Rapid and Multiplexed Nucleic Acid Detection using Programmable Aptamer-Based RNA Switches

Rapid, simple, and low-cost diagnostic technologies are crucial tools for combatting infectious disease. Here, we describe a class of aptamer-based RNA switches called aptaswitches that recognize specific target nucleic acid molecules and respond by initiating folding of a reporter aptamer. Aptaswitches can detect virtually any sequence and provide a fast and intense fluorescent readout, generating signals in as little as 5 minutes and enabling detection by eye with minimal equipment. We demonstrate that aptaswitches can be used to regulate folding of six different fluorescent aptamer/fluorogen pairs, providing a general means of controlling aptamer activity and an array of different reporter colors for multiplexing. By coupling isothermal amplification reactions with aptaswitches, we reach sensitivities down to 1 RNA copy/microL in one-pot reactions. Application of multiplexed one-pot reactions against RNA extracted from clinical saliva samples yields an overall accuracy of 96.67% for detection of SARS-CoV-2 in 30 minutes. Aptaswitches are thus versatile tools for nucleic acid detection that can be readily integrated into rapid diagnostic assays. ### Competing Interest Statement Z.Y., A.A.T., D.M., and A.A.G. have filed patent applications US20190256898A1 and WO2022046978A3 that describe aspects of this technology. A.A.G. is a cofounder of En Carta Diagnostics Inc. ### Funding Statement This work was also supported by an NIH Director New Innovator Award (1DP2GM126892), an NIH U01 award (1U01AI148319-01), an NSF RAPID award (2029532), the Gates Foundation (OPP1160667), Arizona Biomedical Research Centre funds (ADHS16-162400, CTR051763), an Alfred P. Sloan Fellowship (FG-2017-9108), an NIH R21 award (1R21AI136571-01A1), and Canadian Food Inspection Agency funds (39903-200137) to A.A.G, along with funding from Salud Digna Research Council (SDI-20166). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: THE IRB of Arizona State University waived ethical approval for this work on November 3, 2020 because: "The IRB determined that the proposed activity is not research involving human subjects as defined by DHHS and FDA regulations." This decision was reached because the study involved de-identified samples. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The main data supporting the results in this study are available within the paper and the Supplementary Figures. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.