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Journal
Biotechnol. J. 2015, 10, 246–257
DOI 10.1002/biot.201300498
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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, Germany
Biology and Genetics, Department of Biology, Technical University Darmstadt, Germany
2 Synthetic
RNA utilizes many different mechanisms to control gene expression. Among the regulatory elements that respond to external stimuli, riboswitches are a prominent and elegant example. They
consist solely of RNA and couple binding of a small molecule ligand to the so-called “aptamer
domain” with a conformational change in the downstream “expression platform” which then
determines system output. The modular organization of riboswitches and the relative ease with
which ligand-binding RNA aptamers can be selected in vitro against almost any molecule have led
to the rapid and widespread adoption of engineered riboswitches as artificial genetic control
devices in biotechnology and synthetic biology over the past decade. This review highlights proofof-principle applications to demonstrate the versatility and robustness of engineered riboswitches in regulating gene expression in pro- and eukaryotes. It then focuses on strategies and parameters to identify aptamers that can be integrated into synthetic riboswitches that are functional in
vivo, before finishing with a reflection on how to improve the regulatory properties of engineered
riboswitches, so that we can not only further expand riboswitch applicability, but also finally fully
exploit their potential as control elements in regulating gene expression.
Received 14 OCT 2014
Revised 23 DEC 2014
Accepted 15 JAN 2015
Keywords: Engineered riboswitch · Regulatory circuits · RNA aptamer · RNA aptazyme · SELEX
1 Introduction
Working with RNA can be like opening a “goodie bag” –
for one, you never know what you might find until you
look, but once in a while you will discover something
exciting and unknown you never had thought about
before. Similarly, our knowledge of what RNA can do in a
Correspondence: Prof. Dr. Beatrix Suess, Synthetic Biology and Genetics,
Department of Biology, Technical University Darmstadt,
Schnittspahnstr. 10, 64287 Darmstadt, Germany.
E-mail: bsuess@bio.tu-darmstadt.de
Abbreviations: CRISPR, clustered regularly interspaced short palindromic
repeats; GFP, green fluorescent protein; miRNA, micro RNA; mRNA, messenger RNA; PPDA, pyrimido[4,5-d]pyrimidine-2,4-diamine; SD, Shine-Dalgarno; SELEX, systematic evolution of ligands by exponential enrichment;
shRNA, short hairpin RNA; siRNA, short interfering RNA; sRNA, small
RNA; UTR, untranslated leader region
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cell has changed dramatically since the “central dogma of
molecular biology”, in which RNA served primarily as an
information carrier, was proposed about 45 years ago [1].
And fortunately, our perception of what RNA might be
capable of doing in a cell or in a test-tube is still expanding, since new regulatory functions of RNA are continuously being discovered [2]. Circular exonic RNA molecules, for example, are an abundant and differentially
expressed RNA species that have recently been proposed
to serve as molecular “sinks” for trans-acting short RNA
regulators, as mRNA traps in regulating protein expression, or as interaction partners of RNA-binding proteins
[3]. Another newly identified and apparently ubiquitous
RNA species is long noncoding RNAs. This quite heterogeneous population of RNA molecules participates in
many different molecular and cellular processes involved
in regulating nuclear organization, development, viability,
immunity, and disease [4]. CRISPR/Cas systems are used
by various bacteria and archaea as an adaptive immune
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system to mediate defence against bacteriophages and
other foreign nucleic acids. They use a “guide RNA” with
complementarity to a specific DNA target site to direct a
Cas9 nuclease which then initiates a double-strand break
resulting in degradation of the target DNA molecule [5].
Many of these types of RNA-mediated regulation of
gene expression have proven to be very attractive for
biotechnological and even therapeutic applications. RNA
interference [6], using miRNA, shRNA and siRNA, is
employed routinely in the lab for loss-of-function studies
in eukaryotes [7] and also shows potential as therapeutic
molecule in disease treatment [8]. Circular RNAs have
biotechnological potential due to their exceptional intracellular stability and their ability to titrate endogenous
RNA species or to support internal ribosome entry sitemediated translation [3]. Finally, CRISPR/Cas9 systems
are very successful at precisely manipulating the
genomes of both pro- and eukaryotes [9, 10].
