Download Designer nucleic acids to probe and program the cell

TICB-917; No. of Pages 10
Review
Special Issue – Synthetic Cell Biology
Designer nucleic acids to probe and
program the cell
Yamuna Krishnan1 and Mark Bathe2
1
2
National Centre for Biological Sciences, TIFR, GKVK, Bellary Road, Bangalore 560 065, India
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Recent advances in nucleic acid sequencing, structural,
and computational technologies have resulted in dramatic progress in our understanding of nucleic acid structure
and function in the cell. This knowledge, together with the
predictable base-pairing of nucleic acids and powerful
synthesis and expression capabilities now offers the
unique ability to program nucleic acids to form precise
3D architectures with diverse applications in synthetic
and cell biology. The unique modularity of structural
motifs that include aptamers, DNAzymes, and ribozymes,
together with their well-defined construction rules,
enables the synthesis of functional higher-order nucleic
acid complexes from these subcomponents. As we illustrate here, these highly programmable, smart complexes
are increasingly enabling researchers to probe and program the cell in a sophisticated manner that moves well
beyond the use of nucleic acids for conventional genetic
manipulation alone.
Introduction to functional nucleic acid nanostructures
Over 50 years ago, it was discovered that RNA had catalytic activity in addition to its role as an informationcarrying polymer, giving rise to the hypothesis that life
may have evolved from a precursor ‘RNA World’ without
proteins or DNA [1,2]. Today, the diverse structures and
activities adopted by RNAs are revealing a panoply of
cellular functions that were barely imaginable at the time
of this postulation by Alexander Rich and colleagues [1,2].
Unlike polypeptides, polynucleotides are structurally
highly programmable polymers due to the specific, pairwise Watson–Crick interactions of nucleic acid bases that
result in well-defined secondary structures of predictable
form. This, together with a simple set of construction rules
established for DNA in a landmark paper [3], the rapidly
decreasing cost of nucleic acid synthesis, the programmability of RNA expression in living cells, and our increasing
knowledge of higher-order RNA structure and chemical
activity, is resulting in an explosion in the number and
diversity of nucleic acid structures that are being designed
for diverse applications in science and technology, with a
recently emerging focus on designer nucleic acids to probe
and program the cell.
Corresponding authors: Krishnan, Y. ([email protected]);
Bathe, M. ([email protected]).
Keywords: synthetic biology; RNA nanotechnology; DNA nanotechnology.
As with most emerging technologies, with novelty comes
expectation and anticipation. Notwithstanding, a number
of recent works show real promise for transforming our
abilities to measure and manipulate cell function in highly
programmable ways that cannot be achieved with existing
technologies. We review several of these works and provide
our perspective on what we expect to be promising avenues
of research to pursue in this rapidly evolving, largely
uncharted territory.
Programming nucleic acid nanostructure shape via selfassembly
A hallmark of DNA nanotechnology is the ability to program structurally well-defined, thermodynamically stable
3D polyhedra using Watson–Crick base-pairing and construction rules [3,4], where the edges of the polyhedra
comprise individual DNA duplexes. Now, conventional
3D shapes that may be produced with high yield and
sample homogeneity include tetrahedra, icosahedra,
cubes, dodecahedra, and, more recently, larger-scale, considerably more complex wireframe structures [5] using the
DNA origami approach [6], in which approximately 200
short strands are used to ‘staple’ together a longer singlestranded DNA (ssDNA) scaffold strand, reviewed in [7–9]
(Figure 1a–c). Advanced computational tools enable the
rational design of these structures accounting for thermodynamic and mechanical stability key to function [9–12].
The principal advantages of using DNA to design
nucleic acid architectures include its long chemical halflife and highly specific Watson–Crick base-pairing that
offers the ability to design stable nanostructures of precise
3D form [12,9]. By contrast, non-canonical base-pairing
acquired by RNA primary structures results in considerably more diverse, dynamic secondary structures [13,14]
that are additionally subject to hydrolytic instability. This
Glossary
3WJ: a DNA or RNA motif comprising three duplexes radiating from a single
node; for example, the replication fork is a naturally occurring DNA 3WJ.
