Engineered RNA control elements as biosensors for in vitro diagnostics

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110th Anniversary: Engineered RNA control elements as biosensors for in vitro diagnostics Alan Fernando Rodríguez Serrano, and I-Ming Hsing Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.9b03963 • Publication Date (Web): 23 Aug 2019 Downloaded from pubs.acs.org on August 27, 2019

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Industrial & Engineering Chemistry Research

110th Anniversary: Engineered RNA control elements as biosensors for in vitro diagnostics Alan F. Rodríguez-Serrano, I-Ming Hsing*. Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong. Keywords: RNA-based biosensor, RNA aptamer, toehold switch, in vitro diagnostics, synthetic biology.

Abstract Graphic

Abstract

Regulatory mechanisms in biological systems are analogous to process control exerted in many chemical and biomolecular engineering processes. Ribonucleic Acid (RNA) has been identified as 1 ACS Paragon Plus Environment

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a ubiquitous element for tight regulation of cellular activities, such as gene expression, given its high tunability of structures and functions. Enhanced understanding of interactions of RNA with nucleic acids, proteins and small molecules has enabled programming of robust gene circuits for detection of specific target analytes and signal transduction. Bottom up approaches in synthetic biology have accomplished to in silico engineer RNA modules to work as biosensors for nucleic acids in vitro detection. In this review, we describe light-up aptamers and toehold switches as exciting examples of RNA engineered modules that were inspired by evolution and process control in biological organisms. We emphasize how the programmability of these elements has empowered the assembly circuitry to sense and compute information relevant for biomedical applications.

Introduction

The dynamic behavior of a system not only allows but requires process control. In the chemical and biotechnological industries, process control is implemented on physicochemical systems to ensure efficiency, stability and safety.1,2 The core elements of any process control system are an input, a process to control, an output, a sensor, and a controller.3 By optimizing the interplay of these modules, the process operability can increase at the minimum cost penalty at different levels.4 Similarly, biological systems incorporate control processes in many of the simultaneous activities occurring within the cell to deal with disturbances and uncertainty, such as fluctuations in the microenvironment or mutations in the DNA. Additionally, this control extends to intercellular interaction, tissue, and organ levels to maintain equilibrium of the processes, known as homeostasis. The same principles of process control apply to biological organisms, with 2 ACS Paragon Plus Environment

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biomolecules and metabolites, such as nucleic acids, proteins and hormones, acting as inputs, outputs, sensors or controllers, interacting through complex dynamics and nonlinear behavior5,6.

Bacteria, plants, and animals have long been perceived as small bioreactors with internal anticipatory, negative feedback, specialized action and distributed control mechanisms according to the complexity of the system.7 Due to the interconnectivity of the elements, it is challenging to clearly define the processes and the controllers that regulate them. However, efforts in systems biology have allowed separation of the components into functional modules and defined roles from many cellular regulatory networks, such as those that control DNA transcription and protein synthesis.8–10

Synthetic and computational biology have helped us build biomolecules with de novo functions and engineer existing ones to suit specific properties and performance and assemble complex biological circuits. One exciting feature is the possibility to rationally design reactions between biomolecules that mimic (fully or partially) cellular events without using living cells11, such as gene transcription and protein synthesis. These cell-free systems can incorporate individual control elements that are responsive to certain molecules, working as regulation modules composed by a sensing element and a transducer that outputs a measurable signal. In this context, a variety of gene circuits can be generated for in vitro biosensing of DNA and RNA12, which are of high interest in the biomedical field for detection and tracking of diseases biomarkers, like tumor DNA, RNA transcripts and microRNA (miRNA)13–16, which are involved in the dysregulation of many metabolic pathways.