Sophisticated molecular systems that sense external
stimuli and respond to these by modulating the output of
a cellular process are another important application in
biotechnology. Genetically, this problem can be tackled
by several different approaches of which one is based on
allosteric RNA elements termed riboswitches. Typically,
riboswitches are genetic control elements that consist of
two domains – an aptamer domain that binds a small molecule ligand and an expression platform that converts ligand binding into a change in gene expression by adopting an alternative RNA structure [11]. Interestingly, such
allosteric aptamer-containing devices were designed and
shown to be active in vitro [12, 13] and in vivo [14] about
five years before the first naturally occurring riboswitches were discovered [15–17]. Since then, a plethora of different riboswitches have been identified in all three
domains of life and have been reviewed comprehensively
[18, 19]; see also the recent special edition on “Riboswitches” in Biochemica et Biophysica Acta – Gene Regulatory Mechanisms (2014) [133].
There are several reasons why riboswitches are an
attractive tool in biotechnology. Most importantly, they
consist solely of RNA. So far, protein components have not
been found to be necessary for their activity. Therefore,
they are easy to implement, because they involve the
transfer of only a single genetic control element into an
organism. They are modular, so that different aptamer
domains can be combined with different expression platforms [20]. Finally, their use allows spatial, temporal and
dosage control over target gene expression. It is therefore
not astonishing that engineered riboswitches were introduced early to the fields of genetic engineering and synthetic biology [14, 21] and have seen widespread application since then [22–26].
This review is not intended to serve as a comprehensive overview of all aspects of riboswitch engineering. For
example, we do not cover the wide fields of diagnostic
and therapeutic aptamers and riboswitches, which have
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
been addressed elsewhere [23, 27–30]; see also the September 2014 special issue on Aptamers published in
Molecular Therapy – Nucleic Acids. Our aim is rather to
present three topics we consider to be of prime importance for riboswitch biotechnology. We not only demonstrate how far the field has progressed since the early days
of artificial in vitro “riboregulators” [12], but also present
strategies we believe are important for isolating in vivo
active riboswitches, as well as challenges that will be
faced in designing more versatile and effective riboswitches. We hope this format will stimulate discussions
that will lead to new and improved approaches and
selection systems and, ultimately and hopefully, to the
development of new and more efficient synthetic riboswitches.
2 Engineered riboswitches:
An overview of the status quo
2.1 The toolbox of engineered riboswitches
recapitulates the functionality of natural
riboswitches
Natural riboswitches control translation, transcription,
ribozyme activity, and splicing [15–17, 31, 32]. In the
majority of these cases, they act in cis as part of the mRNA
transcript. But they can also regulate in trans either as a
processed, terminated riboswitch that functions like an
sRNA [33] or as a full-length transcript containing a binding site for sequestering an activated RNA-binding protein [34, 35] or by acting as an antisense RNA [36]. In synthetic biology, creating complex and more realistic genetic circuits requires that we eventually identify and design
“BioBricks” that not only reflect the entire spectrum of
genetic regulatory elements, but that can also be tailored
and fine-tuned to obtain the desired output level of the
target gene. The first step towards achieving this goal is
the construction of proof-of-principle applications to
demonstrate that we are able to reproduce these genetic
elements using artificial and/or heterologous components. The following section will provide an overview of
such archetype constructs. More detailed descriptions
can be found in several other reviews [25, 26].
In bacteria, engineered riboswitches most often target
translation initiation, either by controlling access to the
ribosomal binding site through helix slippage [37] or by
sequestering this Shine-Dalgarno (SD) element (Fig. 1A)
[38]. Regulation of gene expression by a theophylline-controlled translational switch was efficient enough to allow
the control of chemotaxis in Escherichia coli [39]. In contrast, transcriptionally-controlled synthetic riboswitches
have only recently been introduced to the toolbox by the
Mörl [40], Batey [41, 42], and Micklefield [43] groups. Here,
a transcriptional terminator is formed depending on the
binding state of the aptamer (Fig. 1B). It will be interest-
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Figure 1. Common mechanisms of
engineered riboswitches in bacteria.
