo -switches is not limited to bacteria. Viral replication wasinhibited in eukaryotic DNA (adenovirus, adeno-associat-ed virus) and RNA viruses (measles) by flanking essentialgenes with theophylline-dependent aptazymes [92, 93].

Taken together, gene expression regulation by ribo -switches has been demonstrated to be stringent,reversible, and dose-dependent in viruses, organelles,archaea, and bacteria from a broad taxonomic range ofspecies, firmly establishing this technology as robust anduniversally applicable.

2.3 Riboswitches can be integrated into complexregulatory circuits

Genetic circuits are frequently more complex than simpleone input/one output type networks. They can have com-plex architectures like toggle switches [94], oscillators[95], Boolean logic gates that integrate several externalsignals [96], and binary or graded responses [97]. Theycan also include positive [98] or negative [99] feedbackloops or linearizers [100]. These examples of engineeredcircuits were all realized with protein-based transcriptionfactors. Consequently, the interesting question is to deter-mine if RNA-based devices can be used similarly toassemble multiple input or complex regulatory circuits.

Nature itself has come up with several combinatorialsolutions to tackle diverse regulatory problems. In B. sub-tilis, the glmS riboswitch probably represents the sim-plest solution to regulation by multiple signals. The lig-and-binding pocket of the aptamer domain not only bindsintracellular glucosamine-6-phosphate as an activatingagent. It also binds glucose-6-phosphate, which acts asan inhibitor of ribozyme activity. The riboswitch, thus,integrates metabolite information to sense the overall





metabolic state of the cell [101]. A second solitary elementthat senses and integrates two different input signals isthe adenine riboswitch encoded by the add gene fromVibrio vulnificus. This translational regulatory elementsenses temperature and ligand concentration to conferefficient regulation over the range of physiologically rele-vant temperatures, allowing gene expression control toadapt to the differing temperatures experienced in thefree-living and in the infectious environments [49]. InBacillus clausii, two different riboswitches, whichresponded independently to S-adenosylmethionine andvitamin B12, respectively, were found in tandem in the 5’untranslated leader region (UTR) of the metE mRNA. Sys-tem output fits a Boolean NOR gate, because transcrip-tion is terminated in the presence of only one or of bothligands [102].

The Yokobayashi group was first to exploit this strate-gy in riboswitch engineering. They embedded aptamersthat recognize theophylline and thiamine pyrophosphate,respectively, in tandem in the 5’-UTR of a bacterial mRNAand selected for translational riboswitches that respond-ed to ligand presence like AND or NAND logic gates.Reporter gene activity was observed either only in thepresence of both ligands (AND gate), or, in the case of theNAND gate, when neither or only one ligand was present[103]. In a subsequent, different experimental setup, theycombined a transcriptional-OFF riboswitch with a trans-lational-ON switch. Because both switches respond to thesame ligand, thiamine pyrophosphate, albeit with differ-ent sensitivity, a band pass filter is the result, with maxi-mum target gene expression obtained at intermediate lig-and concentrations. At low ligand concentrations, geneexpression is repressed translationally, while at high con-centrations, premature transcription termination occurs[104]. Boolean logic gates can also be constructed withaptazymes. The Hartig group [105] combined ribozymeswitches responsive to thiamine pyrophosphate andtheophylline to yield AND, NOR, and ANDNOT gates. Winand Smolke [106] also generated an entire set of Booleanlogic gates by combining theophylline- and tetracycline-responsive aptamers with ON- and OFF-responsiveribozymes. For example, such gates were constructedeither by assembling two different aptazymes in series(AND gate and NOR gate) or by, very ingeniously, exploit-ing the possibility of joining aptamer-responsive controlelements to all stems of the hammerhead ribozyme. Cou-pling two ON-switches responsive to different inputs todifferent ribozyme stems led to an output signal only if noor one of the ligands was present, thus generating aNAND gate [106].