Fluorescence in situ hybridization (FISH): a technique used to visualize
endogenous DNA or RNA molecules in cells and tissues using fluorescently
labeled antisense oligonucleotides.
Hybridization chain reaction (HCR): a process by which metastable hairpins
assemble by a cascade of hybridization events to form a long nicked duplex.
Systematic evolution of ligands by exponential enrichment (SELEX): uses in
vitro evolution to confer specific chemical properties to DNA and RNA
molecules, typically high-affinity binding to a target small molecule.
0962-8924/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tcb.2012.10.001 Trends in Cell Biology xx (2012) 1–10
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Figure 1. (a) Left: schematic of the assembly of four DNA strands to form a DNA tetrahedron via annealing and ligation. Reproduced, with permission, from [18]. Right:
atomic model and cryo-electron microscopy reconstruction of the tetrahedron. Reproduced, with permission, from [102]. (b) Left: overview of the scaffolded DNA origami
concept, showing crossovers formed by staple strands every 21 base pairs to form a 2D rectangle [9]. Right: atomic model of a 52 nm diameter wireframe structure [5,11]
compared with 2D transmission electron microscopy. Reproduced, with permission, from [5]. (c) Top: atomic model of a tetrahedron functionalized with DNA oligomers at
its vertices and cryo-electron microscopy reconstruction. Reproduced, with permission, from [24]. Bottom: schematic of physical encapsulation of fluorescent cargo (yellow
oval) within a DNA icosahedron that binds anionic ligand-binding receptors (ALBRs) to map the pH of endosomes as they mature. Reproduced, with permission, from [22].
(d) Functionalizable RNA nanoring scaffolds (grey) assembled through kissing-loop interactions between RNA modules. Nanoring scaffolds are functionalized with RNAs
(red) that can penetrate the siRNA pathway. Reproduced, with permission, from [28]. (e) RNA cubes constructed from six prefolded RNA building blocks. Reproduced, with
permission, from [27]. (f) Atomic model and 3D cryo-electron microscopy reconstruction of a non-uniform square antiprism constructed from self associating tRNA-based
motifs. Reproduced, with permission, from [31].
relative lack of structural fidelity with respect to DNA,
however, is largely offset by its rich conformational and
catalytic space that may be used to program associations of
high affinity and specificity using aptamers [15], enzymatic
activity using ribozymes, and allostery using riboswitches
[16,17] (Figure 2). Further, the genetic encodability of RNA
offers the exciting opportunity to program cells to express
precise nanoarchitectures for sensing, scaffolding, and
signaling, among other functions, positioning RNA nanotechnology as a powerful medium to probe and program
cell function [18].
Programming DNA architectures for targeted delivery
The tetrahedron has arisen as a popular 3D template due
to its minimal DNA content and high synthetic yield [18]
(Figure 1a,c). An important early advance was the demonstration that a single covalently linked protein molecule
(cytochrome C) could be positioned within the tetrahedral
cavity, opening up the possibility to use the interior space
as a protected cargo hold for delivery [19]. To display such
protected cargo to external receptors, an innovative barrelshaped structure was designed [20] that sequesters T cellstimulating Fab fragments (the cargo) within its interior,
hidden from the external milieu when the barrel is closed
2
(Figure 3a). The hexagonal barrel remains closed with the
help of hasps comprising an aptamer and its complement
that dissociate when specific combinations of ligands bind
the aptameric strand. These act as AND gates for the detection of NKL cells from a mixture of NKL cells and leukocytes,
with the subsequently opened barrel exposing key Fab fragments that trigger specific cellular processes such as T cell
activation or growth arrest. This work demonstrated that
allosteric DNA-based containers may be programmed to
expose cargo to cell-surface receptors based on selective,
specific, logic-based multivalent binding in cell culture.
In cases where the covalent linkage of molecular cargo
to the DNA scaffold is infeasible either due to lack of
reactive groups or because molecular activity would be
compromised, alternative approaches are still needed.
