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 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.

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.

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.

Kaiyue Wu

Department of Biomedical Engineering,
Molecular Biology,
Cell Biology & Biochemistry Program,
Graduate School of Arts and Sciences,
Molecular Biology, Cell Biology & Biochemistry Program, Graduate School of Arts and Sciences

Boston University

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