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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 1 TICB-917; No. of Pages 10 Review Trends in Cell Biology xxx xxxx, Vol. xxx, No. x (a) (b) (c) 2.2–3 nm + + 21 bp + D B D Ligate E C C E F Ligate F A A (e) (d) FP scaffold (f) 8 nm B al ne An siRNA NPs Dicing Funconalizaon siRNAs E A B C D F TRENDS in Cell Biology 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 TICB-917; No. of Pages 10 Review Trends in Cell Biology xxx xxxx, Vol. xxx, No. x Aptamer Aptazyme Mn+ Ribozyme/ DNAzyme Allosteric aptamer + Riboswitches Transcriponal Translaonal Metabolite-binding 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 3 TICB-917; No. of Pages 10 Review (a) Trends in Cell Biology xxx xxxx, Vol. xxx, No. x (b) (c) F H Cargo F Protein aptamer H F H D0 + RNA aptamer Scaffold D0 Epifluorescence 1.8 1.6 Unscaffolded (unorganized) A B 1.4 1.2 1.0 0.8 Scaffolded (organized) A B Discrete Onedimensional Twodimensional TRENDS in Cell Biology 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. TICB-917; No. of Pages 10 Review Trends in Cell Biology xxx xxxx, Vol. xxx, No. x (a) (c) Detecon stage Amplificaon stage mRNA target H1 H2 Probe binding sites HBI (in GFP) Metastable hairpins Spinach H1 Hybridize probes and wash Iniators Fluorescent in situ HCR and wash H2 H1 H2 H1 DMHBI Tethered fluorescent amplificaon polymers DMHBI Spinach-DFHBI fluorescent complex H2 Probe set 13-2 RNA DMHBI + 13-2 RNA DMHBI + control RNA 0 min 15 min 30 min 90 min 120 min 150 min mRNA target Minimal fluorescence Target binding reinforces structure Fluorescent complex formaon (d) (b) Tg(flk1:egfp) tpm3 elavl3 ntla sox10 Donor O1 O2 z = 20 µm D/A OH- 17 min O3 Acceptor 5 min H+ z = 28 µm z = 44 µm z = 52 µm 60 min 5 H+ 7 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), 5 TICB-917; No. of Pages 10 Review 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. 6 Trends in Cell Biology xxx xxxx, Vol. xxx, No. x 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 TICB-917; No. of Pages 10 Review Trends in Cell Biology xxx xxxx, Vol. xxx, No. x (b) (a) AND gate Input A A Input B Output 3′ [SI 1.2] Start RBS 3′ 5′ AB B ON-state OFF-state 5′ A B Output theo tc GFP 0 0 0 0 1 0 1 0 0 1 1 1 AAAAA Output protein (high when AB) 250 Ammeline (μM) 125 5:1 (+) 4:1 3:1 2:1 64 32 NOR gate Input A A A+B Input B Output B [SI 1.3] 1:1 16 1:2 1:3 1:4 1:5 (+) 8 4 0 0 4 8 16 32 64 125 A B Output theo tc GFP 0 0 1 0 1 0 1 0 0 1 1 0 AAAAA 250 2-aminopurine (μM) 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 TICB-917; No. of Pages 10 Review Azy me s Super-resoluon microscopy to rs mo cu lar ule lec o -m s gle sic Sin ophy bi Na no str uct ure s DN DNA M ole Scaffolds Proteomics s ier nsm rotra Neu rce Fo M et ab oli te s Mac ro struc molecu lar ture Trends in Cell Biology xxx xxxx, Vol. xxx, No. x l -cel Cell acons r e int Signaling cascades DNA Sensors Ribozymes Aptamers Delivery RNA Diagn oscs u cs ar -h ht g n s ve Bioreme diaon rap e Lig rgy n Ene versio con esis Biosynth Rib ore gul ao n The Programs Pr ot ei ns A RN A RN mi Rib osw itc he s Ions 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. 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