One means of eliminating chemical conjugation is to use
physical entrapment, whereby the cargo is physically constrained within DNA polyhedra. This strategy was
employed [21] to retain fluorescent dextrans or gold nanoparticles in DNA nanocages, and it was subsequently
shown that these loaded polyhedra could be targeted specifically to single Caenorhabditis elegans cell types that
expressed receptors for the DNA cage without compromising cargo functionality [22] (Figure 1c). Programmed
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Aptamer
Aptazyme
Mn+
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Transcriponal
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TRENDS in Cell Biology
Figure 2. Schematic showing different functional nucleic acid motifs. Red stars and black circles represent reporter fluorophores. Aptamer: a DNA or RNA molecule that
specifically binds a small molecule or biomolecule (green oval). Aptazyme: a DNA or RNA molecule that is comprised of an aptamer domain fused to a catalytic domain,
where the latter becomes functional upon ligand-binding to the aptameric domain. Ribozyme/DNAzyme: an RNA/DNA molecule with catalytic properties, typically
consisting of a self-cleaving reaction in the presence of metal ions (denoted Mn+). Similar to aptazymes, the cleaving reaction is typically followed by strand dissociation.
Allosteric aptamer: aptameric modules sensitive to distinct ligands, typically an analyte and a reporter molecule, fused to one another so that the reporter molecule (red
star) may bind only when the analyte (green oval) is present. Riboswitches: regulatory RNA elements that act in cis by binding a small molecule, switching gene expression
on or off. Switches control either (left, Transcriptional) the formation of transcription terminator hairpins (red), (middle, Translational) accessibility to the Shine-Dalgarno
sequence (blue), or (right, Metabolite-binding) degradation of the transcript. Adapted from [18] and [79].
release of nanoparticles loaded within DNA scaffolds has
also been achieved by altering the pore size of the scaffold
using a complementary DNA strand [23]. This suggests
that it may be possible to both load and release encapsulated molecular cargo from containers in a programmed
manner. Interfacing cell targeting with programmed release of non-tethered cargo, however, remains an open
challenge.
In addition to their ability to encapsulate or sequester
cargo for programmed release, DNA nanostructures have
the capacity to act as pegboards using small-molecule
conjugation to position functional biomolecules precisely
in space due to their well-defined structure and addressable scaffold. One such demonstration was achieved using
DNA tetrahedra, in which short hairpins were grafted onto
the vertices of the scaffold without perturbing the tetrahedral geometry [24]. Such morphology-rich nucleic acid
motifs displayed on DNA tetrahedra were shown to retain
functionality [25] when it was demonstrated that CpG
motifs displayed in a tetrahedral fashion could effect immunostimulation of macrophages by interacting with Tolllike receptor 9.
Importantly, such multivalent CpG motifs showed enhanced immunostimulatory effects, presumably due to
increased ligand density. This increased ligand density
afforded by the immobilization of functional nucleic acid
motifs on a minimal DNA scaffold display was borne out in
vivo [26], when a DNA tetrahedron was designed that
displayed small interfering RNAs (siRNAs) as well as a
targeting ligand on each edge of the tetrahedron projecting
out from the tetrahedral scaffold (Figure 3b). The authors
showed impressive control over dosage by creating stoichiometrically identical particles in bulk with fixed ratios
of the number of siRNA duplexes per tetrahedron. Further, spatial orientation of the targeting ligands affected
the efficiency of RNAi, presumably by affecting the uptake
that is in turn dependent on the surface density of the
ligands. This suggests that aspects of endocytosis, such as
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Figure 3. (a) Functional design of a hexagonal barrel bearing Fab fragments (magenta) internally that opens in response to the binding of a signaling molecule (red circle) to
the aptameric strand (blue) of the hasps keeping the barrel closed. Reproduced, with permission, from [20]. (b) Schematic of tetrahedron assembly and structure obtained
from six DNA strands to which siRNA (orange) is hybridized. The tetrahedron bears one to six strands of siRNA in fixed stoichiometries. siRNA-bearing DNA tetrahedra are
shown to be specifically targeted to a tumor in a mouse model. Reproduced, with permission, from [26]. (c) Schematic of RNA aptamers used to localize protein synthesis
machinery to an RNA scaffold to create a larger-scale synthesis pathway in higher-order 1D and 2D structures. Reproduced, with permission, from [43].
ligand–receptor spacing and distinct uptake mechanisms
that facilitate endosomal escape or lysosomal delivery
may be interrogated using 3D designer DNA scaffolds [22].
