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Comparison of sequence constraints for toehold switches and LIRAs a, The toehold switch contains a 9- to 12-nt stem sequence immediately after the start codon that is defined by the sequence of the input RNA. This clamp region cannot encode an in-frame stop codon, which in turn imposes sequence constraints on the input RNA sequence. The clamp also adds three to four N-terminal residues to the output protein. b, LIRAs employ a 6-bp clamp domain downstream of the start codon to achieve low translational leakage with high ON-state signals. This clamp domain is not correlated with the input RNA sequence but does incorporate two additional residues into the N-terminus of the output protein. c, Experimental study of the effect of different clamp sequences on OFF-state GFP fluorescence of four LIRA H01 variants with different clamp sequences. OFF-state expression is unaffected by clamp sequence. d, Effect of different clamp sequences on the ON-state GFP fluorescence for LIRA H01 variants. e, ON/OFF fluorescence of LIRA H01 clamp variants. Significant variations in ON-state GFP and ON/OFF levels do occur, but all systems show ON/OFF levels greater than 50-fold. These results demonstrate that LIRAs can accommodate changes in clamp sequence and output protein N-terminal residues, while displaying good performance. n = 3 biological replicates, bars represent the geometric mean ± s.d. for c and d and the arithmetic mean ± s.d. for e.
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Applications of RNA-based molecular logic have been hampered by sequence constraints imposed on the input and output of the circuits. Here we show that the sequence constraints can be substantially reduced by appropriately encoded multi-arm junctions of single-stranded RNA structures. To conditionally activate RNA translation, we integrated multi-a...
Citations
... The ability to encode genetic information while executing a diverse range of functions has allowed RNA to gain traction as a powerful molecular platform for the development of therapeutics, diagnostics, and synthetic biological systems [1][2][3][4][5][6][7][8][9][10][11][12] . The quartet of canonical nucleotides and simple rules of Watson-Crick base pairing make the design of specific RNA molecules a far more computationally tractable problem than analogous protein-based systems. ...
... However, there does not exist an equally generalized set of tools for computational engineering of RNA function. As RNA-based systems have become more sophisticated and widely used, a disconnect between deterministic thermodynamic properties and resulting RNA function has grown, often necessitating experimental screening of many RNA sequences 3,5,7,9 . Accordingly, data-driven methods that can decipher the relationship between RNA sequence and function are essential for expediting RNA device design and improving their performance in biomedical applications. ...
... The toehold-discriminator model consisted of 3 spectrally normalized convolutional layers, each followed by a layer normalization operation as a drop-in replacement for batch normalization as suggested by the original WGAN-GP authors 55 . The GARDN-UTR model leveraged transpose convolutional layers in place of upsampling with sizes of (2,5), (2,5), and (1,2) to reach the 50-nt final sequence output. The GARDN-UTR generator also utilized a selfattention layer 67 following the three transposed convolutions and before being passed through the final convolutional output instead of a programmable reverse complementation operation to potentially allow for non-specific, long-range nucleotide dependencies. ...
RNA is a remarkably versatile molecule that has been engineered for applications in therapeutics, diagnostics, and in vivo information-processing systems. However, the complex relationship between the sequence, structure, and function of RNA often necessitates extensive experimental screening of candidate sequences. Here we present a generalized, efficient neural network architecture that utilizes the sequence and structure of RNA molecules (SANDSTORM) to inform functional predictions across a diverse range of settings. We pair these predictive models with generative adversarial RNA design networks (GARDN), allowing the generative modelling of a diverse range of functional RNA molecules with targeted experimental attributes. This approach enables the design of novel sequence candidates that outperform those encountered during training or returned by classical thermodynamic algorithms, and can be deployed using as few as 384 example sequences. SANDSTORM and GARDN thus represent powerful new predictive and generative tools for the development of RNA molecules with improved function.
... 35 (C) OR gate built with two toehold activator RNAs, each toehold activator alone can expose the RBS and start codon of the targeted gene. 79 (D) IMPLY gate constructed using inducible repressors LacI and tetR. LacI represses the target promoter by forming a DNA loop, inhibiting gene expression. ...