(A) Regulation of translation initiation:
In the absence of a ligand, a stem-loop
structure is formed between the aptamer
domain (depicted in green) and a
sequence element complementary to the
Shine-Dalgarno (SD) sequence (magenta). Thus, the SD sequence (orange) is
accessible for 30S binding and translation initiation occurs. As a consequence
of ligand binding (blue circle) and folding of the aptamer domain, an alternative stem-loop is formed which
sequesters the SD sequence, blocking
the binding of the 30S ribosomal subunit. (B) Regulation of transcription termination: The aptamer domain is fused
to a short spacer region (grey), followed
by a sequence complementary to the
3’ part of the aptamer (magenta) and a
U stretch. In the absence of ligand, the
complementary 3’ part is base-paired with
the aptamer forming a terminator structure; thus, RNA polymerase (RNAP) dissociates and transcription is terminated.
Upon ligand binding, terminator structure formation is inhibited and transcription can proceed, resulting in expression
of the reporter gene. (C) Control of gene
expression with catalytic riboswitches:
A ligand-dependent aptazyme is inserted
into the 5’-UTR of an mRNA in such a
way that ligand-induced self-cleavage
liberates a sequestered SD sequence
to induce translation initiation.
ing to see how these devices perform with respect to regulatory efficiency, basal activity, noise, and kinetic parameters, in comparison to analogous, but translationally controlled riboswitches in similar experimental settings.
The cleavage activity of small ribozymes, like the hammerhead ribozyme, was made ligand-dependent by
inserting an aptamer domain into one of its three stemloops [44]. Luckily, all three stem-loops of the hammerhead ribozyme are amenable to aptamer insertion [45–48],
greatly facilitating the applicability of such “aptazymes”,
because the “best fit” between ribozyme and target
sequence can now be exploited. In bacteria, such aptazymes were used to liberate the ribosomal binding site
after ligand-dependent ribozyme cleavage (Fig. 1C). With
respect to the thermally-controlled riboswitches mentioned below in Section 2.3 [49], it will be interesting to
see if the recently introduced “thermozymes” [50] can also
be modified to include an additional aptamer control element. The examples listed here indicate that in bacteria
engineered riboswitches function in regulatory settings
that are also found for natural riboswitches.
In eukaryotes, translational regulation by engineered
riboswitches functions differently. Here, insertion of the
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riboswitch into the 5’-untranslated leader region leads to
inhibition of translation initiation, either by preventing
binding of the small ribosomal subunit to the mRNA cap
structure or by interfering with ribosomal subunit scanning for the AUG start codon (Fig. 2A). Here, aptamers
which specifically recognize a Hoechst dye [14], malachite green [51], tetracycline [21, 52], neomycin [53],
biotin or theophylline [54] (Fig. 2A) have been engineered
to serve as regulatory elements. The riboswitches were
active in yeast, Xenopus oocytes and mammalian cell culture [55]. Cap-independent translation initiation by an
internal ribosome entry site has also been controlled by
engineered riboswitches. However, up to now this has
only been demonstrated in vitro [56].
The only naturally occurring eukaryotic riboswitches
identified so far have been discovered in filamentous fungi, green algae and higher plants where they control gene
expression via regulation of mRNA splicing [57–60]. Analogously, theophylline- or tetracycline-binding aptamers
have been used to control constitutive and alternative
splicing by placing them close to either the 5’-splice site,
the 3’-splice site or the intron internal branch point
(Fig. 2B) [61–63].
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Figure 2. Common mechanisms of
engineered riboswitches in eukaryotes.
(A) Regulation of ribosome scanning:
After insertion of the aptamer (depicted
in green) into the 5’UTR of a mRNA, ligand binding (blue circle) to the aptamer
prevents scanning of the 40S ribosomal
subunit and, consequently, translation
initiation. (B) Regulation of mRNA splicing: An aptamer is integrated into an
intron of a eukaryotic mRNA to control
the accessibility of the 5’ splice site (SS).
Ligand binding inhibits splicing. (C)
Control of gene expression with catalytic
riboswitches: A ligand-dependent
aptazyme is inserted into the 5’-UTR of a
eukaryotic mRNA. Ligand-induced selfcleavage triggers RNA degradation
(magenta pacman). (D) Regulation of
RNA interference: An integrated aptamer
domain (green) interferes in its ligandbound state with the enzymatic activity
of Dicer.