Programming RNA architectures for targeted delivery
Advanced 3D designer RNA scaffolds have also been realized, including nanocubes [27] and nanorings [28], using
the assembly principle ‘RNA tectonics,’ which exploits the
modular nature of RNA molecules to form new architectures [29] (Figure 1d–f). RNA-based particles may be
functionalized with siRNA motifs of controllable stoichiometry and incorporate chemically modified RNA and
DNA analogs due to their synthetic nature, and may
therefore prove valuable for delivery in nanomedicine
[18,30]. Larger-scale RNA nanostructures approaching
the size of DNA origami objects of 5–25 nm have also
recently been achieved based on tectonics of tRNAs [31],
yielding fully addressable polyhedra functionalized with
proteins inside and outside the nanostructure. The bacteriophage phi29 DNA packaging motor pRNA has been used
as an alternative construction module [32], comprising a
117-nucleotide RNA that folds into a three-way junction
and further associates into dimers, trimers, or hexamers
[33,34]. This enabled the construction of functional heterodimeric RNA nanoparticles bearing a targeting moiety
such as folate, as well as an siRNA cargo that brings about
apoptosis in nasopharyngeal carcinomas [35].
One important limitation of RNA nanoparticles assembled through intermolecular tertiary interactions for cellbased applications, however, is the need for metal salts to
stabilize intermolecular junctions. Recently, this limitation
was surmounted by a new thermodynamically stable 3WJ
junction based on the pRNA junction that circumvents this
requirement and retains its structure at physiological concentrations of Mg2+. Further, this junction was programmed
using three distinct cargoes: a targeting moiety, an siRNA
against an antiapoptotic marker, and an aptamer or ribozyme, all of which proved to be functional in vivo [36].
Additionally, the realization of RNase-resistant yet biologically active pRNA [37] suggests that assembly of preformed
RNA components from native biological constructs will
4
remain an attractive alternative to de novo assembly from
synthesized nucleic acids until complex tertiary structures
can reliably be predicted computationally [38,39] or robust
principles of DNA-based assembly can be adopted in the
RNA domain. An alternative approach is to use protein
fragments to guide the assembly of nucleic acid building
blocks into well-defined structures. For example, the L7Ae
protein is known to bind the k-turn motif and force it to adopt
a bent conformation [40]. This has been used to form closed
polygonal shapes, with the protein occupying the vertices of
the associating RNA building blocks [41].
Programming biomolecular spatial arrays for synthesis
and signaling
Organizing biomolecules, in 3D to control, for example,
multi-enzyme or signaling cascades is another attractive
aim for nanostructure design. For example, increasing
local concentrations of reactants by spatially localizing
enzymes in close proximity resulted in increased downstream mevalonate production by approximately 70-fold
[42] using synthetic protein scaffolds in yeast. Limitations
of protein-based scaffolds, however, include propensity for
nonspecific aggregation, lack of scaffold addressability, and
lack of scaffold structural fidelity due to a limited ability to
program precisely higher-order protein nanostructures. As
an alternative, extended 1D and 2D RNA-based scaffolds
were designed [43 to organize catalytic enzymes in bacteria
to achieve enhanced rates of hydrogen synthesis of up to
approximately 50-fold, demonstrating in vivo structural
engineering of the intracellular environment (Figure 3c).
Similarly, increased metabolic flux in Escherichia coli was
achieved using a plasmid-based DNA scaffold and zincfinger binding motifs [44. Synthetic microcompartments
for sequestration of reactants similar to natural organelles,
such as carboxysomes, and scaffolding motifs for signaling
modules with allosteric functionality [45,46] represent
other enticing possibilities. In vitro scaffolded enzyme
cascades [47,48] studied in conjunction with computational
models will be useful in defining the optimal design criteria
for maximizing chemical synthesis yield and efficiency in
the cell.