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These genetic networks are comprised of RNA, DNA and proteins (including enzymes) and can be programmed to work and assemble in a Boolean logic manner.17 In recent years, RNA-based biosensors have shown high tunability due to their single-stranded nature, diverse threedimensional structures that modulate binding affinity to other molecules, and role in the central dogma of biology for being the precursor of proteins18,19, which can be used as intermediates or direct measurable output signals of a given biosensing system. The interaction of individual biological parts and control exerted on the programmed circuits can be rationally designed and directly observed and characterized, which makes an attractive platform to developing biosensors.

Extensive research has been carried out and commercial outputs developed in the field of nucleic acids-based diagnostics, such as quantitative Polymerase Chain Reaction (qPCR). Shortly after its introduction, qPCR revolutionized the way genetic and infectious diseases are detected in clinical samples. However, this tool generally requires specialized equipment, have long turn-around time, and tests are priced around tens to hundreds of U.S dollars. In scenarios where fast and simple detection of nucleic acids markers is necessary and sufficient, RNA-based biosensors possess great potential. Even though they cannot achieve sensitivity levels of (Reverse Transcription-) qPCR, when coupled with isothermal amplification methods and simplified format such as paper-based tests, they overperform other detection methods in terms of practicality, such as electrochemical DNA sensors, at a much lower cost. Additionally, the modular nature and programmability of these devices facilitate the detection of virtually any polynucleotide sequence and the construction of more complex circuits for multiplexing, for example.


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In this review, we will illustrate two types of RNA-based biosensors that have been engineered to serve as in vitro molecular diagnostics tools. These programmable biological systems are analogous to regulatory elements found in dynamic processes. First, we focus on light-up aptamers as a label-free fluorescent biosensor regulated by induction of conformational change upon hybridization with a target sequence. Then, we look into the mechanisms and applications of toehold switches, a recently developed RNA tool for programmed target biosensing with Point of Care (PoC) diagnostics potential. Ultimately, this review will provide an insight into the challenges and future development of programmable RNA-based sensors for next generation diagnostics platforms.

Light-up aptamers

Aptamers are short sequences of nucleic acids (DNA or RNA) folded in specific structures to recognize a target molecule with high affinity.20 These molecules range from ions, such as metals,21,22 to organic compounds like proteins23,24 and small molecules25,26. Within a cell, natural aptamers have been found to regulate transcription and translation via direct interactions with RNA polymerase and metabolite-specific binding, called riboswitches, respectively.27,28 Synthetically, the conventional method for selection of aptamers is Systematic Evolution of Ligands by EXponential enrichment (SELEX), which consists of an iterative process of binding affinity screening of a library of millions of RNA or DNA sequences to exponentially enrich the group of oligonucleotides that are able to strongly bind a target molecule.29,30 Other approaches derived from SELEX, such as cell-SELEX31,32, and magnetic-assisted rapid aptamer selection (MARAS)33 have been reported. Taking advantage of this high-throughput approaches, many research groups 5 ACS Paragon Plus Environment


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have focused their efforts towards the generation of aptamers for both therapeutic and diagnostic applications.34–36

In 2003, Tsien’s group developed a new type of aptamer capable of binding to a fluorogenic dye,37 now known as light-up aptamers. The interaction between the aptamer and the dye enhances the light generated by the latter, an unseen phenomenon when using a fluorescent dye, where it fluoresces in its bound and free form thus introducing high background signal.38 This work laid the foundation for the development of a wide range of aptamers now used for in vitro and in vivo imaging, tracking and detection of many biological analytes. The rationale behind these applications is the design of an RNA sequence with a destabilized dye-binding domain, which rearranges into a stable form upon the targeting of another molecule by other specific domain, allowing to bind to the fluorogen and emit fluorescence. Usually, these structures have improved folding and stabilization when integrated with scaffold structures such as tRNA39 (Figure 1a). Many of the light-up aptamers mostly used are Spinach40, Broccoli41, Mango42, SRB37, among others, which bind to 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), DFHBI-1T, Thiazole Orange (TO-1) and Patent blue, respectively. A comprehensive review of RNA/fluorogenic modules was published by Bouhedda and colleagues.38

Combining the fluorogen-binding domain of an aptamer with a single-stranded domain, called a toehold, in a single RNA molecule allows the hybridization of a complementary nucleic acid sequence and the generation of a fluorescent signal when the aptamer folds to allow the binding of the reporter molecule. In this way, RNA aptamers have been used for detection of microRNA,43 small ncRNA,44 and mRNA45, as well as for imaging in structural46 and assembly analysis.47 A 6 ACS Paragon Plus Environment

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better understanding and increasing use of light-up RNA aptamers have facilitated their applications in in vitro detection of nucleic acids with high potential for molecular diagnostics.

