Rapid, multiplexed, and enzyme-free nucleic acid detection using programmable aptamer-based RNA switches

Abstract

Rapid, simple, and low-cost diagnostic technologies are crucial tools for combating infectious disease. We describe a class of aptamer-based RNA switches, or aptaswitches, that recognize target nucleic acid molecules and initiate the folding of a reporter aptamer. Aptaswitches can detect virtually any sequence and provide an intense fluorescent readout without intervening enzymes, generating signals in as little as 5 min and enabling detection by eye with minimal equipment. Aptaswitches can be used to regulate the folding of seven fluorogenic aptamers, providing a general means of controlling aptamers and an array of multiplexable reporter colors. By coupling isothermal amplification reactions with aptaswitches, we reach sensitivities down to 1 RNA copy/μL in one-pot reactions. Application of multiplexed all-in-one reactions against RNA from clinical saliva samples yields an overall accuracy of 96.67% for detection of SARS-CoV-2 in 30 min. Aptaswitches are thus versatile tools for nucleic acid detection that are readily integrated into rapid diagnostic assays.