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TRENDS in Cell Biology
Figure 4. (a) Hybridization chain reaction (HCR) principle whereby metastable fluorescent RNA hairpins H1 and H2 self-assemble into amplification polymers in the
presence of a specific target RNA initiator. An amplification cascade using fluorescent hairpin pairs results in localized amplification of fluorescence. Reproduced, with
permission, from [55]. (b) HCR applied to the multiplexed detection of five distinct mRNAs in whole-mount zebrafish embryos. Reproduced, with permission, from [55]. (c)
Top: chemical structures of 4-hydroxybenzylidene imidazolinone (HBI) (green) and 3,5-dimethoxy-4-hydroxybenzylidene imidazolinone (DMHBI) (black) are shown in
addition to the RNA aptamer Spinach that binds DFHBI but requires the formation of stem 2. Complexation of DMHBI with its aptamer (13-2) enhances its fluorescence.
Reproduced, with permission, from [58]. Bottom: S-adenosyl methionine (SAM) (magenta hexagon) sensor using RNA aptamer region Spinach (black), a transducer
domain (orange), and a SAM aptamer (blue). SAM binding of the sensor promotes the binding of DFHBI (green circle) to Spinach, leading to fluorescence. Bottom right:
distinct patterns of SAM accumulation after adding methionine to Escherichia coli expressing the sensor. From [60]. Reprinted with permission from AAAS. (d) top left:
Schematic of an i-motif powered, Förster resonance energy transfer (FRET)-based DNA pH sensor, the I-switch, that undergoes a pH triggered conformational change.
Bottom left: the I-switch is internalized by specific receptors present on coelomocytes (yellow cells) in Caenorhabditis elegans and is able to map spatiotemporal pH
changes associated with maturing endosomes on the anionic ligand-binding receptor (ALBR) pathway. Right: pH heat maps of early endosomes, late endosomes and
lysosomes along the ALBR pathway. Reproduced, with permission, from [63].
Probing the cell with fluorescence-based sensors
Biological sensors aim to recognize molecular inputs with
high specificity and sensitivity to transduce them into
readouts with minimal perturbation to the cell. Molecular
beacons are a simple early form of nucleic acid-based
biosensor comprising a single hairpin with a complementary probe sequence and quenched Förster resonance energy transfer (FRET) pair that fluoresces on hybridization
to the target mRNA/DNA [49,50]. Genomic DNA loci and
single RNA transcripts are also now routinely measured in
fixed cells using fluorescence in situ hybridization (FISH)
[51–54], which tiles numerous fluorescent probes along the
target sequence to enhance reporter signal. Although useful for fixed cells and tissue sections that require only
limited imaging depth, FISH still suffers from a limited
signal-to-noise ratio due to the finite number of fluorophores that can be tiled to the target transcript without
amplification and limited probe specificity for short
sequences. To overcome these limitations and enable single-step, multiplexed RNA imaging, a triggered hybridization chain reaction was used [55,56] to dramatically
increase the signal-to-noise ratio (Figure 4a). Here, RNA
probes folded into metastable hairpins harboring fluorescent reporters restructure in the presence of a desired
mRNA sequence into a nicked double-stranded polymer
that forms the basis of localized amplification of the fluorescent signal. This approach enabled multiplexed RNA–
FISH in whole-mount embryos of impressive clarity, albeit
still in fixed samples (Figure 4b).
Directly visualizing RNA molecules in live cells to probe
RNA trafficking, localization, and transcriptional bursting
remains an important challenge [52,57] that currently
limits our understanding of the burgeoning RNA layer of
gene regulation. A promising recent development towards
realizing live-cell mRNA imaging exploits the photophysical properties of the green fluorescent protein (GFP) fluorophore using RNA aptamers to yield multispectral RNA
mimics of GFP [58]. 4-Hydroxybenzylidene imidazolinone
(HBI), the fluorescent moiety in GFP, fluoresces when
caged in the beta-barrel core of GFP due to proper fluorescence emission from the excited fluorophore state, entering
the dark state when exposed to bulk solution. SELEX was
used [58] to obtain an RNA aptamer that binds DMHBI, a
chemical analog of HBI, resulting in fluorescence emission
in the bound state (Figure 4c). Further, the authors synthesized the small molecule DFHBI that is exclusively in
the phenolate form at neutral pH and also obtained an
aptamer, termed Spinach, that binds this target molecule.
