Given the irreversible neurotoxicity and high detection rates of chlorpyrifos (CPF) in agricultural products, developing sensitive and efficient sensors for CPF is highly urgent. Herein, we constructed a novel Locked-Activator-Cas12a (LAC) strategy with process “slimming” capability. It is predicated on a DNA activator that possesses two states acting as a crucial intermediary. The biosensor's efficacy is further enhanced by the copious generation of activators induced by the target, ensuring the unimpeded progression of the entire reaction, thereby facilitating precise and reliable CPF detection in complex matrices. Furthermore, we used bioinformatics resources, including AutodockVina, to explore the molecular interaction mechanism of CPF in binding to its aptamers through molecular docking simulations. Employing a two-step detection workflow, this approach is straightforward and readily scalable for detecting other organophosphorus pesticides by substituting the aptamer sequences. It is envisioned that these biosensors can be applied as a strategy robustness tool in agricultural samples, ultimately contributing to enhanced human health and well-being.
CRISPR-Cas12a, an RNA-guided nuclease, has been repurposed for genome editing and molecular diagnostics due to its capability of cis-cleavage on target DNA and trans-cleavage on non-target single-strand DNA (ssDNA). However, the mechanisms underlying the activation of trans-cleavage activity of Cas12a, particularly in the context of split DNA activators, remain poorly understood. We elucidate the synergistic effect of these activators and introduce the concepts of induced targeting effect and exon-unwinding to describe the phenomenon. We demonstrate that upon binding of split DNA activators adjacent to the Protospacer Adjacent Motif (PAM) to the Cas12a ribonucleoprotein (Cas12a–RNP), a ternary complex form that can capture and interact with distal split DNA activators to achieve synergistic effects. Notably, if the distal activator is double-strand DNA (dsDNA), the complex initiates exon-unwinding, facilitating the RNA-guide sequence's access. Our findings provide a mechanistic insight into action of Cas12a and propose a model that could significantly advance our understanding of its function.
危险品化学品经营许可证（带存储）