Figure 1. Simplified structures and general mechanisms of RNA light-up aptamers for nucleic acid detection. a) Overall structure of a light-up RNA aptamer. The scaffold enhances the structure stability and folding efficiency and protects it from RNases. b) Toehold-mediated activation of light-up RNA aptamer. Upon hybridization of the toehold domain (red color) and the target nucleic acid, branch migration induces refolding into the correct structure for fluorogen binding and activation.

In 2014, Bhadra & Ellington designed a light-up SPINACH RNA sensor that was activated upon recognition of an oligonucleotide sequence, which was termed SPINACH.ST.48 The original SPINACH aptamer was modified to adopt a conformation that prevented the binding of DFHBI. Such modification consisted of extensions on both ends of the aptamer to create a toehold that worked as the sensing element. In this manner, the hybridization of a complementary sequence (target) with the toehold causes a strand displacement reaction and the internally blocked Spinach RNA undergoes a conformational change that activates the DFHBI-binding domain (Figure 1b). Interestingly, the SPINACH.ST was used as a signal transducer for sequence-specific detection of Nucleic Acid Sequence-Based Amplification (NASBA) products in the nanomolar range for a 7 ACS Paragon Plus Environment


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target RNA, and up to picomolar concentration of a target DNA after eight hours of amplification. In this case, the Spinach.ST works as an aptamer-based molecular beacon, with the advantage that chemical conjugation of fluorophores is no longer required.39 Strand displacement-mediated mechanisms have been previously reported for detection of amplicons from other isothermal amplification techniques, using Catalytic Hairpin Assembly in Rolling Circle Amplification (RCA) and Strand Displacement Amplification (SDA).49 The ability of biosensing genetic circuits to be integrated with these methods is crucial for simpler and more sensitive diagnostic assays. However, further work is needed for implementation of strand displacement-activated methods with regard to the detection limit and response time. In this context, the RNA aptamer can be designed to be transcribed in situ from a DNA template to construct a more stable one-pot assay.48 Leveraging an in situ transcription system, signal amplification can be carried out in an interesting way. Recently, Ying and colleagues took advantage of two different DNA probes containing a T7 promoter sequence and a template for RNA aptamer transcription (Figure 2).50 Adjacent hybridization of both probes to a target microRNA (miR-21 or miR-141) triggered the transcription of one of the probes to generate light-up RNA aptamers, which yielded a signal-to-background ratio of ̴200 and improved sensitivity by 3 orders of magnitude compared to systems described above. The selective and sensitive detection of miRNA-21 and miRNA-141 is of biomedical relevance due to their role as apoptotic suppressors and are proposed biomarkers of different kinds of cancer, including lung, breast, stomach, prostate, colon, and pancreas.51,52


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Figure 2. In situ transcription of Spinach-based light-up aptamer for detection of microRNA. Two different single-stranded DNA templates contain complementary regions (yellow color) to a target miRNA (pink color). After co-hybridization, the DNA templates are ligated and the T7 promoter region (purple color) enables the transcription of the RNA aptamer (light blue color). Finally, the Spinach aptamers fold and bind to the fluorogen and generate a signal. Figure reproduced with permission from Ref. 50.

