Rapid, Multiplexed, and Enzyme-Free Nucleic Acid Detection Using Programmable Aptamer-Based RNA Switches

Rapid, simple, and low-cost diagnostic technologies are crucial tools for combatting infectious disease. We describe a class of aptamer-based RNA switches or aptaswitches that recognize target nucleic acid molecules and initiate 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 minutes and enabling detection by eye with minimal equipment. Aptaswitches can be used to regulate folding of seven fluorogenic aptamers, providing a general means of controlling aptamers and an array of multiplexable reporter colors. 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 minutes. Aptaswitches are thus versatile tools for nucleic acid detection that are readily integrated into rapid diagnostic assays.