Engineered RNA biosensors enable ultrasensitive SARS-CoV-2 detection in a simple color and luminescence assay

Abstract

The continued resurgence of the COVID-19 pandemic with multiple variants underlines the need for diagnostics that are adaptable to the virus. We have developed toehold RNA–based sensors across the SARS-CoV-2 genome for direct and ultrasensitive detection of the virus and its prominent variants. Here, isothermal amplification of a fragment of SARS-CoV-2 RNA coupled with activation of our biosensors leads to a conformational switch in the sensor. This leads to translation of a reporter protein, for example, LacZ or nano-lantern that is easily detected using color/luminescence. By optimizing RNA amplification and biosensor design, we have generated a highly sensitive diagnostic assay that is capable of detecting as low as 100 copies of viral RNA with development of bright color. This is easily visualized by the human eye and quantifiable using spectrophotometry. Finally, this PHAsed NASBA-Translation Optical Method (PHANTOM) using our engineered RNA biosensors efficiently detects viral RNA in patient samples. This work presents a powerful and universally accessible strategy for detecting COVID-19 and variants. This strategy is adaptable to further viral evolution and brings RNA bioengineering center-stage.

Diagnostic assay and technology advancement for detecting SARS-CoV-2 infections causing the COVID-19 pandemic

“…Isothermal amplification techniques range in terms of total quantity of primers and enzymes employed, the temperature at which the amplification takes place, and the sorts of templates used. Table 2 [75][76][77][78][79][80][81][82][83][84][85][86][87] summarizes the several isothermal amplification platforms established for detecting SARS-CoV-2 in both laboratory and clinical samples.…”

“…Summary of the several isothermal amplification platforms established for SARS-CoV-2 diagnostics in both laboratory and clinical samples [Refs [75][76][77][78][79][80][81][82][83][84][85][86][87]. …”

Progress and Challenges of Point-of-Need Photonic Biosensors for the Diagnosis of COVID-19 Infections and Immunity

“…To visualize the result, the analyte can be labelled with fluorescent tags. There are also biosensors with different mechanisms developed for nucleic acid detection, such as the fluorescence-based toehold switch sensor for SARS-CoV-2 RNA detection [ 22 ]. The target viral RNA is amplified prior to detection.…”

Update on the Development of Toehold Switch-Based Approach for Molecular Diagnostic Tests of COVID-19

“…The development of toehold switch-based COVID-19 diagnostic devices has been reported since 2021 ( Table 1 ). Chakravarthy et al [ 40 ] developed PHAsed NASBA-translation optical method (PHANTOM), a toehold switch-based biosensor coupled with isothermal NASBA (nucleic acid sequence-based amplification) to detect the SARS-CoV-2 genome ( Figure 2(a) ). In the PHANTOM system, RNA from SARS-CoV-2 in a patient's sample was extracted and then amplified isothermally using NASBA.…”

“… General scheme of the currently developed toehold switch-based diagnostics for COVID-19: (a) a system developed by Chakravarthy et al [ 40 ] which utilized NASBA (nucleic acid sequence-based amplification) for amplifying trigger RNA from patient's nasopharyngeal swab sample and toehold switch-based biosensor with lacZ as a reporter gene, (b) a system developed by Köksaldl et al [ 41 ] which utilized NASBA for amplifying trigger RNA from patient's nasopharyngeal swab sample and toehold switch-based biosensor with superfolder GFP as a reporter gene, (c) a system developed by Park et al [ 42 ] which utilized reverse transcription loop-mediated amplification (RT-LAMP) for amplifying trigger RNA from patient's saliva sample and toehold switch-based biosensor with lacZ as a reporter gene, and (d) a trigger RNA amplification-free system developed by Hunt et al [ 43 ] which detected SARS-CoV-2 RNA from patient's saliva sample using toehold switch-based biosensor with NanoLuc as a reporter gene. …”