Aptazyme-mediated control of gene expression in
yeast and in mammalian cell lines using hammerhead
ribozymes (Fig. 2C) has been demonstrated by several
groups [46, 48, 64–66]. Here, ligand-dependent ribozyme
cleavage leads to mRNA degradation. Besides the hammerhead ribozyme, the hepatitis delta virus ribozyme has
also been successfully applied to RNA engineering [67].
RNA interference is an important and widely used
mechanism in eukaryotes to repress gene expression by
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base-pairing to mRNA molecules. RNA interference controls a broad range of developmental and physiological
processes [68–70]. It is also a standard method in molecular biology with a possible therapeutic option which is
being tested in clinical trials [71]. Consequently, attempts
to control the biogenesis pathway of RNA interference
were made by introducing theophylline-dependent
aptamers [72, 73] or aptazymes [74] at sites of siRNA or
shRNA molecules where their presence can interfere with
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processing by either Drosha or Dicer (Fig. 2D). So far,
extremely high ligand concentrations are needed for efficient regulation. Optimization of the experimental strategies is clearly needed and it will be challenging to see if
endogenous miRNA molecules can also become subject
to riboswitch control.
2.2 Riboswitch regulation is becoming more robust
and versatile
While proof-of-principle applications are important for
demonstrating the functionality of any kind of experimental approach, they do not, per se, demonstrate their
general transferability to other systems, organisms or
even across the kingdoms of life. Undoubtedly, the general applicability and the ease with which system functionality can be achieved is an important parameter for determining success and acceptance of an application as a tool
by the respective community. So far, most of the regulatory strategies presented in the previous section were
tested in the well-characterized model organisms E. coli
for Gram-negative bacteria, Bacillus subtilis for Grampositive bacteria, Saccharomyces cerevisiae for lower
eukaryotes and mammalian cell lines for higher eukaryotes [19]. In developmental biology, biotechnology and in
medicine, other organisms are frequently employed as
physiological, developmental and disease models or as
cell factories for the production of materials [75–80]. Since
endogenous riboswitches have been identified in the
genomes of many different organisms [81], it seem reasonable to assume that engineered riboswitches, based
on similar regulatory principles, should also be active in
these species.
In bacteria, the Gallivan lab, consequently, designed a
set of theophylline-inducible translationally controlled
riboswitches, initially termed A-E, which vary in the
strength of their ribosomal binding sites and the stability
of their secondary structures [38]. Later, riboswitches designated E* (supplemental material in [38]), F (clone 8.1
from [82]), and G (clone D2 from [83]) were added to the
set. These riboswitches were then tested for activity in
diverse bacterial species, including the developmental
model organism and major producer of antibiotics, anticancer and antiviral drugs or enzymes, Streptomyces
coelicolor [84], cyanobacteria, model organisms for photosynthesis and potential material-producing cell factories
[85, 86] or tobacco chloroplasts, which are of interest in
plant biotechnology [87]. The riboswitch set was also
evaluated in important pathogens like Agrobacterium
tumefaciens, Acinetobacter baumannii, Streptococcus
pyogenes [38], Mycobacterium [88] and Francisella [89].
The tetracycline aptamer was shown to be functional in
the anaerobic model organism Methanosarcina acetivorans, an archaeon [90]. In all organisms tested, at least one
riboswitch was active enough to establish a conditional
knockout of an endogenous gene. In some cases, the reg-
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ulatory factors obtained were similar to those obtained
with transcriptional regulation. Effector molecule penetration across several membranes was also effective
enough to permit target gene expression control in chloroplasts and in intracellular infections models.
Theophylline was the preferred ligand of choice. It can
be added at high concentrations (2–4 mM) to the medium
without causing obvious toxicity. In contrast, tetracycline
and neomycin are antibiotics in bacteria and the concentrations needed for riboswitch activity can be close to or
above the respective minimal inhibitory concentrations.
