design-to-implementation-study-for-development-patient-validation

Design to Implementation Study for Development and Patient Validation of Paper-Based Toehold Switch Diagnostics

Access to low-burden molecular diagnostics that can be deployed into the community for testing is increasingly important and has meaningful wider ...

Boston University

The exciton dynamics for an ensemble of individual, suspended (6,5), single-walled carbon nanotubes revealed by single color E(22) resonant pump-probe spectroscopy for a wide range of pump fluences are reported. The optically excited initial exciton population ranges from approximately 5 to 120 excitons per ∼725 nm nanotube. At the higher fluences of this range, the pump-probe signals are no longer linearly dependent on the pump intensity. A single, predictive model is described that fits all data for two decades of pump fluences and three decades of delay times. The model introduces population loss from the optically active zero momentum E(22) state to the rest of the E(22) subband, which is dark due to momentum selection rules. In the single exciton limit, the E(11) dynamics are well described by a stretched exponential, which is a direct consequence of diffusion quenching from an ensemble of nanotubes of different lengths. The observed change in population relaxation dynamics as a function of increasing pump intensity is attributed to exciton-exciton Auger de-excitation in the E(11) subband and, to a lesser extent, in the E(22) subband. From the fit to the model, an average defect density 1/ρ = 150 nm and diffusion constants D(11) = 4 cm(2)/s and D(22) = 0.2 cm(2)/s are determined.

Intensity-dependent exciton dynamics of (6,5) single-walled carbon nanotubes: momentum selection rules, diffusion, and nonlinear interactions.

Efforts to construct synthetic networks in living cells have been hindered by the limited number of regulatory components that provide wide dynamic range and low crosstalk. Here, we report a class of de-novo-designed prokaryotic riboregulators called toehold switches that activate gene expression in response to cognate RNAs with arbitrary sequences. Toehold switches provide a high level of orthogonality and can be forward engineered to provide average dynamic range above 400. We show that switches can be integrated into the genome to regulate endogenous genes and use them as sensors that respond to endogenous RNAs. We exploit the orthogonality of toehold switches to regulate 12 genes independently and to construct a genetic circuit that evaluates 4-input AND logic. Toehold switches, with their wide dynamic range, orthogonality, and programmability, represent a versatile and powerful platform for regulation of translation, offering diverse applications in molecular biology, synthetic biology, and biotechnology.

Toehold switches: de-novo-designed regulators of gene expression.

Synthetic gene networks have wide-ranging uses in reprogramming and rewiring organisms. To date, there has not been a way to harness the vast potential of these networks beyond the constraints of a laboratory or in vivo environment. Here, we present an in vitro paper-based platform that provides an alternate, versatile venue for synthetic biologists to operate and a much-needed medium for the safe deployment of engineered gene circuits beyond the lab. Commercially available cell-free systems are freeze dried onto paper, enabling the inexpensive, sterile, and abiotic distribution of synthetic-biology-based technologies for the clinic, global health, industry, research, and education. For field use, we create circuits with colorimetric outputs for detection by eye and fabricate a low-cost, electronic optical interface. We demonstrate this technology with small-molecule and RNA actuation of genetic switches, rapid prototyping of complex gene circuits, and programmable in vitro diagnostics, including glucose sensors and strain-specific Ebola virus sensors.

Paper-based synthetic gene networks.

The recent Zika virus outbreak highlights the need for low-cost diagnostics that can be rapidly developed for distribution and use in pandemic regions. Here, we report a pipeline for the rapid design, assembly, and validation of cell-free, paper-based sensors for the detection of the Zika virus RNA genome. By linking isothermal RNA amplification to toehold switch RNA sensors, we detect clinically relevant concentrations of Zika virus sequences and demonstrate specificity against closely related Dengue virus sequences. When coupled with a novel CRISPR/Cas9-based module, our sensors can discriminate between viral strains with single-base resolution. We successfully demonstrate a simple, field-ready sample-processing workflow and detect Zika virus from the plasma of a viremic macaque. Our freeze-dried biomolecular platform resolves important practical limitations to the deployment of molecular diagnostics in the field and demonstrates how synthetic biology can be used to develop diagnostic tools for confronting global health crises. PAPERCLIP. 

Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components.

