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.

SUMMARY Synthetic biology aims to develop engineering-driven approaches to program cellular function that could yield transformative technologies1. Synthetic gene circuits that combine DNA, protein, and RNA components have demonstrated a range of functions including bistability2, oscillation3,4, feedback5,6, and logic capabilities7–15. However, scaling up these circuits remains challenging due to the limited number of designable, orthogonal, high-performance parts, the empirical and often tedious composition rules, and substantial resource requirements for encoding and operation. Here, we report a strategy for constructing RNA-only nanodevices to evaluate complex logic in living cells. Such ‘ribocomputing’ systems are composed of de-novo-designed parts and operate via predictable and designable base-pairing rules, allowing for effective in silico design of computing devices with prescribed configurations and functions in complex cellular environments. These devices operate at the post-transcriptional level and use an extended RNA transcript to co-localize all circuit sensing, computation, signal transduction, and output elements in the same self-assembled molecular complex, which reduces diffusion-mediated signal losses, lowers metabolic cost, and improves circuit reliability. We demonstrate that ribocomputing devices in E. coli can evaluate two-input logic with dynamic range up to 900-fold and scale them to four-input AND, six-input OR, and a complex 12-input expression (A1 AND A2 AND NOT A1*) OR (B1 AND B2 AND NOT B2*) OR (C1 AND C2) OR (D1 AND D2) OR (E1 AND E2). Successful operation of ribocomputing devices based on programmable RNA interactions suggests that systems employing the same design principles could be implemented in other host organisms or in extracellular settings.

Two-dimensional semiconducting transition metal dichalcogenides (TMDCs) like molybdenum disulfide (MoS 2 ) are generating significant excitement due to their unique electronic, chemical, and optical properties. Covalent chemical functionalization represents a critical tool for tuning the properties of TMDCs for use in many applications. However, the chemical inertness of semiconducting TMDCs has thus far hindered the robust chemical functionalization of these materials. Previous reports have required harsh chemical treatments or converting TMDCs into metallic phases prior to covalent attachment. Here, we demonstrate the direct covalent functionalization of the basal planes of unmodified semiconducting MoS 2 using aryl diazonium salts without any pretreatments. Our approach preserves the semiconducting properties of MoS 2 , results in covalent C-S bonds, is applicable to MoS 2 derived from a range of different synthesis methods, and enables a range of different functional groups to be tethered directly to the MoS 2 surface. Using density functional theory calculations including van der Waals interactions and atomic-scale scanning probe microscopy studies, we demonstrate a novel reaction mechanism in which cooperative interactions enable the functionalization to propagate along the MoS 2 basal plane. The flexibility of this covalent chemistry employing the diverse aryl diazonium salt family is further exploited to tether active proteins to MoS 2 , suggesting future biological applications and demonstrating its use as a versatile and powerful chemical platform for enhancing the utility of semiconducting TMDCs.

Noroviruses are a primary cause of gastroenteritis and foodborne illness with cases that affect millions of people worldwide each year. Inexpensive tests for norovirus that do not require sophisticated laboratory equipment are important tools for ensuring that patients receive timely treatment and for containing outbreaks. Herein, we demonstrate a low-cost colorimetric assay that detects norovirus from clinical samples by combining paper-based cell-free transcription–translation systems, isothermal amplification and virus enrichment by synbodies. Using isothermal amplification and cell-free RNA sensing with toehold switches, we demonstrate that the assay enables detection of norovirus GII.4 Sydney from stool down to concentrations of 270 aM in reactions that can be directly read by eye. Furthermore, norovirus-binding synbodies and magnetic beads are used to concentrate the virus and provide a 1000-fold increase in assay sensitivity extending its detection limit to 270 zM. These results demonstrate the utility of paper-based cell-free diagnostic systems for identification of foodborne pathogens and provide a versatile diagnostic assay that can be applied to the concentration, amplification and detection of a broad range of infectious agents.

No abstract

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.

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.

The ability to identify single-nucleotide mutations is critical for probing cell biology and for precise detection of disease. However, the small differences in hybridization energy provided by single-base changes makes identification of these mutations challenging in living cells and complex reaction environments. Here, we report a class of de novodesigned prokaryotic riboregulators that provide ultraspecific RNA detection capabilities in vivo and in cell-free transcription-translation reactions. These single-nucleotide-specific programmable riboregulators (SNIPRs) provide over 100-fold differences in gene expression in response to target RNAs differing by a single nucleotide in E. coli and resolve single epitranscriptomic marks in vitro. By exploiting the programmable SNIPR design, we implement an automated design algorithm to develop riboregulators for a range of mutations associated with cancer, drug resistance, and genetic disorders. Integrating SNIPRs with portable paper-based cell-free reactions enables convenient isothermal detection of cancer-associated mutations from clinical samples and identification of Zika strains through unambiguous colorimetric reactions.