Unfortunately, anhydrotetracycline, a less toxic derivative
of tetracycline, does not bind to the tetracycline aptamer
[91]. Translational ON-switches are the most frequently
used type of expression platform. They appear the easiest
to integrate into the sequence of the target gene. And
finally, there is no single “optimal” riboswitch. Depending
on the species and the specific target gene, different
riboswitches displayed the best regulatory properties. So
far, several constructs have to be tested to find the right
combination for a given application.
Regulation of gene expression by synthetic riboswitches is not limited to bacteria. Viral replication was
inhibited in eukaryotic DNA (adenovirus, adeno-associated virus) and RNA viruses (measles) by flanking essential
genes with theophylline-dependent aptazymes [92, 93].
Taken together, gene expression regulation by riboswitches has been demonstrated to be stringent,
reversible, and dose-dependent in viruses, organelles,
archaea, and bacteria from a broad taxonomic range of
species, firmly establishing this technology as robust and
universally applicable.
2.3 Riboswitches can be integrated into complex
regulatory circuits
Genetic circuits are frequently more complex than simple
one input/one output type networks. They can have complex architectures like toggle switches [94], oscillators
[95], Boolean logic gates that integrate several external
signals [96], and binary or graded responses [97]. They
can also include positive [98] or negative [99] feedback
loops or linearizers [100]. These examples of engineered
circuits were all realized with protein-based transcription
factors. Consequently, the interesting question is to determine if RNA-based devices can be used similarly to
assemble multiple input or complex regulatory circuits.
Nature itself has come up with several combinatorial
solutions to tackle diverse regulatory problems. In B. subtilis, the glmS riboswitch probably represents the simplest solution to regulation by multiple signals. The ligand-binding pocket of the aptamer domain not only binds
intracellular glucosamine-6-phosphate as an activating
agent. It also binds glucose-6-phosphate, which acts as
an inhibitor of ribozyme activity. The riboswitch, thus,
integrates metabolite information to sense the overall
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metabolic state of the cell [101]. A second solitary element
that senses and integrates two different input signals is
the adenine riboswitch encoded by the add gene from
Vibrio vulnificus. This translational regulatory element
senses temperature and ligand concentration to confer
efficient regulation over the range of physiologically relevant temperatures, allowing gene expression control to
adapt to the differing temperatures experienced in the
free-living and in the infectious environments [49]. In
Bacillus clausii, two different riboswitches, which
responded independently to S-adenosylmethionine and
vitamin B12, respectively, were found in tandem in the 5’
untranslated leader region (UTR) of the metE mRNA. System output fits a Boolean NOR gate, because transcription is terminated in the presence of only one or of both
ligands [102].
The Yokobayashi group was first to exploit this strategy in riboswitch engineering. They embedded aptamers
that recognize theophylline and thiamine pyrophosphate,
respectively, in tandem in the 5’-UTR of a bacterial mRNA
and selected for translational riboswitches that responded to ligand presence like AND or NAND logic gates.
Reporter gene activity was observed either only in the
presence of both ligands (AND gate), or, in the case of the
NAND gate, when neither or only one ligand was present
[103]. In a subsequent, different experimental setup, they
combined a transcriptional-OFF riboswitch with a translational-ON switch. Because both switches respond to the
same ligand, thiamine pyrophosphate, albeit with different sensitivity, a band pass filter is the result, with maximum target gene expression obtained at intermediate ligand concentrations. At low ligand concentrations, gene
expression is repressed translationally, while at high concentrations, premature transcription termination occurs
[104]. Boolean logic gates can also be constructed with
aptazymes. The Hartig group [105] combined ribozyme
switches responsive to thiamine pyrophosphate and
theophylline to yield AND, NOR, and ANDNOT gates. Win
and Smolke [106] also generated an entire set of Boolean
logic gates by combining theophylline- and tetracyclineresponsive aptamers with ON- and OFF-responsive
ribozymes. For example, such gates were constructed
either by assembling two different aptazymes in series
(AND gate and NOR gate) or by, very ingeniously, exploiting the possibility of joining aptamer-responsive control
elements to all stems of the hammerhead ribozyme. Coupling two ON-switches responsive to different inputs to
different ribozyme stems led to an output signal only if no
or one of the ligands was present, thus generating a
NAND gate [106].
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3 What makes a riboswitch
out of an aptamer?