Non-van der Waals (non-vdW) solids are emerging sources of two-dimensional (2D) nanosheets that can be produced via liquid-phase exfoliation (LPE), and are beginning to expand our understanding of 2D and quasi-2D materials. Recently, nanosheets formed by LPE processing of bulk metal diborides, a diverse family of layered non-vdW ceramic materials, have been reported. However, detailed knowledge of the exfoliation efficiency of these nanomaterials is lacking, and is important for their effective solution-phase processing and for understanding their fundamental surface chemistry, since they have significant differences from more conventional nanosheets produced from layered vdW compounds. Here in this paper we use Hansen solubility theory to investigate nanosheets of the metal borides CrB and MgB derived from LPE. By preparing dispersions in 33 different solvents, we determine Hansen solubility parameters (, , ) for both these metal diborides. We find that they exhibit notably higher  and  values compared to conventional vdW materials such as graphene and MoS, likely as a result of the types of bonds broken in such materials from exfoliation which allows for more favorable interactions with more polar and hydrogen-bonding solvents. We apply the solubility parameters to identify cosolvent blends suitable for CrB and MgB that produce dispersions with concentrations that match or exceed those of the top-performing individual solvents for each material and that have markedly higher stability compared to the constituent solvents of the blends alone. This work provides insight into the exfoliation effectiveness of different solvents for preparation of nanosheets from metal diborides and non-vdW materials in general. Such knowledge will be crucial for developing liquid-phase exfoliation strategies for incorporating these materials in applications such as nanocomposites, inks, and coatings.

Evaluating the Exfoliation Efficiency of Quasi-2D Metal Diboride Nanosheets Using Hansen Solubility Parameters.

Recent advances in cell-free synthetic biology have given rise to gene circuit-based sensors with the potential to provide decentralized and low-cost molecular diagnostics. However, it remains a challenge to deliver this sensing capacity into the hands of users in a practical manner. Here, we leverage the glucose meter, one of the most widely available point-of-care sensing devices, to serve as a universal reader for these decentralized diagnostics. We describe a molecular translator that can convert the activation of conventional gene circuit-based sensors into a glucose output that can be read by off-the-shelf glucose meters. We show the development of new glucogenic reporter systems, multiplexed reporter outputs and detection of nucleic acid targets down to the low attomolar range. Using this glucose-meter interface, we demonstrate the detection of a small-molecule analyte; sample-to-result diagnostics for typhoid, paratyphoid A/B; and show the potential for pandemic response with nucleic acid sensors for SARS-CoV-2.

A glucose meter interface for point-of-care gene circuit-based diagnostics.

Antibiotic-resistant bacteria are a significant and growing threat to human health. Recently, two-dimensional (2D) nanomaterials have shown antimicrobial activity and have the potential to be used as new approaches to treating antibiotic resistant bacteria. In this Research Article, we exfoliate transition metal dichalcogenide (TMDC) nanosheets using synthetic single-stranded DNA (ssDNA) sequences, and demonstrate the broad-spectrum antibacterial activity of MoSe encapsulated by the T ssDNA sequence in eliminating several multidrug-resistant (MDR) bacteria. The MoSe/T is able to eradicate Gram-positive  and Gram-positive  at much lower concentrations than graphene-based nanomaterials. Eradication of MDR strains of methicillin-resistant  (MRSA), , , , and  are shown to occur at at 75 μg mL concentration of MoSe/T, and  at 150 μg mL. Molecular dynamics simulations show that the thymine bases in the T sequence lie flat on the MoSe surface and can, thus, form a very good conformal coating and allow the MoSe to act as a sharp nanoknife. Electron microscopy shows the MoSe nanosheets cutting through the cell membranes, resulting in significant cellular damage and the formation of interior voids. Further assays show the change in membrane potential and reactive oxygen species (ROS) formation as mechanisms of antimicrobial activity of MoSe/T. The cellular death pathways are also examined by mRNA expression. This work shows that biocompatible TMDCs, specifically MoSe/T, is a potent antimicrobial agent against MDR bacteria and has potential for clinical settings.

Elimination of Multidrug-Resistant Bacteria by Transition Metal Dichalcogenides Encapsulated by Synthetic Single-Stranded DNA.

The ability to tune RNA and gene expression dynamics is greatly needed for biotechnological applications. Native RNA stabilizers or engineered 5' stability hairpins have been used to regulate transcript half-life to control recombinant protein expression. However, these methods have been mostly ad hoc and hence lack predictability and modularity. Here, we report a library of RNA modules called degradation-tuning RNAs (dtRNAs) that can increase or decrease transcript stability in vivo and in vitro. dtRNAs enable modulation of transcript stability over a 40-fold dynamic range in Escherichia coli with minimal influence on translation initiation. We harness dtRNAs in messenger RNAs and noncoding RNAs to tune gene circuit dynamics and enhance CRISPR interference in vivo. Use of stabilizing dtRNAs in cell-free transcription-translation reactions also tunes gene and RNA aptamer production. Finally, we combine dtRNAs with toehold switch sensors to enhance the performance of paper-based norovirus diagnostics, illustrating the potential of dtRNAs for biotechnological applications.