... Ma et al. develop a toehold regulatory mechanism so that regulated genes can be activated by only one of the two activator sequences independently ( Figure 5C), thus achieving the OR gate function. 79 Toehold switches can also achieve a complex AND gate with up to 4 inputs. 44 An IMPLY gate was constructed by combining two inducible repressors. ...
Cell-free systems are advancing synthetic biology through fast prototyping and modularity. Complex regulatory networks can now be implemented in cell-free systems enabling various applications, such as diagnostic tool development, gene circuit prototyping, and metabolic engineering. As functional complexity increases, the need for regulatory components also grows. This review provides a comprehensive overview of native as well as engineered regulatory components and their use in bacterial cell-free systems.
... The type of toehold-mediated switches that we exploited, are incapable of distinguishing mismatches less than 4 nucleotides. However, other engineered systems show promise to identify single mismatches in a given RNA sequence [45,46]. ...
While many proposals of paper-based diagnostics utilize cell-free gene expression systems, these assays oftentimes suffer from the need for temperature cycling and high operational costs, particularly for developing countries. Here, we explore and report the experimental conditions for the colorimetric detection of viral RNA with an in vitro transcription/translation assay that uses crude E. coli extracts at room temperature where the signal amplification is aided by body heat. Clinically-relevant concentrations of RNA (ca. 600 copies/test) were detected from synthetic RNA samples. The activation of cell-free gene expression was achieved using toehold-switch-mediated riboregulatory elements that are specific to RNA sequences. The colorimetric output was generated by the α-complementation of β-galactosidase ω-fragment (LacZω) with cell-free expressed LacZα, using an X-gal analogue as a substrate. The estimated cost of a single reaction is as low as ~ 0.26 euro/test, which may help to facilitate the accessibility of the diagnostic kit in developing countries. With future optimizations and bacterial strain engineering, production costs can be even further brought down, and the test times can be shortened.
Graphical Abstract
... The toehold-mediated mechanism has been adapted to de novo design eukaryotic translational activators 55 , bacterial translational repressors 56 , transcriptional activators (STAR) 57 , and gRNA regulators 58 . Other riboregulators leverage loop-linear interactions 59 and three-way junctions (3WJs) 56 to transform RNA inputs into protein outputs. The riboregulators have been integrated into sophisticated ribocomputing circuits by designing RNA self-assembly and co-localization 11,56,57 . ...
As synthetic biology permeates society, the signal processing circuits in engineered living systems must be customized to meet practical demands. Towards this mission, novel regulatory mechanisms and genetic circuits with unprecedented complexity have been implemented over the past decade. These regulatory mechanisms, such as transcription and translation control, could be integrated into hybrid circuits termed “multi-level circuits”. The multi-level circuit design will tremendously benefit the current genetic circuit design paradigm, from modifying basic circuit dynamics to facilitating real-world applications, unleashing our capabilities to customize cellular signal processing and address global challenges through synthetic biology.
... To ensure the miRNA-Argonaute (Ago) complex only binds to miRBS without cleavage, we designed miRBS as miRNA sponge 17 with 2-nucleotide mismatches with miRNA at positions 10 and 11. Enlighten by toehold switch 18 and other riboregulator design 19 , we constructed three types of MITAs ( Fig. 2a-c), namely toehold-like (TL, Fig. 2a), stem-loop (SL, Fig. 2b), and 3-arm-junction (AJ, Fig. 2c). We hypothesized that, in the absence of complementary miRNA, the 13-nt RS was sequestered in the designed structure, and IRES-mediated tagBFP translation was inhibited by the 20-nt DS (OFF state). ...
Artificial RNA translation modulation usually relies on multiple components, such as RNA binding proteins (RBPs) or microRNAs (miRNAs) for off-switches and double-inverter cascades for on-switches. Recently, translational circular RNAs (circRNAs) were developed as promising alternatives for linear messenger RNAs (mRNAs). However, circRNAs still lack straightforward and programmable translation control strategies. Here, we rationally design a programmable miRNA-responsive internal ribosome entry site (IRES) translation activation and repression (PROMITAR) platform capable of implementing miRNA-based translation upregulation and downregulation in a single RNA construct. Based on the PROMITAR platform, we construct logic gates and cell-type classifier circRNAs and successfully identify desired mammalian cell types. We also demonstrate the potential therapeutic application of our platform for targeted cancer cell killing by encoding a cytotoxic protein in our engineered circRNAs. We expect our platform to expand the toolbox for RNA synthetic biology and provide an approach for potential biomedical applications in the future.
