Dual-site responsive module-triggered CRISPR/Cas12a switch with enhanced reaction kinetics for specific detection of PSA

Abstract

Despite advances in DNA-based nanodevices for tumor biomarker detection, the pursuit of enhanced sensitivity and specificity remains a profound challenge. In this study, we designed an integrated dual-site responsive module as the AND logic gate that responded to both prostate-specific antigen (PSA) and apurinic/apyrimidinic endonuclease 1 (APE1), triggering a tetrahedral DNA nanostructure (TDN)-accelerated CRISPR/Cas12a signal switch for PSA detection. In detail, the dual-site responsive module was a T-shaped probe comprised of three DNA strands, which contained PSA aptamer and apurinic/apyrimidinic (AP) sites, significantly improving the specificity of prostate cancer screening by orthogonal recognition of PSA and APE1. When both PSA and APE1 were present, PSA could specifically bind with the aptamer, while APE1 catalyzed and cleaved the AP sites, releasing a part of one auxiliary strand as the mimic target (MT). To enhance the trans-cleavage efficiency of the CRISPR/Cas12a signal switch, we parallelly constructed the TDN with extended non-target strands (NTS) at each vertex, which could capture the MT to form double-stranded DNA (dsDNA) activators at the TDN vertices (TDN-activators) for triggering the CRISPR/Cas12a signal switch. Benefiting from the rigid framework of TDN, the TDN-activators not only increased the local concentration of Cas12a but also enabled a dominant conformation that was more favorable for recognizing and activating the Cas12a-crRNA complex, which demonstrated a 4.06-fold increase in trans-cleavage efficiency compared to that of dsDNA-activated Cas12a. This work applies the T-shaped probes and TDN-accelerated CRISPR/Cas12a signal switch to achieve the specific and sensitive electrochemiluminescence (ECL) detection of PSA from 1.0 × 10−5 to 10 μg/mL with a low limit of detection (LOD) of 4.5 × 10−6 μg/mL, offering a promising tool for early clinical screening of prostate cancer.