Quality Control Certification of RNA Aptamer‐Based Detection

Aptamers are single-stranded DNA or RNA molecules with a defined tertiary structure for molecular recognition. Numerous RNA aptamers with excellent binding affinity and specificity have been reported; they constitute an attractive reservoir of molecular recognition elements for biosensor development. However, RNA is relatively unstable owing to spontaneous hydrolysis and nuclease degradation. Thus, RNA aptamer-based biosensors are prone to producing false-positive signals. Here, we present an RNA aptamer biosensor design strategy that utilises an internal control to distinguish target binding from false-positive signals. The sequence of a chosen RNA aptamer is expanded so that it can form three consecutive short RNA-DNA duplexes with 1) a quencher-labelled DNA strand (Q(1)DNA), 2) a dual-fluorophore-labelled DNA strand (F(1)DNAF(2)) and 3) another quencher-labelled DNA strand (Q(2)DNA). The addition of a target releases Q(2)DNA from the duplex assembly, and produces the expected positive signal from F(2). However, the authenticity of target recognition is validated only if no signal is generated from F(1). We have successfully engineered two fluorescent reporters by using an RNA aptamer that binds thrombin and one that binds theophylline. Both reporters show the expected binding affinity and specificity, and are capable of reporting system malfunction when treated with nucleases and chemical denaturants. This strategy provides a simple and reliable way to ensure high-quality detection when RNA aptamers are employed as molecular-recognition elements.