... The ability to encode genetic information while executing a diverse range of functions have allowed RNA to gain traction as a powerful molecular platform for the development of human therapeutics, diagnostics, and synthetic biological systems [1][2][3][4][5][6][7][8][9][10][11] . The quartet of canonical nucleotides . ...
RNA is a remarkably versatile molecule that has been engineered for applications in therapeutics, diagnostics, and in vivo information-processing systems. However, the complex relationship between the sequence and structural properties of an RNA molecule and its ability to perform specific functions often necessitates extensive experimental screening of candidate sequences. Here we present a generalized neural network architecture that utilizes the s equence and s tructure o f R NA m olecules (SANDSTORM) to inform functional predictions. We demonstrate that this approach achieves state-of-the-art performance across several distinct RNA prediction tasks, while learning interpretable abstractions of RNA secondary structure. We paired these predictive models with g enerative a dversarial R NA d esign n etworks (GARDN), allowing the generative modelling of novel mRNA 5’ untranslated regions and toehold switch riboregulators exhibiting a predetermined fitness. This approach enabled the design of novel toehold switches with a 43-fold increase in experimentally characterized dynamic range compared to those designed using classic thermodynamic algorithms. SANDSTORM and GARDN thus represent powerful new predictive and generative tools for the development of diagnostic and therapeutic RNA molecules with improved function.
... CRISPR-based diagnostic assays 20 that employ cognate guide RNAs and collateral cleavage to verify the sequence of products and generate readout signals have been developed [21][22][23][24][25] . Similarly, paperbased cell-free transcription-translation systems have made use of sequence-specific riboregulators to confirm amplicon sequences down to the single-nucleotide level while providing a convenient color-based readout using protein reporters 19,[26][27][28][29][30] . ...
Rapid, simple, and low-cost diagnostic technologies are crucial tools for combatting infectious disease. Here, we describe a class of aptamer-based RNA switches called aptaswitches that recognize specific target nucleic acid molecules and respond by initiating folding of a reporter aptamer. Aptaswitches can detect virtually any sequence and provide a fast and intense fluorescent readout, generating signals in as little as 5 minutes and enabling detection by eye with minimal equipment. We demonstrate that aptaswitches can be used to regulate folding of six different fluorescent aptamer/fluorogen pairs, providing a general means of controlling aptamer activity and an array of different reporter colors for multiplexing. By coupling isothermal amplification reactions with aptaswitches, we reach sensitivities down to 1 RNA copy/microL in one-pot reactions. Application of multiplexed one-pot reactions against RNA extracted from clinical saliva samples yields an overall accuracy of 96.67% for detection of SARS-CoV-2 in 30 minutes. Aptaswitches are thus versatile tools for nucleic acid detection that can be readily integrated into rapid diagnostic assays.
... A potential approach to extend our two-input gates to larger numbers of inputs could be the utilization of multi-arm junctions as input modules, as previously demonstrated for translational toehold riboregulators. [29] Figure 5. Design and characterization of two-input logic gates based on toehold-Spinach and guanine-Spinach switches. a) A two-input AND gate is realized with two toehold-Spinach activators at the 5' and 3' termini of the Spinach aptamer. ...
Fluorescent light‐up RNA aptamers (FLAPs) such as Spinach or Mango can bind small fluorogens and activate their fluorescence. Here, we adopt a switching mechanism otherwise found in riboswitches and use it to engineer switchable FLAPs that can be activated or repressed by trigger oligonucleotides or small metabolites. The fluorophore binding pocket of the FLAPs comprises guanine (G) quadruplexes, whose critical nucleotides can be sequestered by corresponding anti‐FLAP sequences, leading to an inactive conformation and thus preventing association with the fluorophore. We modified the FLAPs with designed toehold hairpins that carry either an anti‐FLAP or an anti‐anti‐FLAP sequence within the loop region. The addition of an input RNA molecule triggers a toehold‐mediated strand invasion process that refolds the FLAP into an active or inactive configuration. Several of our designs display close‐to‐zero leak signals and correspondingly high ON/OFF fluorescence ratios. We also modified purine aptamers to sequester a partial anti‐FLAP or an anti‐anti‐FLAP sequence to control the formation of the fluorogen‐binding conformation, resulting in FLAPs whose fluorescence is activated or deactivated in the presence of guanine or adenine. We demonstrate that switching modules can be easily combined to generate FLAPs whose fluorescence depends on several inputs with different types of input logic.