Another simple yet elegant approach to signal amplification is through target recycling, which resembles a closed-loop system. As its name suggests, the same analyte is used several times by identical recognition molecules, hence potentially increasing the sensitivity and reducing the turnaround time of the assay. An in vitro signal amplification RNA circuit using Spinach RNA and a fuel strand for strand displacement-dependent recycling of RNA input strand was reported by Akter & Yokobayashi53 (Figure 3). Considering the simplicity of the feedback circuit, the catalytic turnover of the system of approximately 5 activated aptamer molecules per each molecule of catalytic input strand in 3 hours supports the robustness of nonenzymatic signal amplification circuits. Although the selectivity of this approach is constrained by the inner sequences necessary for aptamer refolding, it is a proof of the robustness achieved by RNA circuits. This represents a 9 ACS Paragon Plus Environment


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large area of opportunity for optimization through rational design to integrate the target-binding domains in order to expand the potential of applications in molecular diagnostics, even allowing the design of reversible molecular light-up switches via the use of toehold-containing “kleptamer” sequences for aptamer-ligand dissociation.54

Figure 3. RNA signal amplification circuit using a fuel-strand (F) to recycle the trigger-strand (C). A blocked light-up aptamer (Sp-1) is activated by hybridization of C and a new toehold is formed. Strand displacement of C is carried out by F, so the former is released and can hybridize with a new inactivated SP-1. Figure reproduced with permission from Ref. 53.

Rational modelling, structural analysis, and enhanced screening methodologies have allowed the engineering of aptamers with higher stability and ability to perform consistently over a range of in vitro conditions. For instance, the Spinach2 aptamer showed greater thermal stability and folding efficiency than its predecessor, the original Spinach aptamer.55 Also, another derivative of the same aptamer, called the iSpinach, was designed to take these advantages to in vitro applications by increasing its affinity for DFHBI.56 This is of particular interest for in vitro testing, due to an enhanced sensitivity of fluorogenic assays and improved robustness of the biosensors’ microenvironment because of a wider salt-tolerance. Additionally, aptamers have been engineered for the direct detection of small RNA57,58 molecules, such as miRNA and siRNA, despite their 10 ACS Paragon Plus Environment

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short sequences. Identification of small RNAs have potential biomedical applications. For instance, dysregulation of many miRNAs has been linked to tumor metastasis, development of cardiomyopathies and nervous system disorders.59–61

It is important to consider the mechanism of strand displacement to improve its kinetics and efficiency. The entire process can be divided into two steps: first, the target hybridizes to the toehold, which acts as the nucleation site; and second, the invading strand hybridizes to the rest of the complementary DNA sequence, called the branch migration step. The length and sequence of the toehold are factors that determine its strength, and the hybridization with the target is a limiting step.62 Hence, it directly affects the rate of the strand displacement reactions. Besides careful design of the abovementioned factors to alleviate the constrains of the dynamic stranddisplacement steps, optimization of the dye-binding domain of the aptamer should primarily focus on enhancing the refolding of the aptamer, affecting both its conformational change rate and quantum yield of the aptamer/fluorogen modules after activation.

Specificity of sensing probes is crucial for reliable and reproducible detection of nucleic acids. To increase the specificity, an aptamer-based AND Boolean gate can be integrated into a genetic circuit in the form of binary (split) probes.63,64 In this system, two input non-functional RNA moieties co-localize and hybridize a complimentary strand to assemble into a functional structure containing the dye-binding domain and output a fluorescent signal. This approach leverages a three-way junction (3WJ) architecture47,65,66 (Figure 4). Similar to a single aptamer probe, in vitro co-transcription can be used for production of aptamer moieties.67



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Figure 4. Split aptamer probes contain a toehold domain (red color) complementary to the target RNA. When the target is present, it serves as a scaffold for the aptamer’s correct alignment and folding into a full functional conformation. If the scaffold strand is not complementary to the toehold domain of one or both probes, co-localization does not occur and the AND gate cannot be true, resulting in no output observed.