So far, in vivo active riboswitches rely on a small set of ligands – theophylline, tetracycline, neomycin [53], 2,4-dinitrotoluene [107], ammeline, 5-aza-cytosine [108] or pyrimido[4,5-d]pyrimidine-2,4-diamine (PPDA) [43], although
several dozen small molecule-binding aptamers have
been selected by a process called SELEX (systematic evolution of ligands by exponential enrichment), an in vitro
technology invented about a decade before the discovery
of natural riboswitches [109, 110]. During SELEX, a huge
combinatorial library of RNA (and sometimes DNA) molecules is subjected to affinity of a small RNA chromatography to the ligand of choice. Non-binding sequences are
removed during washing steps, whereas sequences with
a certain binding affinity to the target of interest are eluted. These molecules are amplified and subjected to further rounds of selection. During these iterative cycles, the
selection pressure can be increased steadily, finally resulting in molecules with sometimes extraordinarily high
binding affinities and specificities, the so-called aptamers
(Fig. 3A). However, as stated above, only a handful of such
small molecule-binding aptamers have made it into the
toolbox 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 be
used for riboswitch engineering.
A detailed analysis of neomycin-binding aptamers
could shed some light on the properties such aptamers
need in order to become regulatory molecules that are
active in vivo. The neomycin-binding aptamer R23 was
isolated by in vitro selection [111] and shows both high
affinity and specificity for its ligand [111]. However, this
aptamer – like many other small molecule-binding
aptamers, too – showed no ligand-dependent regulation
when tested for riboswitch activity in an assay for translational regulation (like in Fig. 2A) [53]. The subsequent
analysis of the enriched aptamer pool (after seven rounds
of selection) for sequences with regulatory activity using
an 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 new
neomycin aptamer is active as riboswitch when inserted
into the 5’-UTR of a reporter gene (Fig. 2A) [53], but also
as sensing domain in the context of an allosteric ribozyme
(Fig. 2C) [66]. Interestingly, N1 was not among the
sequenced aptamers isolated after in vitro selection indicating that this aptamer was underrepresented in the
pool enriched for binding aptamers [111].
A detailed genetic, biochemical and structural analysis of the aptamer N1 unravelled the molecular basis of
this regulation. While the aptamer shows tight ligand
binding with a Kd in the low nanomolar range, this affinity was still very similar to that of the originally identified
in vivo inactive aptamer R23 [112]. However, substantial
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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-dependent
changes in GFP expression. (B) The regulating neomycin-binding aptamer N1. The secondary structures of the ground- and ligand-bound states of the
neomycin-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.
conformational changes concomitant to ligand binding
were detected in N1, which were completely absent in
the inactive aptamer R23 [113]. A short intervening
sequence separating two short helical elements was
identified as being important for regulation and therefore called “switching element”. It allows the ground
state of the aptamer to retain an open, less structured
conformation. Ligand binding then stabilizes a highly
structured conformation, in which additional base pairs
are formed. These elongate the two short helices and
promote stacking of the upper onto the lower helix
resulting in a long α-helical element (Fig. 3B). The ligand
neomycin then acts like a clamp holding both the upper
and the lower helix together. This conformation then is
able to efficiently interfere with ribosomal scanning in
yeast. It seems that the interplay between an open
ground state and a highly structured ligand-bound state
is important for the regulatory activity of the aptamer.
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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 regulating aptamers have to possess two properties: first, high
affinity ligand binding, and second, significant conformational changes and stabilization upon ligand binding. The
first point can be addressed in any classical SELEX experiment. By increasing the stringency of the selection
process, high affinity binding aptamers can be obtained
with binding constants even in the picomolar range [116].
Tight binding, however, is necessary but not sufficient for
regulation. The second requirement – the conformational
changes within the aptamer upon ligand binding – cannot be addressed by in vitro selection. An additional cellular screening step, preferably directly in the organism of
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choice, is necessary to identify aptamers with regulatory
activity.
Several approaches have been undertaken which not
only support the success, but also reinforce the importance of cellular screening for the identification of in vivo
active regulatory devices. Gallivan and colleagues applied
different screens to identify in vivo active riboswitches
[83, 117], which proved to be extremely powerful in bacteria (see also Section 2.2). In vivo screening was also
applied to identify aptamer-controlled ribozymes. Here,
the communication module connecting the sensing
domain (aptamer) with the catalytic domain (ribozyme)
was randomized [118, 119]. Together with the neomycin
screen described above, these examples underline the
importance of this approach.