DFHBI exhibits fluorescence when bound to Spinach that
is approximately half as bright as enhanced GFP (eGFP),
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yet brighter than many other fluorescent proteins, considerably more photo-stable than GFP, and does not require
the maturation time of GFP. Impressive live-cell imaging
of 5S RNA translocation in mammalian cells was achieved
when incorporated into the 30 UTR, which does not affect
localization.
To achieve conditional functionality, first described [59]
using the cytotoxic malachite green (MG), a metabolitebinding aptamer was fused [60] to Spinach using a communication module that confers proper folding of Spinach and
subsequent fluorescence in the presence of DFHBI and the
metabolite in a manner that is dependent on concentration of
the latter (Figure 4c). This enabled visualization of S-adenosyl methionine distribution in living bacteria, which, together with the ability to evolve aptamers to bind nearly any
small molecule, provides an exciting opportunity to image
bioactive small molecules and RNAs in living systems.
In addition to RNA-based switches, DNA-based switches
may also exhibit large-scale conformational rearrangements in response to molecular cues including pH that offer
the unique ability to map the latter in live cells. Spatiotemporal maps of pH in endocytic compartments within living
cells have provided important insights into the stages of
endosomal maturation, viral entry mechanisms, and their
crosstalk with secretory pathways [61]. Imaging endosomal
maturation within a multicellular, live organism was recently achieved using a FRET-based, pH-sensitive DNA
switch called the I-switch [62] that undergoes a conformational change under acidic conditions (Figure 4d). This
conformational change was mediated by i-motif formation
of C-rich sequences on the I-switch and the device was first
validated in cell culture [63]. Importantly, the functionality
of this pH-triggered device was quantitatively recapitulated
from the in vitro to the in vivo context, providing such
bistable DNA devices with a new role inside living systems
as probes of the intracellular chemical environments of
specific proteins.
Programming the cell to perform desired functions
Synthetic biology seeks to program novel functions into
living systems, for example, convert or synthesize biomolecules including biofuels, sense and destroy biological material including pathogens, harvest, convert, and store solar
energy, and test working hypotheses on the function of
complex living systems [64,65]. Engineered biological systems must therefore have the capacity to receive molecular
cues, perform context-dependent computation, and produce
outputs that lead to desired changes in cell state or function,
and achieve all of this under the noisy, heterogeneous conditions in which cells operate. Unlike solid-state machines
that principally seek to store and compute large amounts of
information, typically containing only a single mode of input
and output, biological computation exploits an opposing side
of the computational spectrum, typically comprising only a
few ‘states’ with large numbers of inputs comprising small
molecules, metabolites, RNAs, and protein, and outputs at
the levels of transcription, translation, ligation, cleavage,
and small-molecule signaling [17,64–67]. Here, we focus on
selected recent findings of relevance to a breadth of cell
biological problems and refer the reader to the preceding indepth reviews for additional background.
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Digital and analog controls of protein expression in
living systems are essential tools for probing the role of
protein function in determining cell state and behavior.
Controlling translation using small-molecule inducers is
of major interest for this purpose, both for the reversible
control intrinsic to such methods per se, and specifically for
the study, for example, of processes and pathogens not
amenable to conventional genetic tools, such as the malarial parasite Plasmodium falciparum. The adenine
riboswitch is one such ‘translational ON’ switch that
allows translation to occur in the presence of adenine
by restructuring the riboswitch to unmask the ShineDalgarno sequence. This switch was reengineered [68]
to produce two different riboswitches that are responsive
either to ammeline or to 2-aminopurine. In subsequent
work [69], the authors went on to independently control in
the same bacterium the coexpression of eGFP and DsRed
induced by ammeline and 2-aminopurine, respectively, in
a dose-dependent manner (Figure 5a). A major advantage
of these approaches over analogous protein-based systems
is the elimination of crosstalk and/or the ability to control
protein expression profiles more finely. Further, protein
stoichiometries may possibly be sculpted in a multiplexed
fashion using this approach with a cocktail of inducers at
different concentrations. Such systems thereby facilitate
quantitative approaches to probing and understanding
protein function in live cells. In this respect, a recent
ion-sensitive fluoride riboswitch represents a valuable
addition for temporal control of gene expression using
orthogonal triggers, because fluoride does not result in
metabolites that could potentially crosstalk with the trigger, is cell-permeable, and may be washed out post-induction [70].