3 What makes a riboswitch out of an aptamer?

So far, in vivo active riboswitches rely on a small set of lig-ands – theophylline, tetracycline, neomycin [53], 2,4-dini-trotoluene [107], ammeline, 5-aza-cytosine [108] or pyrim-ido[4,5-d]pyrimidine-2,4-diamine (PPDA) [43], althoughseveral dozen small molecule-binding aptamers havebeen selected by a process called SELEX (systematic evo-lution of ligands by exponential enrichment), an in vitrotechnology invented about a decade before the discoveryof natural riboswitches [109, 110]. During SELEX, a hugecombinatorial library of RNA (and sometimes DNA) mole-cules is subjected to affinity of a small RNA chromatogra-phy to the ligand of choice. Non-binding sequences areremoved during washing steps, whereas sequences witha certain binding affinity to the target of interest are elut-ed. These molecules are amplified and subjected to fur-ther rounds of selection. During these iterative cycles, theselection pressure can be increased steadily, finally result-ing in molecules with sometimes extraordinarily highbinding affinities and specificities, the so-called aptamers(Fig. 3A). However, as stated above, only a handful of suchsmall molecule-binding aptamers have made it into thetoolbox of RNA engineers. This raises the questions (i) what makes a riboswitch out of an aptamer; and (ii) how can we identify further aptamers which can beused for riboswitch engineering.

A detailed analysis of neomycin-binding aptamerscould shed some light on the properties such aptamersneed in order to become regulatory molecules that areactive in vivo. The neomycin-binding aptamer R23 wasisolated by in vitro selection [111] and shows both highaffinity and specificity for its ligand [111]. However, thisaptamer – like many other small molecule-bindingaptamers, too – showed no ligand-dependent regulationwhen tested for riboswitch activity in an assay for trans-lational regulation (like in Fig. 2A) [53]. The subsequentanalysis of the enriched aptamer pool (after seven roundsof selection) for sequences with regulatory activity usingan in vivo screen, such as the one displayed in Fig. 3A,resulted in the isolation of a completely different aptamer,designated N1 and shown in Fig. 3B [53]. This newneomycin aptamer is active as riboswitch when insertedinto the 5’-UTR of a reporter gene (Fig. 2A) [53], but alsoas sensing domain in the context of an allosteric ribozyme(Fig. 2C) [66]. Interestingly, N1 was not among thesequenced aptamers isolated after in vitro selection indi-cating that this aptamer was underrepresented in thepool enriched for binding aptamers [111].

A detailed genetic, biochemical and structural analy-sis of the aptamer N1 unravelled the molecular basis ofthis regulation. While the aptamer shows tight ligandbinding with a Kd in the low nanomolar range, this affin-ity was still very similar to that of the originally identifiedin vivo inactive aptamer R23 [112]. However, substantial





conformational changes concomitant to ligand bindingwere detected in N1, which were completely absent inthe inactive aptamer R23 [113]. A short interveningsequence separating two short helical elements wasidentified as being important for regulation and there-fore called “switching element”. It allows the groundstate of the aptamer to retain an open, less structuredconformation. Ligand binding then stabilizes a highlystructured conformation, in which additional base pairsare formed. These elongate the two short helices andpromote stacking of the upper onto the lower helixresulting in a long α-helical element (Fig. 3B). The ligandneomycin then acts like a clamp holding both the upperand the lower helix together. This conformation then isable to efficiently interfere with ribosomal scanning inyeast. It seems that the interplay between an openground state and a highly structured ligand-bound stateis important for the regulatory activity of the aptamer.

Further aptamers with confirmed riboswitch activity,like the malachite green or the theophylline aptamers,show a similar open, less structured ground state and a highly structured ligand-bound conformation [114,115].

