LNA-Modified DNAzyme Nanomachines Enable Physiological-Temperature Recognition of Double-Stranded DNA

Sequence-specific detection of double-stranded DNA (dsDNA) under physiological conditions remains challenging because the complementary strand is inaccessible to conventional oligonucleotide probes, often requiring thermal denaturation or protein-assisted strand separation. Here, we developed a binary DNAzyme (BiDz) sensor for sequence-specific dsDNA detection at 37 °C. While the all-DNA BiDz failed to recognize dsDNA efficiently, incorporating alternating locked nucleic acid (LNA)/DNA residues into the analyte-binding arms enabled efficient strand invasion and markedly improved signal generation. Integration of the optimized BiDz into a multivalent DNAzyme nanomachine (DNM) further enhanced target binding and reduced the detection limit for dsDNA amplicons to 25 pM, a 3.5-fold improvement over BiDz alone. The DNM also detected long plasmid dsDNA at nanomolar concentrations while maintaining excellent discrimination of centrally located single-base mismatches. Both BiDz and DNM demonstrated excellent discrimination of both A-C and the most challenging G-T mismatches. These findings demonstrate that combining alternating LNA/DNA-modified binding arms with a multivalent DNM architecture enables sensitive and highly specific dsDNA detection under physiological conditions, providing a promising platform for isothermal nucleic acid analysis.