4 How can we further improve riboswitches?
Chris Berens studied biology in Erlangen 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 Greifswald and Erlangen where she received
Despite of the success engineered riboswitches are
enjoying as regulatory devices, there are still two important problems that have to be overcome to allow us to
build even better riboswitches. One is the lack of a large
number of alternative aptamers as ligand-binding modules and the second is the large discrepancy between the
in vitro affinities of aptamers for their ligands and the high
concentrations of ligand needed in vivo to flip the corresponding switches.
Besides introducing an additional cellular screening
step which allows the identification of regulating aptamers
as specified above, reengineering the aptamer domain
scaffolds of existing functional riboswitches to recognize
novel ligands is also a promising approach to extend the
ligand toolbox. The Micklefield group mutated a purine
riboswitch to not recognize its natural ligand anymore
and then screened this mutant pool for binding to synthetic, heterocyclic molecules. In the end, they obtained
a ligand, PPDA, which controlled riboswitch activity efficiently [43, 108]. It will be interesting to see if this
approach can be extended successfully to other natural
riboswitches and, how different any potential novel ligands can be structurally from the respective native ligands. The many approaches to identify inhibitors of
riboswitch activity for therapeutic applications might
serve as starting points for finding such alternative ligands [120–122]. More extensive mutagenesis might contribute to this process, because natural sequence variants
of riboswitches already display functional diversity,
which is thought to reflect the adaptation to the idiosyncratic regulatory requirements of a specific gene [123].
Correspondingly, mutagenesis of residues that are not
directly involved in ligand binding affect the regulatory
properties of riboswitches [124, 125].
The ligand-binding site of the tetracycline aptamer is
formed by a three-way-junction that is stabilized by longrange interactions and almost completely envelops the
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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-University Frankfurt, Main. Since 2012, she is full professor for Synthetic
Biology at the Technical University Darmstadt.
ligand [126]. This is reminiscent of natural riboswitches
and distinguishes the tetracycline aptamer from the ligand-binding pockets of many aptamers which are much
more open and solvent-exposed [127]. It might suggest
that alternative aptamer selection protocols, like capillary
electrophoresis, that do not require coupling of the ligand
to a matrix and, thus, do not force surface exposure might
be more advantageous for isolating in vivo functional
aptamer domains [128–130].
A discrepancy between high affinity binding and efficient regulation has not only been observed for engineered riboswitches, but also for the tetrahydrofolate
riboswitch. Although several ligand analogs and adenine
derivatives were found to bind with high affinity to the
riboswitch, they were not able to regulate its activity
[131]. The mechanism(s) responsible for this effect are not
clear at the moment, although for synthetic riboswitches,
the fusion of an aptamer domain to an expression platform
might result in a suboptimal interface between the two
elements. Here, some optimization by mutagenesis and
selection might be needed. A recent publication by the
Gallivan lab might aid in this optimization process [132].
Mishler & Gallivan were able to recapitulate the approximately 1000-fold discrepancy between the concentrations needed for efficient binding of theophylline to its
aptamer and the concentration needed for activating the
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riboswitch in E. coli in vitro in an S30 cell extract. This
might finally allow in vitro screening and selection for
more efficient switches requiring less ligand [132].
5 Conclusions
Riboswitch engineering has come a long way since the
first applications were introduced approximately fifteen
years ago. This technology has finally reached a stage in
which it can not only complement other established mechanisms and strategies to regulate gene expression, but in
which there are applications where we believe that it can
even effectively replace these. The riboswitch engineer’s
toolbox now provides a broad platform which should be
able to offer flexible solutions to most synthetic biology
applications requiring regulation of gene expression.
Advances in selection technology and further improvement of our understanding of riboswitch structure and
function will guide the design of better riboswitches to
harness the full regulatory, diagnostic and therapeutic
potential of aptamer-controlled regulatory devices.
We would like to thank the Deutsche Forschungsgemeinschaft (SFB902/A2) and Loewe CGT for financial support.