The conclusion drawn from this analysis is that regu-lating aptamers have to possess two properties: first, highaffinity ligand binding, and second, significant conforma-tional changes and stabilization upon ligand binding. Thefirst point can be addressed in any classical SELEX exper-iment. By increasing the stringency of the selectionprocess, high affinity binding aptamers can be obtainedwith binding constants even in the picomolar range [116].Tight binding, however, is necessary but not sufficient forregulation. The second requirement – the conformationalchanges within the aptamer upon ligand binding – can-not be addressed by in vitro selection. An additional cel-lular screening step, preferably directly in the organism of


Figure 3. (A) Finding regulating aptamers. In the first step, several rounds of in vitro selection (SELEX) result in an enriched pool of aptamers that bind to the ligand. This pool is then cloned into the 5’-UTR of a GFP reporter gene. Yeast cells transformed with this library are screened for ligand-dependentchanges in GFP expression. (B) The regulating neomycin-binding aptamer N1. The secondary structures of the ground- and ligand-bound states of theneomycin-binding aptamer N1 are shown. Nucleotides which form new base pairs in the bound state resulting in the stabilization of the RNA structure are marked in red.




choice, is necessary to identify aptamers with regulatoryactivity.

Several approaches have been undertaken which notonly support the success, but also reinforce the impor-tance of cellular screening for the identification of in vivoactive regulatory devices. Gallivan and colleagues applieddifferent screens to identify in vivo active riboswitches[83, 117], which proved to be extremely powerful in bac-teria (see also Section 2.2). In vivo screening was alsoapplied to identify aptamer-controlled ribozymes. Here,the communication module connecting the sensingdomain (aptamer) with the catalytic domain (ribozyme)was randomized [118, 119]. Together with the neomycinscreen described above, these examples underline theimportance of this approach.

4 How can we further improve riboswitches?

Despite of the success engineered riboswitches areenjoying as regulatory devices, there are still two impor-tant problems that have to be overcome to allow us tobuild even better riboswitches. One is the lack of a largenumber of alternative aptamers as ligand-binding mod-ules and the second is the large discrepancy between thein vitro affinities of aptamers for their ligands and the highconcentrations of ligand needed in vivo to flip the corre-sponding switches.

Besides introducing an additional cellular screeningstep which allows the identification of regulating aptamersas specified above, reengineering the aptamer domainscaffolds of existing functional ribo switches to recognizenovel ligands is also a promising approach to extend theligand toolbox. The Micklefield group mutated a purineriboswitch to not recognize its natural ligand anymoreand then screened this mutant pool for binding to syn-thetic, heterocyclic molecules. In the end, they obtaineda ligand, PPDA, which controlled riboswitch activity effi-ciently [43, 108]. It will be interesting to see if thisapproach can be extended successfully to other naturalriboswitches and, how different any potential novel lig-ands can be structurally from the respective native lig-ands. The many approaches to identify inhibitors ofriboswitch activity for therapeutic applications mightserve as starting points for finding such alternative lig-ands [120–122]. More extensive mutagenesis might con-tribute to this process, because natural sequence variantsof riboswitches already display functional diversity,which is thought to reflect the adaptation to the idiosyn-cratic regulatory requirements of a specific gene [123].Correspondingly, mutagenesis of residues that are notdirectly involved in ligand binding affect the regulatoryproperties of riboswitches [124, 125].

The ligand-binding site of the tetracycline aptamer isformed by a three-way-junction that is stabilized by long-range interactions and almost completely envelops the

ligand [126]. This is reminiscent of natural riboswitchesand distinguishes the tetracycline aptamer from the lig-and-binding pockets of many aptamers which are muchmore open and solvent-exposed [127]. It might suggestthat alternative aptamer selection protocols, like capillaryelectrophoresis, that do not require coupling of the ligandto a matrix and, thus, do not force surface exposure mightbe more advantageous for isolating in vivo functionalaptamer domains [128–130].

