Triplex DNA Clamp Regulates Cas12a Activation for ssDNA and RNA Sensing

We present a molecular strategy that enables the programmable activation of CRISPR-Cas12a system in response to Triplex DNA formation triggered by single stranded DNA (ssDNA) or RNA inputs. This assay, called Triplex Regulated Activation of Cas12a for ssDNA and RNA sensing (TRACR), leverages the highly specificity of clamp-like triplex structures to control a strand displacement mechanism within a rationally designed DNA hairpin (PAM-Switch). Upon displacement and PAM complementation, the Cas12a ribonucleoprotein (RNP) is activated, initiating trans-cleavage and producing a concentration-dependent fluorescent signal. By decoupling target recognition (via triplex formation) from direct hybridization with the Cas12a-crRNA complex, TRACR eliminates the need for target-specific crRNAs. This design also allows multiple, spatially resolved detection of distinct nucleic acid targets within a single Cas12a reaction. Through the use of triplex-based clamps, TRACR achieves high specificity for single-nucleotide variants and supports the detection of both ssDNA and RNA targets across a broad range of lengths (10–20 nucleotides). TRACR addresses key limitations in current Cas12-based diagnostics and opens new avenues for nucleic acid sensing.