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

Abstract

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.

Optimization of RNA Pepper Sensors for the Detection of Arbitrary RNA Targets

“…The first design strategy (Figure A), named the toehold-mediated Pepper sensor, contains a 5′ toehold sequence as part of the target-binding region (orange), the core hairpin structure, and the Pepper aptamer sequence at the 3′ end (blue and green), following a strategy proposed in refs − . The Pepper stem is initially sequestered with the b * domain of the core hairpin, thereby preventing binding of the fluorogen HBC 530 to the sensor RNA and resulting in a nonfluorescent (OFF) state in the absence of the target RNA.…”

“…We demonstrated the in vitro detection of RNA targets of lengths varying between 20 and 35 nt. We devised approaches to optimize toehold- and loop-mediated sensors’ performances for arbitrary target RNA, building upon previous work that established such RNA sensors for diagnostic applications using other aptamers. − Our toehold-mediated sensor design originally included two 2-nt bulges in the hairpin structure to prevent premature transcription termination and increase the thermodynamic stability of target–sensor interactions. However, our results suggest that the ON/OFF ratio of these sensors can be improved by approximately 10-fold through the removal of the bottom bulge in the hairpin structure and the incorporation of two clamps (Figure D).…”

“…In this study, we introduce and demonstrate design approaches and generalizable optimization strategies for Pepper-based RNA sensors that can achieve up to 300-fold fluorescence enhancement in vitro . We demonstrate two types of Pepper sensor designs adapted from previous work demonstrating switchable RNA sensors ,− that take inspiration from RNA riboswitches developed for translation control in bacteria. , In both designs, the 5′ end of the Pepper stabilizing stem is sequestered in the stem-loop structure of the sensor, which destabilizes Pepper and forces it to adopt a nonfluorescent configuration (Figure B,C) . The first design, named the toehold-mediated sensor, employs a 5′ toehold domain to initiate the interaction with the target RNA strand and induce refolding of the aptamer into its fluorogenic conformation (Figure B).…”

Clinical and laboratory diagnosis of Mayaro virus (MAYV): Current status and opportunities for further development

“…By coupling isothermal amplification reactions (e.g., RT-LAMP) with aptaswitches, it was found a great sensitivity (down to 1 RNA copy/μL). 99 Lastly, the authors validated the aptaswitches for SARS-CoV-2 detection using 60 patient samples and they found a diagnostic accuracy of 96.7%, 99 highlighting the potential of aptaswitches for rapid, high-capacity, and low-cost diagnostic assays. This class of PoC tool can promote access to health care through diagnosis and has great potential for producing reliable and rapid results to assist physicians during the treatment of patients.…”

“…99 After hybridisation with a target sequence, aptaswitches exploit a toehold-mediated interaction strategy to release this repression and promote fast activation (e.g., in as little as 5 min). 99 Using this technology, the authors have developed aptaswitches for 10 human pathogens. By coupling isothermal amplification reactions (e.g., RT-LAMP) with aptaswitches, it was found a great sensitivity (down to 1 RNA copy/μL).…”