Split aptamers have been demonstrated to detect RNA and DNA in the low nanomolar concentration with single-mismatch selectivity and immediate output upon mixing of reagents at ambient temperatures.68 These properties are desired for the proof-of-concept study to target the inhA gene from Mycobacterium tuberculosis (Mtb), which point mutations are related to drug resistance. Tests for detecting antimicrobial strains are urgently needed as resistance-associated mutations from many bacteria have been described for first- and second-line drugs and is a global health concern.69,70 Importantly, one predominant characteristic of this system is the low background fluorescence, restricted by both the low fluorescent dye in free form and the AND gate.68,71 Important factors for the assessment and design of split aptamers for nucleic acids detection in vitro should include stability of the 3D structure at different temperatures and composition of the matrix containing the target (presence of RNases, ions concentration, etc.).67 Also, special care should be paid to the mechanistic modelling of RNA structures by structure12 ACS Paragon Plus Environment

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prediction algorithms. While computational design is useful for thermodynamic calculations for prediction of folding and binding, non-Watson-Crick base pairing and pseudoknots calculations are not adequately integrated, for which practical in vitro screening is still required for optimization steps.72

In conclusion, the better understanding and recently spread use of light-up RNA aptamers has facilitated its applications in in vitro detection of nucleic acid molecules with high potential for molecular diagnostics. This usefulness resides on the simplicity of the genetic circuit as a whole. Recapitulating, the main merits are its label-free and modular design and the ability to perform in homogeneous reactions39, which figure as the principal advantages against other fluorescent probes.73,74 Nevertheless, further work is necessary to optimize the sensitivity of the systems to clinically relevant ranges if wider and feasible real-world applications are desired. Finally, an interesting unexplored field could derive from the integration of in vitro transcription of light-up aptamers with more complex gene circuits for multiplexed assays in a logic gated manner, which would explode the intrinsic advantages of purification-free and reproducible generation of complex sensors.

RNA Toehold switches

In spite of the simplicity of bacteria relative to higher-ordered organisms, many of the most exciting mechanisms in nucleic acids biology have been discovered in these living systems. One of the major control elements of gene expression found in bacteria are riboswitches, which affect protein synthesis initiation from an RNA transcript.75 A riboswitch is a naturally-occurring 13 ACS Paragon Plus Environment


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aptamer domain located in the 5’ untranslated region of mRNA.76 In translational control, the upstream three-dimensional conformation of the aptamer domain comprises the Ribosomal Binding Site (RBS), which is released only when the aptamer binds to a specific ligand.77 Similarly, a riboregulator relies on the sequestration of the RBS via a cis-repression sequence, which is released by a trans-acting RNA for further translation of the downstream mRNA78 (Figure 5a). Under this rationale, a riboregulator serves as a scaffold for detection of single-stranded nucleic acids that act as the trans-activator. However, in natural and engineered riboregulators, the trans-activator must include the RBS sequence in order to displace the RBS-sequestering strand and release this site for translation79 (Figure 5a), which limits the use of the switches to a much reduced number of target sequences. To circumvent this limitation, Green et al designed a riboregulator with an alternative RNA structure called toehold switch.80 This switch relies on the sequestration of the translational start site and RBS in a hairpin structure and the addition of a toehold sequence upstream of these regions, which is not constrained by the RBS sequence. It hybridizes a single-stranded target via linear interactions, which are more favorable than looplinear interactions present in riboregulators.

The flexible design and multi-step mechanism of the toehold switches permit the actuator to translate a reporter protein for detection of any single-stranded polynucleotide trigger (Figure 5b). In this context, RNA plays both the feed stream and the central component of the translational machinery.81 While RNA switches get momentum as a modular control element in complex genetic circuits,82 it can be used in bottom-up engineered systems for simple and orthogonal diagnostic platforms. The rational design of toehold sensors is a crucial approach that enables the analysis of secondary structures to select the most appropriate sequences to conform the switch in 14 ACS Paragon Plus Environment

