A Self-Locking DNAzyme Platform for Highly Specific Sensing of Low-Abundance Single-Base Mutations

Abstract

Analysis of low-abundance single-base mutations remains technically challenging because of the difficulty in distinguishing minor sequence variations from wild-type backgrounds. Herein, we report a self-locking DNAzyme (SLOD) biosensor that suppresses spontaneous activation through a thermodynamically stabilized intramolecular inactive conformation. Unlike conventional hairpin DNAzymes that rely on direct target-induced activation and often exhibit limited capability for single-base discrimination, the proposed SLOD platform operates through a decoupled mechanism in which single-base mutation recognition is performed through flap endonuclease 1 (FEN1), whereas DNAzyme activation is driven by polymerase-generated competitive unlocking strands. This design separates mutation discrimination from signal generation, thereby improving specificity against wild-type background. The platform enabled quantitative detection of EGFR and KRAS mutations at mutant allele fractions as low as 0.1%–0.5%. A dual-color format was further established for the simultaneous detection of EGFR L858R and T790M in clinical samples. In a cohort of 35 clinical samples, the SLOD platform achieved an overall qualitative concordance of 97.1% (34/35) with the ARMS-PCR. In addition, the programmability of the biosensor was demonstrated through the AND, OR, and INHIBIT logic operations. This work establishes a low-background, single-base-discriminating, and modular DNAzyme biosensor platform for single-base mutation detection and clinically relevant nucleic acid sensing.