A discrepancy between high affinity binding and effi-cient regulation has not only been observed for engi-neered riboswitches, but also for the tetrahydrofolateriboswitch. Although several ligand analogs and adeninederivatives were found to bind with high affinity to theriboswitch, they were not able to regulate its activity[131]. The mechanism(s) responsible for this effect are notclear at the moment, although for synthetic riboswitches,the fusion of an aptamer domain to an expression platformmight result in a suboptimal interface between the twoelements. Here, some optimization by mutagenesis andselection might be needed. A recent publication by theGallivan lab might aid in this optimization process [132].Mishler & Gallivan were able to recapitulate the approxi-mately 1000-fold discrepancy between the concentra-tions needed for efficient binding of theophylline to itsaptamer and the concentration needed for activating the


Chris Berens studied biology in Erlan-

gen where he received his Ph. D. in

microbiology. After postdoctoral work

in the laboratory of Prof. Renée

Schroeder on tetracycline-binding

aptamers and metal ion binding sites

in RNA, he went back to Erlangen to

work on the development of inducible

regulatory systems for application in

both pro- and eukaryotes with a focus on studying cell death. He is

currently at the Institute of Molecular Pathogenesis which is located

at the Jena site of the Friedrich-Loeffler-Institut.

Beatrix Suess studied biology in Greifs -

wald and Erlangen where she received

her Ph.D.. After postdoctoral work in

the lab of Prof. Dr. Wolfgang Hillen she

started working on RNA aptamers and

their use as synthetic control devices.

In 2007, she was appointed as Aventis

foundation-endowed professor for

Chemical Biology at the Goethe-Univer-

sity Frankfurt, Main. Since 2012, she is full professor for Synthetic

Biology at the Technical University Darmstadt.




riboswitch in E. coli in vitro in an S30 cell extract. Thismight finally allow in vitro screening and selection formore efficient switches requiring less ligand [132].

5 Conclusions

Riboswitch engineering has come a long way since thefirst applications were introduced approximately fifteenyears ago. This technology has finally reached a stage inwhich it can not only complement other established mech-anisms and strategies to regulate gene expression, but inwhich there are applications where we believe that it caneven effectively replace these. The riboswitch engineer’stoolbox now provides a broad platform which should beable to offer flexible solutions to most synthetic biologyapplications requiring regulation of gene expression.Advances in selection technology and further improve-ment of our understanding of riboswitch structure andfunction will guide the design of better riboswitches toharness the full regulatory, diagnostic and therapeuticpotential of aptamer-controlled regulatory devices.

We would like to thank the Deutsche Forschungsgemein-schaft (SFB902/A2) and Loewe CGT for financial support.EU FP7-KBBE-2013-7 no. 613745, Promys

The authors declare no financial or commercial conflict ofinterest.

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Research ArticleSynthetic secondary chromosomes in Escherichia coli based on the replication origin of chromosome II in Vibrio choleraeSonja J. Messerschmidt, Franziska S. Kemter, Daniel Schindler and Torsten Waldminghaus


Research Article Bacterial XylRs and synthetic promoters function as geneticallyencoded xylose biosensors in Saccharomyces cerevisiaeWei Suong Teo and Matthew Wook Chang


Biotech MethodSingle-cell analysis reveals heterogeneity in onset of transgeneexpression from synthetic tetracycline-dependent promotersUlfert Rand, Jan Riedel, Upneet Hillebrand, Danim Shin, Steffi Willenberg, Sara Behme, Frank Klawonn, Mario Köster,Hansjörg Hauser and Dagmar Wirth


Biotech MethodEngineering connectivity by multiscale micropatterning of individual populations of neuronsJonas Albers, Koji Toma and Andreas Offenhäusser


Biotechnology Journal – list of articles published in the February 2015 issue.

Special issue: Synthetic Biology.Synthetic biology has become more application oriented, by designing and implementing synthetic pathways in industrial biotechnology. This Special issue, edited by Roland Eils (German Cancer Research Center, DKFZ, and University of Heidelberg), Julia Ritzerfeld (German Cancer Research Center, Heidelberg) and Wolfgang Wiechert (IBG-1: Biotechnology, Forschungszentrum Jülich), includes contributions from the Helmholtz Initiative on Synthetic Biology and focusses on applications of synthetic biology in biotechnology.Image: © Sergey Nivens Fotolia.com

Systems & Synthetic Biology ·Nanobiotech · Medicine

ISSN 1860-6768 · BJIOAM 10 (2) 227–338 (2015) · Vol. 10 · February 2015

2/2015Minimal cellsArtificial chromosomesExpression control

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