Phosphorothioate-Modified Hairpin G-Triplex Reporter-Assisted Split CRISPR/Cas12a-Powered Biosensor for “Turn-On” Fluorescent Detection of Nucleic Acid and Non-Nucleic Acid Targets

CRISPR/Cas12a-powered biosensors with guanine (G)-rich sequence reporters (e.g., G-quadruplex and G-triplex) are widely used in detection applications due to their simplicity and sensitivity. However, when these biosensors are employed for molecular detection in complex samples, they may encounter difficulties such as high background signal and susceptibility to interference because of the "turn-off" signal output. Herein, we explore, for the first time, a set of phosphorothioate (ps)-modified G-quadruplex (G4) and G-triplex (G3) sequences that can bind with thioflavin T (ThT) in an active split CRISPR/Cas12a system (SCas12a) to generate a "turn-on" fluorescent signal. To apply this new phenomenon, we develop a universal SCas12a-powered biosensor for "turn-on" fluorescent detection of nucleic acid (miRNA-21) and non-nucleic acid (kanamycin) targets by using ps-modified hairpin G3 as a reporter (SCas12a/psHG3). Target recognition activates SCas12a's <i>trans</i>-cleavage activity, leading to cleavage at the loop region of the psHG3 reporter. The released prelocked psG3 DNA binds ThT to produce a strong fluorescence signal. Without preamplification, this strategy can detect miRNA-21 with a detection limit of 100 fM. Moreover, the SCas12a/psHG3 system was further utilized for detecting kanamycin by incorporating its aptamers, enabling the detection of kanamycin at concentrations as low as 100 pM. This work is the first to develop a "turn-on" SCas12a/psHG3 system, showcasing its improved performance and wide range of applications in synthetic biology-based sensing technology.