The RNA motifs and small molecules described above
may be used not only to modulate the expression of any
unrelated gene, but also to turn on a pathway that metabolizes the chemical trigger. It is thus feasible to engineer
synthetic functions into living systems, such as sensing
and responding to a non-natural molecule. As an example,
a synthetic riboswitch was used to control motility in E. coli
by introducing an atrazine aptamer in the 50 UTR of the
cheZ gene, a key player in the chemotactic E. coli motility
machinery, in the background of an atrazine-metabolizing
gene [71]. This resulted in ‘seek-and-destroy’ bacteria that
were programmed to seek out and degrade the toxic herbicide atrazine. This approach to controlling chemotaxis is
potentially generalizable to myriad chemicals where E. coli
may be engineered to clear unwanted chemicals in the
environment or other systems [72].
Apart from ON/OFF riboswitches that respond to a
unique molecular cue, there are rare natural examples of
complex logical control over gene expression through tandem riboswitches on a single transcript [73]. Such control
was realized synthetically using tandem riboswitches coupled to the hammerhead ribozyme (HHR) on the 30 UTR of a
given mRNA transcript that mediated logical control over
mRNA cleavage and degradation (Figure 5b). Thus, yeastenhanced GFP (yeGFP) was expressed as a function of AND,
OR, NAND, and NOR logic by positionally coupling multiple
aptamers via crucial connecting modules on the mRNA
transcript such that the addition of two small-molecule
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Output protein (high when A+B)
TRENDS in Cell Biology
Figure 5. (a) Top: translational mechanism of gene regulation by the engineered adenine riboswitch responsive to either ammeline or 2-aminopurine (2AP) (blue hexagon).
Binding to the inducer releases the normally sequestered ribosome-binding site (RBS) and start codon, allowing mRNA translation. Bottom: contour plots showing relative
coexpression ratios of enhanced green fluorescent protein (eGFP): DsRed for the full range of ammeline and 2-aminopurine. Reproduced, with permission, from [69]. (b)
Top: an RNA device incorporated into an mRNA that expresses yeast-enhanced GFP (yeGFP) according to AND logic and the associated truth table. In the absence of either
A or B, at least one functional hammerhead ribozyme (HHR) motif cleaves the transcript. In the presence of both A and B, both HHR motifs are inactivated and the yeGFP is
produced. Reproduced, with permission, from [74]. Bottom: an RNA device incorporating two analogous inactivated HHR motifs that become activated on addition of A or B
serves as a NOR gate. Reproduced, with permission, from [74].
inputs resulted in activation or deactivation of mRNA
cleavage by HHR [74].
Examples of implementation of logical control of cell
function in mammalian systems are limited [75,76,77],
representing a major challenge of interest going forward.
In a recent success, specific signatures of microRNAs
characteristic of cancer cells were read into a logic-gated
computer and used to induce apoptosis using network logic
[76]. The logic framework implemented is completely general for use in detecting specific gene expression profiles in
mammalian cells [78], utilizing a combination of microRNA inputs together with AND logic different logic gates
such as AND and NOT to produce a specific programmed
cellular output. Various means of using riboswitches, aptamers, and allosteric aptamers to switch on or off gene
expression are comprehensively discussed in [79].
The aforementioned studies have largely used manual
approaches to achieve RNA-based programs that modulate
gene expression. In an important development, a highthroughput approach was delineated [80] that screens
endogenous RNA interactions to identify orthogonal
mRNA-regulatory RNA pairs. Using an integrated modeling–experimental framework based on the free energies of
RNA hybridization, the authors realized a library of engineered RNA regulators for predictable interactors. Such
high-throughput approaches are likely to play an important future role in identifying suitable candidates for RNAbased function.