EU FP7-KBBE-2013-7 no. 613745, Promys
The authors declare no financial or commercial conflict of
interest.
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ISSN 1860-6768 · BJIOAM 10 (2) 227–338 (2015) · Vol. 10 · February 2015
Systems & Synthetic Biology ·
Nanobiotech · Medicine
2/2015
Minimal cells
Artificial chromosomes
Expression control
Synthetic
Biology
www.biotechnology-journal.com
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
Biotechnology Journal – list of articles published in the February 2015 issue.
Editorial: Synthetic biology – ready for application
Roland Eils, Julia Ritzerfeld, Wolfgang Wiechert
http://dx.doi.org/10.1002/biot.201400842
Forum
Synthetic biology’s self-fulfilling prophecy –
dangers of confinement from within and outside
Daniel Frank, Reinhard Heil, Christopher Coenen,
Harald König
http://dx.doi.org/10.1002/biot.201400477
Forum
Synthetic biology and intellectual property rights:
Six recommendations
Timo Minssen, Berthold Rutz, Esther van Zimmeren
http://dx.doi.org/10.1002/biot.201400604
Commentary
Fully glycerol-independent microbial production
of 1,3-propanediol via non-natural pathway:
Paving the way to success with synthetic tiles
Ewelina Celińska
http://dx.doi.org/10.1002/biot.201400360
Commentary
Chassis organism from Corynebacterium glutamicum: The way
towards biotechnological domestication of Corynebacteria
Víctor de Lorenzo
http://dx.doi.org/10.1002/biot.201400493
Review
RNA aptamers as genetic control devices: The potential
of riboswitches as synthetic elements for regulating gene
expression
Christian Berens, Florian Groher and Beatrix Suess
http://dx.doi.org/10.1002/biot.201300498
Review
CRISPR genome engineering and viral gene delivery:
A case of mutual attraction
Florian Schmidt and Dirk Grimm
http://dx.doi.org/10.1002/biot.201400529
Review
Optogenetic control of signaling in mammalian cells
Rapid Communication
Protein design and engineering of a de novo pathway
for microbial production of 1,3-propanediol from glucose
Zhen Chen, Feng Geng and An-Ping Zeng
http://dx.doi.org/10.1002/biot.201400235
Research Article
Chassis organism from Corynebacterium glutamicum –
a top-down approach to identify and delete irrelevant
gene clusters
Simon Unthan, Meike Baumgart, Andreas Radek,
Marius Herbst, Daniel Siebert, Natalie Brühl, Anna Bartsch,
Michael Bott, Wolfgang Wiechert, Kay Marin, Stephan Hans,
Reinhard Krämer, Gerd Seibold, Julia Frunzke,
Jörn Kalinowski, Christian Rückert, Volker F. Wendisch
and Stephan Noack
http://dx.doi.org/10.1002/biot.201400041
Research Article
Synthetic secondary chromosomes in Escherichia coli based
on the replication origin of chromosome II in Vibrio cholerae
Sonja J. Messerschmidt, Franziska S. Kemter,
Daniel Schindler and Torsten Waldminghaus
http://dx.doi.org/10.1002/biot.201400031
Research Article
Bacterial XylRs and synthetic promoters function as genetically
encoded xylose biosensors in Saccharomyces cerevisiae
Wei Suong Teo and Matthew Wook Chang
http://dx.doi.org/10.1002/biot.201400159
Biotech Method
Single-cell analysis reveals heterogeneity in onset of transgene
expression from synthetic tetracycline-dependent promoters
Ulfert Rand, Jan Riedel, Upneet Hillebrand, Danim Shin,
Steffi Willenberg, Sara Behme, Frank Klawonn, Mario Köster,
Hansjörg Hauser and Dagmar Wirth
http://dx.doi.org/10.1002/biot.201400076
Biotech Method
Engineering connectivity by multiscale micropatterning
of individual populations of neurons
Jonas Albers, Koji Toma and Andreas Offenhäusser
http://dx.doi.org/10.1002/biot.201400609
Hannes M. Beyer, Sebastian Naumann, Wilfried Weber
and Gerald Radziwill
http://dx.doi.org/10.1002/biot.201400077
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.biotechnology-journal.com