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silico. In silico-enhanced designs shorten the time to engineer sensor prototypes to a matter of hours with the sequence information being the only major requirement. Pardee et al programmed a toehold switch as a cell-free sensor and modulator of the expression of a transducer LacZ mRNA for an Ebola paper-based prototype biosensor,83 with sensors developed in under 12 hours. The colorimetric output showed a maximum ON/OFF ratio of 77 after 90 minutes of reaction, detecting trigger RNA at 30 nM. It is worth mentioning that the paper-based biosensor relies on the freezedrying of a commercially available expression system on paper discs for further reconstitution with solutions containing the toehold switch and target. Particularly, this modality disrupts the way synthetic gene circuits had been developed for diagnostics applications, providing an inexpensive, safe and deployable alternative for Point of Care (PoC) applications. Paper-based circuits have the potential to bridge diagnosis of infectious diseases with reliable and affordable technology in regions with limited resources. The integration of paper in microfluidic tests is an area that holds promise and work should focus on the development of sample-to-result platforms for further push into commercialization.

Figure 5. Ribocomputing devices. a) Riboregulators are translational control elements. They prevent the access of the ribosome by sequestering the Ribosome Binding Site (RBS). Loop-linear or loop-loop interactions between a trans-activator RNA (taRNA) are constrained by specific 15 ACS Paragon Plus Environment


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sequences that remain constant (yellow and blue regions). b) The toehold switch relies on the sequestration of the translational start site (methionine codon - AUG) and the RBS by a hairpin loop. A toehold sequence is located upstream of the RBS and hybridization of a complementary strand to the toehold initiates RNA-RNA linear interactions for further disruption of the hairpin, exposing the RBS accessible to the ribosome for translation of a reporter gene, e.g. LacZ or Green Fluorescent Protein (GFP). The trigger RNA sequence is variable as it interacts with nonconserved regions (grey color). Figure adapted with permission from Ref. 80.

Increased sensitivity by several orders of magnitude and reduced time of switch-ON induction can be achieved if coupled with target amplification. Recently, Pardee and colleagues coupled NASBA with toehold switches to detect Zika virus with a remarkable sensitivity in the low femtomolar range in matter of minutes using the same strategy of paper-based colorimetric sensors.84 Additionally, genotyping with Cas9 protein allowed distinguishing between two strains of the virus, taking as target of the endonuclease Cas9 the dsDNA intermediate during NASBA, which cleavage halted the reaction and further activation of toehold switches. This was the first example of integration of CRISPR/Cas9 systems with an isothermal amplification strategy and opens the door to exciting possibilities where single nucleotide specificity is required, such as virus subtyping. The increased sensitivity, orthogonal selectivity, and deployable modality exhibit major advantages over current field-ready diagnostic devices. Additionally, the microvolume required for these paper-based tests allows to carry out isothermal amplification and colorimetric output for just a fraction of a U.S. dollar.84


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Computational design for the assessment of programmed RNA secondary structure before and after activation of a trigger RNA enhances orthogonality and maximizes the ON/OFF ratio of the biosensor. Sensors for a quantitative panel of 10 bacteria present in gut microbiota, which exhibited exceptional orthogonality, have been developed by Collins’ group.85 Species-specific detection of mRNA was well executed in the low femtomolar range after NASBA. This platform can be devised as a rapid and simple proof-of-concept to monitor diseases and efficacy of treatments. Notably, this on-demand alternative offers several advantages over 1) the traditional deep sequencing for gene expression profiling, which involves data processing, and is generally time-consuming and expensive; and 2) RT-qPCR, which costs more and is limited by the number of RNA targets analyzed. In the same study, the mRNA of bacterial toxins and host transcripts markers were detected, which provide actionable information for the identification of latent and active infections, which is indistinguishable using DNA qPCR, and account for better clinical sensitivity for diagnosing Clostridium difficile infection (CDI). For relevant clinical usefulness, future work should focus on simplifying sample collection, storage and preparation steps, which currently hinders the direct use of toehold sensors in an integrated platform.

