In an unexpected experiment, we discovered a novel property of λ Exo. An experiment originally intended to utilize λ Exo for constructing DNA circuits unexpectedly observed the degradation of DNA. Upon further investigation of this phenomenon, we found that when 5'-phosphorylated single-stranded DNA (pDNA) coexisted with double-stranded DNA (dsDNA) containing complementary sequences of pDNA, the pDNA was digested into nucleotides by λ Exo.

Through single-molecule binding analysis and enzymatic assays, we have confirmed this new property of λ Exo. The experiments demonstrated that λ Exo can bind 5'-phosphorylated single-stranded DNA to the complementary regions of double-stranded DNA and DNA-RNA duplexes at room temperature, without the need for PAM-like motifs. After binding, in the presence of Mg²⁺, λ Exo digests the pDNA into nucleotides. This characteristic enables the direct detection of dsDNA and DNA-RNA duplexes through a simple fluorescence quenching assay.

The discovery of the λ Exo-pDNA system not only allows for rapid, isothermal detection of pathogen genes and single nucleotide mutations but also supports logic and amplification operations of ds-nucleic acid responsive DNA circuits. Moreover, the system permits fluorescent imaging in situ of genomic loci, demonstrating excellent single-mismatch sensitivity and the ability to recognize a broad range of mutations.

The discovery represents a significant advancement over existing technologies and is expected to pave the way for the development of next-generation molecular diagnostics, DNA computing, and genetic imaging tools. Additionally, variants of λ Exo that retain target binding activity in the presence of Mg²⁺ while eliminating nuclease activity could enable in situ imaging in living cells. These variants also hold promise for the development of gene expression interference tools similar to CRISPRi, and the creation of delivery tools to introduce the λ Exo-pDNA system into living cells could further expand its application prospects in the biomedical field.