... In this study, we introduce and demonstrate design approaches and generalizable optimization strategies for Pepper-based RNA sensors that can achieve up to 300-fold fluorescence enhancement in vitro. We demonstrate two types of Pepper sensor designs that take inspiration from RNA riboswitches developed for translation control in bacteria (21)(22)(23). In both designs, the 5' end of the Pepper stabilizing stem is sequestered in the stem-loop structure of the sensor, which destabilizes Pepper and forces it to adopt a non-fluorescent configuration ( Fig. 1B and C). ...
... In addition, these sensors require a long, single-stranded linear overhang, which is not well-suited for RNA circularization techniques that are commonly used to enhance RNA stability and expression in mammalian cells (25). To overcome these limitations, we developed an alternative design of the Pepper sensor that takes inspiration from loop-initiated RNA activator (LIRA) (21). By placing the target-binding region within the loop of the hairpin structure, the stem sequence of Pepper (b/b* domain) in the sensor becomes completely independent of the sequence of the cognate RNA target. ...
... By placing the target-binding region within the loop of the hairpin structure, the stem sequence of Pepper (b/b* domain) in the sensor becomes completely independent of the sequence of the cognate RNA target. Moreover, LIRAs have lower translational leakage compared to toehold-initiated riboregulators (21). Therefore, if they could provide comparable ON signals to the toehold-mediated sensors, we anticipated that the loop-mediated sensors would exhibit higher dynamic range. ...
The development of fluorescent light up RNA aptamers (FLAPs) has paved the way for the creation of sensors to track RNA in live cells. A major challenge with FLAP sensors is their brightness and their limited signal-to-background ratio both in vivo and in vitro. To address this, we develop sensors using the Pepper aptamer, which exhibits superior brightness and photostability when compared to other FLAPs. The sensors are designed to fold into a low fluorescence conformation, and to switch to a high fluorescence conformation through toehold or loop-mediated interactions with their RNA target. Our sensors detect RNA targets as short as 20 nucleotides in length with a wide dynamic range over 300-fold in vitro, and we describe strategies for optimizing the sensor's performance for any given RNA targets. To demonstrate the versatility of our design approach, we generate Pepper sensors for a range of specific, biologically relevant RNA sequences. Our design and optimization strategies are portable to other FLAPs, and offer a promising foundation for future development of RNA sensors with high specificity and sensitivity for detecting RNA biomarkers with multiple applications.
... A different approach to realize AND and OR logic functions in the input domain of translational riboregulators is based on multi-arm junctions [52] and was recently demonstrated for applications in cell-free diagnostics. In contrast to toehold switches, strand invasion is initiated within the loop region of a hairpin loop (termed LIRA for loop-initiated RNA activator). ...
Nucleic acid strand displacement reactions involve the competition of two or more DNA or RNA strands of similar sequence for binding to a complementary strand, and facilitate the isothermal replacement of an incumbent strand by an invader. The process can be biased by augmenting the duplex comprising the incumbent with a single-stranded extension, which can act as a toehold for a complementary invader. The toehold gives the invader a thermodynamic advantage over the incumbent, and can be programmed as a unique label to activate a specific strand displacement process. Toehold-mediated strand displacement processes have been extensively utilized for the operation of DNA-based molecular machines and devices as well as for the design of DNA-based chemical reaction networks. More recently, principles developed initially in the context of DNA nanotechnology have been applied for the de novo design of gene regulatory switches that can operate inside living cells. The article specifically focuses on the design of RNA-based translational regulators termed toehold switches. Toehold switches utilize toehold-mediated strand invasion to either activate or repress translation of an mRNA in response to the binding of a trigger RNA molecule. The basic operation principles of toehold switches will be discussed as well as their applications in sensing and biocomputing. Finally, strategies for their optimization will be described as well as challenges for their operation in vivo.