Predictable control of RNA lifetime using engineered degradation-tuning RNAs.

Norovirus infections are the leading cause of foodborne illness and human gastroenteritis, afflicting hundreds of millions of people each year. Molecular assays with the capacity to detect norovirus without expensive equipment and with high sensitivity and specificity represent useful tools to track and contain future outbreaks. Here we describe how norovirus can be detected in low-cost paper-based cell-free reactions. These assays combine freeze-dried, thermostable cell-free transcription-translation reactions with toehold switch riboregulators designed to target the norovirus genome, enabling convenient colorimetric assay readouts. Coupling cell-free reactions with synbody-based viral enrichment and isothermal amplification enables detection of norovirus from clinical samples down to concentrations as low as 270 zM. These diagnostic tests are promising assays for confronting norovirus outbreaks and can be adapted to a variety of other human pathogens.

Detection of Norovirus Using Paper-Based Cell-Free Systems.

The COVID-19 pandemic has emphasized the importance of widespread testing to control the spread of infectious diseases. The rapid development, scale-up, and deployment of viral and antibody detection methods since the beginning of the pandemic have greatly increased testing capacity. Desirable attributes of detection methods are low product costs, self-administered protocols, and the ability to be mailed in sealed envelopes for the safe analysis and subsequent logging to public health databases. Herein, such a platform is demonstrated with a screen-printed, inductor-capacitor (LC) resonator as a transducer and a toehold switch coupled with cell-free expression as the biological selective recognition element. In the presence of the N-gene from SARS-CoV-2, the toehold switch relaxes, protease enzyme is expressed, and it degrades a gelatin switch that ultimately shifts the resonant frequency of the planar resonant sensor. The gelatin switch resonator (GSR) can be analyzed through a sealed envelope allowing for assessment without the need for careful sample handling with personal protective equipment or the need for workup with other reagents. The toehold switch used in this sensor demonstrated selectivity to SARS-CoV-2 virus over three seasonal coronaviruses and SARS-CoV-1, with a limit of detection of 100 copies/μL. The functionality of the platform and assessment in a sealed envelope with an automated scanner is shown with overnight shipment, and further improvements are discussed to increase signal stability and further simplify user protocols toward a mail-in platform.

Toward Mail-in-Sensors for SARS-CoV-2 Detection: Interfacing Gel Switch Resonators with Cell-Free Toehold Switches.

In low-resource settings, resilience to infectious disease outbreaks can be hindered by limited access to diagnostic tests. Here we report the results of double-blinded studies of the performance of paper-based diagnostic tests for the Zika and chikungunya viruses in a field setting in Latin America. The tests involved a cell-free expression system relying on isothermal amplification and toehold-switch reactions, a purpose-built portable reader and onboard software for computer vision-enabled image analysis. In patients suspected of infection, the accuracies and sensitivities of the tests for the Zika and chikungunya viruses were, respectively, 98.5% (95% confidence interval, 96.2-99.6%, 268 serum samples) and 98.5% (95% confidence interval, 91.7-100%, 65 serum samples) and approximately 2 aM and 5 fM (both concentrations are within clinically relevant ranges). The analytical specificities and sensitivities of the tests for cultured samples of the viruses were equivalent to those of the real-time quantitative PCR. Cell-free synthetic biology tools and companion hardware can provide de-centralized, high-capacity and low-cost diagnostics for use in low-resource settings.

Field validation of the performance of paper-based tests for the detection of the Zika and chikungunya viruses in serum samples.

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-arm junctions, self-assembled upstream of a regulated gene and designed to unfold sequentially in response to different RNA inputs, with motifs of loop-initiated RNA activators that function independently of the sequence of the input RNAs and that reduce interference with the output gene. We used the integrated RNA system and sequence-independent input RNAs to execute two-input and three-input OR and AND logic in Escherichia coli, and designed paper-based cell-free colourimetric assays that accurately identified two human immunodeficiency virus (HIV) subtypes (by executing OR logic) in amplified synthetic HIV RNA as well as severe acute respiratory syndrome coronavirus-2 (via two-input AND logic) in amplified RNA from saliva samples. The sequence-independent molecular logic enabled by the integration of multi-arm junction RNAs with motifs for loop-initiated RNA activators may be broadly applicable in biotechnology.

Multi-arm RNA junctions encoding molecular logic unconstrained by input sequence for versatile cell-free diagnostics.

Enzyme assemblies on demand.