Toehold switches promise a great range of applications. Tunability of toehold linear-linear interactions renders it as an adaptable module for detection of nucleic acids from bacteria and virus, and very likely fungi and other biological sources. Besides, when combined with other synthetic biology-based tools, such as isothermal amplification and Cas9, 12a or 13, many possibilities arise for robust assays, which can either tolerate single nucleotide differences or discriminate among targets for genotyping purposes, e.g. influenza or zika virus subtyping. Ribocomputing devices foster exciting opportunities for the development of multiplex in vitro 17 ACS Paragon Plus Environment


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nucleic acid logic computation. However, due to variation of viral RNA loads in clinical samples for different diseases, work is needed to improve the sensitivity of the sensor, by coupling it either with enhanced amplification techniques or with other cascades of logic-gated reactions that do not compromise its selectivity.

Concluding remarks

Engineered control elements found in biological organisms are analogous to those implemented in process control systems in the biotechnological and chemical industries. By refining and combining these components from a bottom-up approach, high levels of modularity and programmability can be achieved to arrange specific genetic circuits. These properties are extremely valuable in diagnostics. In the two approaches that we presented here, the toehold domains that act as trigger RNA binding sites are free to adopt practically any polynucleotide sequence independently from the transducing design, be it the folding of a light-up aptamer or the expression of a reporter gene for colorimetric or fluorescent output. This flexibility of design can be leveraged to construct a wide variety of RNA-based sensors for DNA and RNA targets for genetic or infectious diseases and deploy these in different formats, such as paper-based or solution-based.

Biosensing circuits with target and signal amplification can be applied to logical decisionmaking and noise cancellation in diagnostics. Amplification of the target nucleic acid is preferred over signal amplification-only, which are strategies used on protein-based and immunoassays,


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along with sample enrichment. However, increased sensitivity of the biosensing system alone is also favorable.

Combination of tools, such as in vitro transcription/translation and CRISPR-Cas systems, will play an important role in the rapid development of next generation diagnostics for PoC applications. Interrogation of specific features such as genotyping and expression level determination through simple tests expands the possibilities to implement RNA-based circuits in routine clinical practices, saving time and resources without trading off accuracy and actionable information. One exciting application of this approach is the development of cell-free systems with improved robustness to work properly in a wider range of conditions, such as tolerance to certain contaminants or inhibitors present in the sample matrix.

RNA aptamers and toehold switches are suitable alternatives for deployment in the PoC when integrated with an isothermal amplification method. In this regard, there are still big challenges to address until we can see these technologies applied in real-life scenarios. Even though paper-based advances have yielded promising results, development of sample-to-result devices, in a microfluidics system for example, is highly desired. Compatibility of these proofs of concept with clinical samples and integrated sample processing steps represent a major limitation in the creation of simple and affordable assays.

Arrangement of input, sensing and controller biomolecules can mimic logic gated systems with precise design and control on their execution. The last couple decades have witnessed a tremendous effort to develop programming languages for nucleic acids and nucleic acids-based 19 ACS Paragon Plus Environment


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circuitries computing, considering not only Boolean logic but the biological nonlinear behavior. Mechanistic models for kinetics and structure prediction software are essential tools for the rational design of biosensing networks. Even though practical in vitro screening is still recommended for optimization purposes, we believe computational design of diagnostic circuitries for initial in silico assessment is a powerful tool that will shape the way the development pipeline of bioassays is conducted to achieve increased robustness, high tunability, faster dynamics and increased specificity.

AUTHOR INFORMATION Corresponding Author *Correspondence should be addressed to I-Ming Hsing (Email: [email protected]) Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources Funding support from the Research Grants Council of Hong Kong SAR Government (Project # GRF 16301817 & GRF 16306218) is acknowledged. ACKNOWLEDGMENT A.F.R.-S. would like to thank the Hong Kong PhD Fellowship Scheme from Hong Kong SAR Government and support from Conacyt-Mexico. REFERENCES 20 ACS Paragon Plus Environment

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