Concluding remarks
Fifty years after the postulate that RNA stands at the
crossroads of the biocatalytic and information-bearing
molecules, proteins and DNA, our rapidly advancing understanding of nucleic acid form and function, together
with our ability to program these polymers for custom
purposes, offer immense room for their application to basic
and applied cell biology.
We envision an eventual palette of made-to-order RNAbased devices to probe cellular machinery including levels
and localization of metabolites, small molecules, ions, and
diverse coding and non-coding RNAs in a multiplexed
manner with sensitivities that are inaccessible to existing
technologies that are largely protein-based (Figure 6). Exploration of intracellular spaces in an organelle-specific
manner to probe their proteomic composition in individual,
living cells, as well as intercellular spaces such as the
neuronal or immunological synapse [81,82], with such
designer devices represent intriguing uncharted territories. Utilizing the ability to precisely engineer higher-order
structural scaffolds and nucleic acid architectures from
7
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TRENDS in Cell Biology
Figure 6. Overview showing the distinct layers of tools based on DNA and RNA and their diverse applications to cellular and synthetic biology.
DNA and RNA templates at the scale of macromolecular
assemblies such as the nuclear pore complex, chaperones,
and the ribosome further offers the unique ability to probe
the effect of the specific higher-order organization of diverse macromolecular structures, including relative positioning and orientation, on their function.
Active scaffolds integrated with precisely positioned proteins and protein domains may additionally be designed
with allosteric response to probe signaling cascades [45],
interrogate cell circuits, probe the RNA transcriptome [83],
modulate enzyme cascades and metabolic pathways [42–
44], perform single-molecule manipulations, and provide
platforms for structural biology [84,85] as well as superresolution imaging [86,87]. Extrinsic control of nucleic acid
duplex stability and conformation with light, as demonstrated extensively in vitro [88–90], will offer novel means to
spatially and temporally regulate the activity of scaffolds,
sensors, and inhibitors in living systems from individual
cells to developing embryos [91]. Finally, construction of
extracellular origami using DNA to position cells in 3D
[92,93] to probe their interaction with their environment
in artificial tissue scaffolds and on 2D substrates [94,95] will
provide insight into their function in organizing higherorder tissues. Such studies, together with an understanding
8
of force sensation and feedback on binding interactions
mediated by focal adhesions [96] and cell–cell junctions
[97], will be key to mechanistically understanding morphogenesis, development, and regeneration.
As evidenced by the macromolecular architectures and
programs presented in this review, nucleic acids offer the
unique capability to design scaffolds with high precision for
diverse applications in cell biology. Among the many challenges we face in bringing these structures to bear broadly
on the study of cell biology are an understanding of how to
control and circumvent crosstalk between thousands of
molecules and pathways in the cell to leverage sophisticated nucleic acid-based in vitro programs [66,98–100], as well
as the immense heterogeneity and variability in molecular
levels that feed back to determine cell state and response,
particularly in complex mammalian systems [17,64,66,75–
78,101]. Although the challenge is great, the ever-increasing arsenal of new tools and technologies at our disposal
ensures that meaningful progress will be achieved in the
near term. Along the way, developing and applying nucleic
acid-based tools to address problems that are currently
intractable using conventional technologies alone will undoubtedly aid the advance of synthetic biology in its aim to
engineer biological systems for the benefit of society.
TICB-917; No. of Pages 10
Review
Acknowledgments
M.B. acknowledges the Office of Naval Research (ONR N000141210621),
the Army Research Office (MURI W911NF-12-1-0420), and MIT Faculty
Start-up Funds for financial support. Y.K. acknowledges the Innovative
Young Biotechnologist Award and the Wellcome Trust-DBT India
Alliance Senior Research Fellowship. The authors are grateful to
Camille Delebecque, Hendrik Dietz, Shawn Douglas, Luc Jaeger,
Takayuki Kato, Hyukjin Lee, Chengde Mao, Jason Micklefield, Niles
Pierce, Christina Smolke, and Andrew Turberfield, for providing highresolution versions of published figures that are reproduced here, as well
as Matthew Adendorff for help with rendering figures. The authors
appreciate discussions and feedback from numerous colleagues in the
preparation of this work, and sincerely apologize to those authors whose
work was not included due to space limitations.
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