Translation activators are an important class of riboregulators that respond to nucleic acid signals by activating gene expression. Toehold switches and single-nucleotide-specific programmable riboregulators (SNIPRs) are two types of translation activators that can detect nearly any nucleic acid sequence using interactions initiated by single-stranded domains known as toeholds. Toehold switches operate with high dynamic range, orthogonality, and programmability, making them capable of detecting a variety of pathogens in paper-based cell-free diagnostic assays. SNIPRs are designed to enable the accurate detection of single-nucleotide mutations, making them valuable tools for mutation and drug-resistance assays. Here we describe the computational design process for generating toehold switches and SNIPRs active against different pathogens and mutations of interest. Such riboregulators can be deployed in paper-based diagnostic assays to enable rapid and low-cost disease detection.

Computational Design of RNA Toehold-Mediated Translation Activators.

The toehold switch is an RNA-based riboregulator that activates translation in response to a cognate trigger RNA and provides high ON/OFF ratios, excellent orthogonality, and logic capabilities. Riboregulators that provide the inverse function - turning off translation in response to a trigger RNA - are also versatile tools for sensing and efficiently implementing logic gates such as NAND or NOR. Toehold and three-way junction (3WJ) repressors are two de novo designed translational repressors devised to provide NOT functions with an easily programmable and intuitive structural design. Toehold and 3WJ repressors repress translation upon binding to cognate trigger RNAs by forming strong hairpin and three-way junction structures, respectively. These two translational repressors can be incorporated into multi-input NAND and NOR gates. This chapter provides methods for designing these translational repressors and protocols for in vivo characterization in E. coli.

Design of RNA-Based Translational Repressors.

The ability to control cell function is a critical goal for synthetic biology and motivates the development of ever-improving methods for precise regulation of gene expression. RNA-based systems represent powerful tools for this purpose since they can take full advantage of the predictable and programmable base pairing properties of RNA to control gene expression. This chapter is focused on the computational design of RNA-only biological circuits that can execute complex Boolean logic expressions in living cells. These ribocomputing devices use toehold switches as building blocks for circuit construction, integrating sensing, computation, and signal generation functions within a gate RNA transcript that regulates expression of a gene of interest. The gate RNA in turn assesses the assembly state of networks of interacting input RNAs to execute AND, OR, and NOT operations with high dynamic range in E. coli. Harnessing in silico tools for device design facilitates scaling of the circuits to complex logic expressions, including four-input AND, six-input OR, and disjunctive normal form expressions with up to 12 inputs. This molecular architecture provides an intuitive and modular strategy for devising logic systems that can be readily engineered using RNA sequence design software and applied in vivo and in vitro. In this chapter, we describe the process for designing ribocomputing devices from the generation of orthogonal toehold switch libraries through to their use as building blocks for AND, OR, and NOT circuitry.

Article	Year
Intensity-dependent exciton dynamics of (6,5) single-walled carbon nanotubes: momentum selection rules, diffusion, and nonlinear interactions. ACS nano\| PubMed ID: 22077149	2011
Toehold switches: de-novo-designed regulators of gene expression. Cell\| PubMed ID: 25417166	2014
Paper-based synthetic gene networks. Cell\| PubMed ID: 25417167	2014
Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell\| PubMed ID: 27160350	2016
Evaluating the Exfoliation Efficiency of Quasi-2D Metal Diboride Nanosheets Using Hansen Solubility Parameters. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 33423497	2021
A glucose meter interface for point-of-care gene circuit-based diagnostics. Nature communications\| PubMed ID: 33526784	2021
Elimination of Multidrug-Resistant Bacteria by Transition Metal Dichalcogenides Encapsulated by Synthetic Single-Stranded DNA. ACS applied materials & interfaces\| PubMed ID: 33570927	2021
Predictable control of RNA lifetime using engineered degradation-tuning RNAs. Nature chemical biology\| PubMed ID: 34155402	2021
Detection of Norovirus Using Paper-Based Cell-Free Systems. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 34985757	2022
Toward Mail-in-Sensors for SARS-CoV-2 Detection: Interfacing Gel Switch Resonators with Cell-Free Toehold Switches. ACS sensors\| PubMed ID: 35254055	2022
Field validation of the performance of paper-based tests for the detection of the Zika and chikungunya viruses in serum samples. Nature biomedical engineering\| PubMed ID: 35256758	2022
Multi-arm RNA junctions encoding molecular logic unconstrained by input sequence for versatile cell-free diagnostics. Nature biomedical engineering\| PubMed ID: 35288660	2022
Enzyme assemblies on demand. Nature chemical biology\| PubMed ID: 35468951	2022
Computational Design of RNA Toehold-Mediated Translation Activators. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 35666437	2022
Design of RNA-Based Translational Repressors. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 35666438	2022
Design of Ribocomputing Devices for Complex Cellular Logic. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 35666439	2022
Closing the loop on crowdsourced science. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 35687665	2022

Alexander A. Green

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